«2 United States Patent Green et al. US007968321B] (10) Patent No. 4s) Date of Patent: US 7,968,321 BI Jun, 28, 2011 ETHANOL PRODUCTION BY MICROORGANISMS oa (75) Investors: Brian Green, Cambridge, MA (US): ‘Nikos Reppas, Cambridge, MA (US) Dan Robertson, Cambridge, MA (US) (73) Assignee: Joule Unlimited, Ine, Cambridge, MA ws) (4) Notice: Subject to any diselaimer, the tem of this patent is extended or adjusted under 35 USC. 154(b) by 0 days. an @y Appl.Nos 1384470 PCT Filed: Sep. 3, 2009 (86) PCT No. $371 (0), (2).(4) Date PCT/USZ009/055949 Jan, 21, 2014 (87) PCT Pub, Nos WO2010/044960 PCT Pub, Date: Ape. 22,2010 Related US. Application Data Provisional application No. 61/184,757, filed on Jun. 5, 2008, provisional application No. 61/121,532, fled ‘on Dee. 10, 2008, provisional application No. 61/106,583, fled on Oxt. 17, 208, (60) G0) Foreign Application Priority Data Mar.3,2009 (vo) PCT/US2009035937, GD Inc. C12 706 erp vo RN 10 (2006.01) us.cl. 435/161; 435/41; 435/245 Field of Classification Search None See pplication ile for complete search history. (2006.01) (2005.01), (32) 68) 66) References Cited USS. PATENT DOCUMENTS 4920487 A188 Armstong etal 62.516 A 111992 Ingram ea, Sapea7s A 411904 Kime a 6306639 BI 102001, Woods ea {640.006 Bi" 2002. Pomo et 632631 BI 102003 Shuster etal ‘100.606 B2 "33004, Woods etal ‘T026827 2 412006 Fale etal 722331 BL 102006 Elseneich ta 2oosdoiazat AL “N2005 Chen amps0124010 AL 62005 Shon eal 20080239179 AL 102005. Srayet al doowor7eior AL "7/2008 Lee dovoo00y?39 AL 1/2010 doroosve6s? AL 122010 zo.vooossel AL ‘1/2011 aL BI BL 2 BD BI Al AI A al FOREIGN PATENT DOCUMENTS wo wo2m70s77 12007 WO WO2007 136769 2007 WO WO 2007 139995 2007 WO — Wo2m9082100 52009 WO —Wo2moKns —- 009 WO Worumst0s714 £009 WO WO2I004O 42010, (OTHER PUBLICATIONS (Cho, H. eal, “Broerihia col Thiostrse I, Molecular Cloning and Sequencing ofthe Stuctral Gene and Identfeation a8 & Perplsmic Enzyme" The fournal of Biological Chemisty. May 8, 1903, pp. 0238-9248, vol. 268, No. 13 CCobca, Rc al, "Functional expression of rat GLUT 1 glucose transporter in Dicttelium dicotdeum,” Biachem. J. 198, Bp. oHPIs, ol 31S Deng. Met al. “Ethanol Synthesis by Genetic Engineering in Cynnibactetar” Apple aid Emiroimental Mierbitogy, Fe, 1909, pp. £23-528, ol. 6S, No.2 niga, NU. etal, “Gone laetivation inthe Cyanobactrium Shmechococeus sp. BCC 7002 and the Giese Sul Bacterium Chlootiun tepam Using in Viuoomade DNA Consirits ad Natural Transfonmation” Meds tn ioleruar Biology, 2008, 3256, vol 274 Genin Seta, "Ralsion solanaccarum: Scores of Major Patho pon Unveiled by Analysis of ls Geneore,” Molecular Plant Patho 19),2002,pp. LIL-IIS, wo. 3.No.3 bo, N-ctal Genetically Enginsred Sacchoromces Yeast Capable of Esfosive Cofemmentaion of Glncoe and Xslose? applied and Environmental Microbiology, May 199, pp 182-1859, 0 64, No, 5 Tnokuma, K. ct al, “Characterization of Enzymes Involved inthe thane reduction of Moorella sp. HUCDS-L Arch Mikrobiol, 2007, pp. 37-48, ol 188 vais (ME ot al. “Improved. Genetic Trusformation of the Thermopliie Cyanobactert, Thermosynechovoceur elongata BP-L?" Plant and Cell Physiology, 2004, 9p. 171-175, vo. 45. No.2. Kalncniks, etal "NADH Dehydrogenase Deficiency Rests in Tow Respation Ravand Improved Acrobie Growth of Z)momonas mobs" Mirabole, 2008, p. 988-994, ol 134 Kalnenieks, Ue ab “Respracory Behaviour of a Zymomonas ‘mots adh kar” Mutant Suppntsthe Hypothesis of Two Alcohol etyogenase Ioerzymes Catalysing Oppsite Reactions.” FEBS eters, S00. pp SOS4SO8%, OL SBD. Ralachae eet a“ ‘Novel bifuncdonal Wax Ester Synthase Aeyl-CoA-Diyalysero] Acyliranferse Mattes Wax Fter and Triacyelyera Blosyahess in Aemerahacrer”—_caleoueteus DPI" The Journal of Biological Ohmi, 2003, 9. 8075-8083, wo 278 No.0, Kalschever, Rc al, “Miroisc:Exohrihia coll Engineered for Fuel Production crbiolgy, 2008p. 2529-2836 so. 182 Kimura Av et al, "A High TemportureSenstive Mutant of Synechococcus sp PCC 7003 with Modifications in the Endogenous Plasmid pAQL” Plan aa Col Physology. 2002, pp 217-233 Yo. 443 No. (absttact nly) (Online) [Reieved Nov. 28, 2009] Reine fromthe internet URL ip pepoxtondoumal sea conteabaract 482317 (Continved) Primary Examiner — Suzanne Noakes Assistant Examiner — Jae W Lee (4) Attornes, Agent or Frm Wos LLP on ABSTRACT The present disclosure relates to methods and compositions {or engineering pholoautolrphic onganismis to convert a= bon dioxide and ight into fatty acid esters and other mol- ecules, including biofuels. The molecules are then secreted by the onzanism into a growth meson, 11 Claims, 38 Drawing Sheets ‘Chang B. Hong; Fenwie &US 7,968,321 BI Page 2 OTHER PUBLICATIONS Hoy, A et al, “Topology ofthe Esceriohia coll wip Suga Phosphate Transpnte Analyzed by Using Tp Fasions.” nal ‘of Bacteriology, Ap. 1990, pp 1688-1633, vol. 172, No. Marraehi Hee als > Mechanic Diversity and Regulation of Type 1 Fatty Acid yates” Biochemical Sotey Thamacons, 2003. pp. 1080-1088, vol. 30, Pat 6 Moon. J. IL et al. “Structues of oaDependest Alcohol Delgiogemse 2 trom Zimomanas nobls 28 with and wihout ADs Colston? Journal of Moeculr Biology, 2011, pp 41323, ol. 407 (doi 10-10165 jn 2011 01.088). PCT Internation Search Report and Writen Opinion, PCT Appl ‘ation Nov PCTIUSD009°039937, Aug. 3.2009, 15 pages PCT International Search Report and Writen Opinon, PCT Appl ‘ation No. PCTUS2000/058949, Mar. 1S, 2010, sven pages. Rock Cet a, Increased Unsaturated Fatty Acid Proton Asso- ‘ated with a Suppressor ofthe 4s) Mutation in Escherichia ‘ol urna of Bacteriology, 196, pp. S382-5987, 178 NO. 18 United States Office Action, US. App. No. 12867.738, Jan. 27 201. ten pages United States Ofce Action, US. Appl No, 121867738, Mar 30, 2011, sc popes. US. Appl No. 61065,292, filed Fes 8, 2008, US. Appl No. 285,712 file Ang. 6, 2010, US. Appl No. 12368160, ied Fe 9, 2009, US. Appl No. 12368060, ile Fes 9, 2000, ‘Weiser, Pe a, “Funtina Expression ofthe Glucose Transporter of Zsmomanas mois Leadsto Restoration of Gloose and Fructose Uptake in Hscerichia coli Mutants and Provides Evidence for Is Faclifator Action” Jourel of Bacterolgy, Jun. 1995, pp. 3381- 3354, 0 177,No- I ‘Woodger. et a, Inorganic Caron Limitation Induces Teanscips Encoding Components of the CO,-Concenating Mechanism in Synerhovoceus sp. PCCTDA2 through & Redolntependent Pathe ‘¥a" Plan Physolog, Dee 2003, pp. 2069-2080, vo. 133. No.4US 7,968,321 BI Sheet 1 of 38 Jun, 28, 2011 US. Patent ve sunols covrnpetier=cowitoe| sizre | ssotuommnes | eset dowitoe | _ssvs cov 78 = TaCeN HOWN) + dOWaFOUe Teo eyenowees | emmrpaaowrem |e sample ov ious = gov ioehiainhue joomouoicey | dowvonhomfire | vee OV TORTOAI-C = HAOWN + aOW FORDE 0 BiUoUae| eaROApR cORTEROIE | _—_ Og) - ray fowoiwrs +200 = dowiuow + sovvee | Lv V'eT| soo mUeNoWeES | oper dovifowciere | age) en2/e5ue5 ye0+ davinwoiewe doy veovuoew | get ee | wo muauMNaeS aowweo-nioeu | aoe YO Kiera = 209 eal vooveS | evra | woe mutieyoRs | SeNTboqie yoO¥ieDe | —GVORDO ere/oesuea wor dowsnce= dove woriece | seve | soo manos doves | Hoe ove00 Joueind-+ = (HaGWN HAWN) + leueInG Teurind = HOWN + = wire geo eyav0WISS PT eT TOIT sBuO60/PAUP ONO ‘sBUebOPISP SPRIRDR ogra yoo i208 + eyeing = siesoe + yoo4uang | ez aquojsiey | _oscisuea-yoo sxeuhing pd ‘uraytingojsoe ‘sesueBODT RD Hilal + voo-stna = yoo Auoio.s unpis0%9 ‘wooing Peg "yoo? uqoI = yoo RIANAKCIPIA WPaewopewsy | —_eseiephy yoorioue | osi woo ‘eqdenne “Wuinaivointie = HAGWN + woothisoen|908 aqunsiey | eseronpas yoosmisseojene | gous ‘yaouine yoo + yoo Hs80}808 = yoo MiB0e z enue eseowoieyeien | youd roueng “qrgeur roueuja = HOWN = epfuaprsoe seuowowtz | exeuebaiphyeo oyone | guns ger 200 + epkuepreeoe = syennuhd stuowowsz | _aseyixoqieoap oyennukd ond owes wsyuet0 wonseoy aoa eydwexg ewszva| ewe onpoid stsqueSuoo19q}y Suronposd-sonposd poseg-noqaes Supxy-409 Bupeauysuy 20) sau LawiduaxyUS 7,968,321 BI Sheet 2 of 38 Jun, 28, 2011 US. Patent s}aunol Tee ond : - a compan -omcoee-coud| ewers| emetic woes a vane SS eee = —— . oagnons200 supa mesreaerd| —wryvy| anit = "7 cae om emipeomdcmaneoerd| —yevve| smile re — coe Adipeoias geez] seuowopnas, yea eo byes ds ‘woo-veipeaies = yoo-vusons + yooHMae| —pyy-yez| —snosccopoway | PSUS YEO-KEpeCa | Jeo vay —= ee a Ee eee ee conte cmunwantiererommtanivere| —orvzr| mowmora| —_emunaenienes | aoe ey ae ee ee ee 7 eee ree Sages asa eee el ieereee I ee concen = a emo «bso HaYA + muneo selling | eanatantep mere | tay ae a ramon | eartoniaep enuope | tune) ete a ~——LoiUS 7,968,321 BI Sheet 3 of 38 Jun, 28, 2011 US. Patent otsunold eyes YoorKeoe + sienine = eteiee + yooioKce | we eT, equ9jieej |_eseiesueinyog sieKine ped yoonKoiine = yao-vucdodmophre| —arrer| foo euouowey | ssoisphy yoo-feue | goed are eudarne YOO + lid = WoOrKinaAoNA-e uo eqn eseyuts vind | oeud veo -Kukinahvouphire = He¥N + vooMeDeoI00e asepnparyeo-Kysoeaeoe | ged vy09 + voo-misep0ig09 = yoo-tane z sseoiayereg | youd Hd asenpaiopx0 IHL = HOWN + Fe reppauedosd-e'; | eu P= HOWN +8 ‘sausboiphuep ephyepie He ‘opKyyeTUNs URONE + eeouiuesuan srewern = sieranBom-n + aeshnaouwe-r | gi-pe2 | goo euououdes ayeitingouwey | 1908 opr aenqouuEy = seUERE| ——siTTe| joensen | SseNOMeDEP SOWA) ——_yped SRUEIIS= HAN + CHN+ HBIEINBCIDD | yy | yoo eMenoUDe | oseuebaipKyep eyewerns | YunO Ser “BURY ‘eseusbosniuep SP=HAGWN + opAyopewes queore| erry | —_sedopgany sreuknaheorphiry | HOSHe "¥00 + sphiopreques BARE ‘eseuBo:piyop HAGWN's voo-Kuons | ween | wns ‘voowunens | —_gons| —_epauenay's ‘Seounoud ‘eeponpaiopxe opovedoxt-e't = HOYN + euedaidheompiire| zor EL ‘syeisaay repouedoie-e'y | eup “aeuouunaUd rmucdeusxouphie = 0048 syoisgapy | __eseerotyepjcison | e-igoup ‘evo 018046 = a-gossasf-us seafuiaraynars | ssejeydsous-eyo.e2K8us | zdd0) apq08oK6 ‘eesni0 ‘esevebospiuen | 28 = (HQWN HOWN} + a euoie9eKvo,phyp seofuaiey996s gesonKus | rava| _opavedaxte'y Tees os eseus8iKO ZO+HOWN + airenudass=1aa| srziris| _snoocaopouy ceveiweuudes | avedt wsyueBv0 uoRoee #03 orduexy owkzua| ous ponpoldUS 7,968,321 BI Sheet 4 of 38 Jun, 28, 2011 US. Patent quaunois seeauts iio + dd-onfo-y'zsomaiera-Ayouro-2 syeudeouciponso-s'2 |_stmatecvueuoedes eupvorrcz| zi've'y| yoo movoynss | eunahncviuouro-e | adr aeeUby ouaoraniwouo2iad's eupvovira z= lowsaro-crfinaui-g-z make chuous zed sounmorr=div| eb | yoo enenousg 309) lomrie-crvinaw-otdds supnioly= er PMMLODKNeUDeeald | ovLLe| Heo eouoyoss | + os) er foInhio-g Kyou sot= aay + dsesomiccrteoem-t | L9e' | yoo muevenaes ae -crinoops = geephyenreleoieg + eeanskd oo eucuates sm overdon ‘eojaeidea = ejeouexouGoIpAre wueraro eyeouENeoinhirn = 20+ HdQYN+POE MIE | eer yy | sevowapnady | se=vaBivoouow poe | ——_aMe ‘aupsna7e0 cov+ peeks = dove | pi zive| seatuoiewoes aspoiphy govetoe | ssw LOVIBET TiaGvNHOWN) + dow Mowe | Torerve+ | soo enpuerseg | eaeonpes aov-nele |e) aamepivep covitous= dovitoetiontre | ope | soo mucus eovitoesvonnsie | yaey gov eomwoinhie = HaGWN = cov R=SHIOE yoo enouewesa | esepnpe: dow-roeawirt | ove) ov =fo2e10F8 +200 » dOvinuowl + dOW' Ow seo enovewres | evemuse sovvoraie | age) Yyo0 + dO KuoIet = dO¥ + YOO"AuOeUL 109 ouoo4oe3 90) ‘woorKuoew = 203+ dive woes | zi ve | woo BRRUeIES avoame ese oesue yoo + dowinieoe= dove voorwieoe | ge's'e | yoo nouowss ovveoviieon| _yaqy|__evorede> 2 wstweBuo wonseoy oa] rcwexs eukea| 9499 ronpouaUS 7,968,321 BI Sheet 5 of 38 Jun, 28, 2011 US. Patent s43unold aude Topeae sweouruedhorphury = HidJOWN + eeuinAet suotsea! | eseronpa: voor feseaieoe 29 _sreumal = ejeouewwodeoupfirg-Axoaueo-t ‘eouRvadiaciph -rhvoaieee = eejeuhine ze) eo + seu ‘easels ~ Ziw=veoWere's eiousinamoz | geez | sechuoimyaoes | oseputs semurno-2 YOO z * YOO + soHEINGERO “umueaased szepens7+200 +yeqwuoiod | cacy | wmeuram | seemuks eungoxo-2 euopeionren yoo + sievodordxasphy ‘snoeqveme oy “C= HEWN 2 + Voo-Kuojeus smxeyovoiua | voo-ssuoreujeu020un} a Sy veorhuojeu=zoo+aiv+veotieoe | zi've| sosmuoueyoss | oteVroqiea yoormsoe | ayoRvoe 0 ‘pura aly + suojsorheainhup=covormetoinsap | ezize| _se/e9090 yeu via ‘eanskd + HOWN = ewepera | ezii+| fee BuEHeNSS Wa aire ‘suse Jagan = de huswadost eo) sean _dauowodest = de-ienKuoup + adoAteyiaup = Hcy saeit aoe rahunere-tepte rt) ae OWN + da-vuewedos: = Hey + adi use vinous Roptirts) Hest dat if-|-ue-zncyhipoure voip =a) = aaropto-yziouuhie-o-Aueus2z| ewer | moo envoys | -enakgougmopkir ods) wsjueBs0 wonoeou #03 order outa ous aonpoidUS 7,968,321 BI Sheet 6 of 38 Jun, 28, 2011 US. Patent srmunod WoO + aIpUNdOIeN9-ounle-2-uRone “N= woorusons +o jeni| artes | yoo sueweuses op a ene) = HAQWN + stu 21| eee | yoo ewevoyses op paphup 27-1 ophuopiewns-omundseys arent | 2e'ye'y | yoo muueseg wep ‘qeudsoud fuBdse “1+HeQyN=ephvepiewes-oievedse | uvEzy| yoo mwavenses se crriuedso-= div cmueder[ _vreez | noo euaneuey ‘384 een 22 ojeuedso-7 = geweIn6-7+ eimeneo|@o | ez | Hoo eumueYoeT ose urs ‘naibansou auojecien = eieouruedioniiy| —szsTe snaer 2seu0;er4L (oo) ‘ie OUE UeTTROIBAIy + YO ‘ron ooersuen “hove =cience + yoovouRuediopiuy | veez| —_unpusa | _voorKisinatxaIRhiy 20 oon coor Yyoo-Kourwedtxciphity = yoo-Kouueds | eees | wnpuiso19 saveowieoenun | age ‘spodouore ry yoo Kouawed-e = yoorKirenouphire) savoweuwex | _yoorwsnatrosnhire vo "woo Vaerentvainhy ‘ydoane “ee = HAGWN + ¥09 Morena e euasiens | eseionpes yoo-Weoeoie0e | gourd Yoo + yoo ‘adanne “Hosrenoiee = yoo i908 + oo~Kuardord enrsiens eseornoiovera| gna Buyer ‘yoo furdaxd = zHavd + yoo"Konine ssdopgeiy |_eeeuetorphvep voovoe | osasaosiy ‘yoo oiioe » voorhuodardioinhu-s wesayouowes | _ovcieiphy voo-Kove | eed _| "WoO + eBuoMordcunky snosnueane eeemrpe! son HdQWN Z + ¥Oo-AvOReU saxoyaiow9 | _ yoo-Muojeu ouosourye (ou) ‘WOO HURIRUI= 200 + dW + WOO-ViB0E Tee eroverosg | s8eihacie> voo-Ihece | GvORTDe ‘sroyBens0u uoieausiend = aeouEedhncsDhury snes essu0;er eo) ‘wsjueB10 onoven #93 ‘ieluoxg owkruz| ovo ronporsUS 7,968,321 BI Sheet 7 of 38 Jun, 28, 2011 US. Patent SRanot HOWN + a= nena osepnper + 2ushin = oush-iKdosshroaieon Lyi. | seotuergaces | sieimntoxez-eureti| 1947 > ayeansAd aK ; odd sfemuyjed jOUeY]e UMOUY ~¢——___ aqeyoe} pajajep 0q 0) eueb <—Y¥—. dad quesaid Apeaye eueb <———— | pesseidxeleno 0g 0) ue <———— 7 09 S@AI}EAWEP pue s}eulgonsUS 7,968,321 BL Sheet 17 of 38 Jun, 28, 2011 U.S, Patent e ‘Bid 09 % eyeujoons: eusAyje <— oy-p ajo § * ayeno wo yoo-\Ajs0e oe ayeanskd pejejep eq 0} euaB <—Y¥— —- dad quesaud Apeaye eus6 <——— passesdxaseao aq dv9o ——- Z090 “ dv5 wou Aemujed susjAuyUS 7,968,321 BL Sheet 18 of 38 Jun, 28, 2011 U.S, Patent foyooje Aye} 108} vy Bid spioe Aye s8UB1y t apayaple Aye) <—-yoo-\oe <—- plo Me) <—-dov-lhoe “A Loe 1109 eueye-u —— jueseid Apeayje aue6 <——— passaidxaiano aq 0} auebi ages east, dOv-Aous t dOW-Moehkxouphy-e t doy-hoeojay-¢ tL dovifese = doy-\Auojew seyeydsoud |Aoe wou} seuey|y-uUS 7,968,321 BL Sheet 19 of 38 Jun, 28, 2011 U.S, Patent $614 1 ' oreydsoud-g, ~. : ! ! a WO — dad €== apf: ier) <= sojepipueyd ajdwexy \ ateydsoud-g asOInQUy E |- B® ; oyeydsoudsig-¢' Dine res92K\Boudsoude 9 Fooe9 ‘614 sia}se ayeAuoe aseso]so pioe oljAuoe. eseyesphyep — US 7,968,321 BL Sseulwesuey aplweliioe — -sseueboupAyap _ [ eyeuo|dordxxorphus 2 $ jolpeuedoid-¢' |, sesejonpel Z 2 Z ° ueanjoupAyene} Ayng-A = aseeipkyep f auoyoejosAing ‘ jepouemna- omnes aE 5 cosepnncn ayeiAynahxoupku-p Cacongaiz . 5 2 z so|dwexg uolsiaAuoy sUS 7,968,321 BL Sheet 21 of 38 Jun, 28, 2011 U.S, Patent 2614 «_. Saseuluesued Z ek ee ee SAse}jONpal Z uesnjoupAyesje} “ NE ep | POUEING-p' |-OW-Z eseyeapAyep, i —_— auoyejouking-/-e\-p Jo-g <——_—-Sasejonpei ¢ asese}so ploe q1u0sey QUOJORIOID|EA-Q (ploe opweyn)6)Ajod Raggouuts oseiesof esejonpes lopeuejuad-c'} ~<——— ‘¢—___ poe oueyn)6 syeweyn|6 sesejonpel z sesejonpel Z aseyeipAyapaseulwesuen s9jduexy UolsseAuogU.S, Patent ‘alycerol3- Green "x" Sheet 22 of 38 Fig. 8 US 7,968,321 BL Red “x” ‘Green arowU.S. Patent Jun, 28, 2011 Sheet 23 of 38 US 7,968,321 B1 FIG.9 Signal: 20080326-08,D\FID1A.ch 48000 46000) View Mode: Integration 44000) 42000) 40000) 38000! 36000) 34000} 32000) 1 30000: 28000: 26000. 24000) 22000) 20000" iI 18000 16000 oe pepe a ee TT 0.00 0.50 1.00 1.50 200 250 3.00 3.50 4.00 4.50U.S. Patent Jun, 28, 2011 Sheet 24 of 38 US 7,968,321 B1 FIG. 10 Signal: 20080326-10.D\FID1A.ch 37000] 36000] as000} View Mode: integration 31000) | secee | 2oone | 20000 | acca | 180001 0.00 0.50U.S. Patent Jun, 28, 2011 Sheet 25 of 38 US 7,968,321 B1 C136 mie 100137 00 (730nm) —ceaas a CCAde | | |U.S. Patent Jun, 28, 2011 Sheet 26 of 38 US 7,968,321 B1 occ 2CC136 en 00137 Ethanol (mg/L) seuccaas scca4eU.S. Patent Jun, 28, 2011 Sheet 27 of 38 US 7,968,321 B1 FIG. 13 icc ot JCC136 C0137 se icoaas Acetaldehdye (mg/L) eH ceaaeU.S. Patent Jun, 28, 2011 Sheet 28 of 38, US 7,968,321 BI A1ICC136 WICC137 wuccaas miccaag 2 a 3 ; z 2 =U.S. Patent Jun, 28, 2011 Sheet 29 of 38 US 7,968,321 B1 = \ 8 | vices | 3 8 wiccis7 | 3 wyceaas | ? i wiccaas |U.S. Patent Jun, 28, 2011 Sheet 30 of 38 US 7,968,321 B1 10000 | 10054772 Bo-siowe, 6 V7 WS M9 WO 384 382 182 YEA TS IRS WAT IRE MES Aetentlon tie in)U.S. Patent Jun, 28, 2011 Sheet 31 of 38 US 7,968,321 B1 FIG. 17 2500000 + 2000000 + 1500000 + ‘Totalion counts 1000000 00000 | Retention time (min)U.S. Patent Jun, 28, 2011 Sheet 32 of 38 US 7,968,321 B1 FiG.18U.S. Patent Jun, 28, 2011 Sheet 33 of 38 US 7,968,321 B1 FiG.19 Tacomas actor 1) erection eromotar AmYSs¢g=ne raperer|ODyy| PTE ma) | ceed rom GCvaker nemalied to OO a a 7 7s] ges [amen en] woe a @ aT te_[ en [wea [ase] —o- a od aT a a ce ae 2 Gn | wea [es] 0 o | c ve] eee —[ apm ex ° 2 < 73r| 8 [sa eR o e 7 Be ae rc rn o ae a 733] eso | apm ae seh o 7 a 7g | essen [apt [se ies @ @ @U.S. Patent Jun, 28, 2011 Sheet 34 of 38 US 7,968,321 B1 FIG. 20 respon: —— caso s0000001 2000000) ‘8000004 10000004 ‘s00000: 1750. 1800. 1850 1900 1950 2000" 2050" 2100 2150,U.S. Patent Jun, 28, 2011 Sheet 35 of 38 US 7,968,321 B1 FIG. 21. | 0.5% MeOH ~omn ‘ther embodiments, sugars are produced by expressing ‘enzymes in a selected host cel producing 3-phosphioglycer- ‘aldehyde GPGAL) and aetively transported sing tespoet- fers. In yet other embodiments, photosynthetic organisms unetionally lak cellulose, glycogen, or suerose synthesis “The resulting photosynthetic products. eg, sugars produced from the photosynthetic organisms, can be sed as feedstock ‘or as a carbon source ta prodice additional carbon-based products oF interest In another aspect ofthe invention, the invention provides ‘engineered photosynthetic organisms For producing maltose TIneertain embodiments, the invention provides cloned gones or glyeogen hydrolyzing enzymes which allow the engi- ncered cells to hydrolyze glycogen ta glucose and/or maltose an transport maltose and glueose from te ell, Enzymes for transporting maltose from the cell ince the maltose ffhix pump ftom chloroplast for maltose tansport: MEXI glucose permeases, low and high Km, glucose:H+ symporter, pli- ‘cose! iuctose permease, general sugar-1+ antiporter for glu ‘ose transport; and glucose 6-phosphte:Pi antiporter, rose phosphate:phosphate antiporter for_glucose-6-phosphate Transport are contemplated transport mechanisms af the present invention, In another embodiment, hydrocarbons are produced by ‘enginoering various organisms capable of CO, fixation oF ‘engincered to fix CO,, In one embodiment, the mieroorgan- Jsms are introduced with one or more exogenous nuclei acid sequences encoding aeetyLCoA:ACP transacylase activity (fabH), acetyl-CoA carboxylase activity (aceBCAD), malo- nayl-CoA:ACP transaeylase ativity(TahD), 3-ketoaeyl-ACP sythase activity (fb), 3-Ketoaeyl-ACP reductase activity GahG),. Shydroxyaeyl-ACP debsdeatase activity (LabA\) ‘enoyl-ACP reductase. sctivty (fabl), aeyACP hydrolase ‘setivity (FASD), aldehyde dehydrogenase activity (adh, sadhB), alcohol dehydrogenase activity (ADHD, alkane I-monooxygenase activity alkB). ‘Additional genes tht ean be over-expressed forthe pro- ‘oF pyrivate decarboxylase. In yet another embodiment, the method ofelaim 4, wherein said expression of alcohol dety- “drogenase is criven by promoters on at least wo distinct plasmids, ga plasmid selected from the group of plasmids ‘consisting of QI, pAQ3. PAQS, pAQS, PAS, and pAQT. In ‘yet another embodinent, the activity is varied by controlling ihe level of o-factor required by alcohol debydrogenase oF pynivate decarboxylase. In certain embodiments ofthe method for increasing the production of ethanol by an engincered cyanobacterium, the measured lovel of acetaldehyde released into said culture medium by said engineered organism is Tess than about 7 igiL after approximately 10 days of culture. In certain othee ‘embodiments, thecumulative amountof ethanol released ato said culture medium by suid engineered organism i equal t0 ‘or greater than about 4 g/L. after approximately $80 hours, Ia yet other embodiments, the measured level of ethanol Teleased into said culture medium by said engineered organ- jm i atleast about 1750 mgs. Instill her embodiments, the measured concentration (mg/ml) of ethanol released into said culture medium by said engineered organism is 100, 200 ‘0r300 fold higher than the measured level af acetaldehyde in, sad culture medi ‘The invention also provides an enginoered eyanobacterium ‘comprising alcohol dehydrogenase under the contol of the lambda el promote. In related embodiments the engineered ‘cyanobacterium isan engineered Synechococcus sian com Prising atleast two engincered plasmids. For example, the plasmies could be selected from the group consisting of PAQI, pAQ3, PAQS, PAQS, pAQG, and pAQT. In related ‘embodiments, the two engineered plasmids separately ‘encode a recombinant aleobiol dehydrogenase activity. In yet ‘ther related embodiments, the recombinant aleoiel dehy’ “drogenase activity is adhy- In one embodiment, the eyano- bacterium licks a functioning lctate dehydrogenase gene oF Tucate dehydrogenase enzyme activity, In yet another ‘embodiment, the eyanobacterium is engineered to express 3 recombinant pyruvate decarboxylase activity in addition t0 the alcohol dehydrogenase activity. In yet another embod ment, the recombinant pyruvate decarboxylase activity is 0 14 encoded by one ofthe least wo engineered plasmids encod- ‘ng the recombinant alcohol dehydrogenase aetvity BRIPP DESCRIPTION OF THE FIGURES FIG. 1(A-O) provides various genes identified thst ean be expressed, upregulated, attenuated or knocked out in engi- neering carbon dioxide fixing microorganisms of the inven- ‘ion in the production of earbon-based products of interest. FIG, 2 provides an example of pathways to produce etha- ol, succinate and other derivates, FIG. 3 provides a schematic diagram to produce ethylene irom GAP. FIG. 4 provides an example of a pathway for nalkane and fatty alcoho! synthesis FIG. 5 provides an example of pathways to produce several different chemicals: succinate, glutamate, itaconie acid and S-hydroxypropionate FIG, 6 provides a schematie to convert succinate or 3 1 g) hypogeav= fay (eg. < g) tolerant onganisms are aloo contemplated. ‘Vacuum tolerant organisms include tardigrades, insects, microbes and seeds. Dessicant tolerant and anbytdrobiotic ‘organisms inclade xerophiles such as rtemia salina; nem todes, mierobes, fungi and Tichens. Salt tolerant onzanisms inchude halophiles (eg. 2-5 M NaCl) Halobacteriacea and unalielia salina. pH tolerant onmanisms include alkaliphiles such as Narmonobacterim, Bacillus firmus OF4, Spirulina spp. (ea, pH>9) and acidophiles sich as Cyanidium cal- drum, Fermoplasma sp. (€3. low pH). Anaerobes, which ‘cannot tolerate O; such as Methanococcus jammaschil, microaerophils, which tolerate some O, such as Clostridium and aerobes, which require O, are also contemplated. Gas tolerant omganisms, which tolemte pare CO. include Cya- nidium caldarium and metal tolerant organismsinelude meta- fotolerants such as Ferroplasma acidarmanus (eg. Ce, As, CA, Zn), Ralstonia sp. CH34 (ex, Zn, Co, Cd, Hig, Pb) ‘Gross, Michael. Life on the Edge: Amazing Creatures Tri. ing in Extreme Environments. New York: Plenum (1998) and Seekbach, J. "Search for Life in the Universe with Terestral “Microbes Which Thrive Under Extreme Conditions" In Crs- tiano Batali Cosmoviei, Stuart Bowyer, and Dan Werthe- mer, eds, Astronomical and Biochemical Origins and the ‘Search for Life inthe Universe, p. 511. Milan: Féitrice Com- positon (1997), Plaats include but are not limited to the following genera Arabidopsis, Beta, Glycine Jaropha, Miscanthus, Panleum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea Algae and cyanobacteria include but ate not limited to the following gener Acanthoceras, Acamthococcus, Acaryochloris, Aehnan- thes, dchnanthiiam, Actinastrum, Actinochloris, Actinacy- ‘lus, ctinotacniumn, Amphichrysis Amphidiniu, Amphibr- os, Amphipleura, Amphiprora, Amphithrix, Amphora ‘Anabaena, Anabaenopsis, Ancumastus, Ankistrodesmus, “Anksra, dnomoconeis, dpatococeus, Aphanizomenon, pha hnocapsa, Aphanochacte, Aphanothece, Apiocystis, Apis- ‘onema, Arthrodesmus, Artherospira, Ascocklors, Asteri- ‘nella, Asterococeus, dudouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachosper- num, Binuclearia, Biichia, Blidingia, Bordiopsis, Botry- dium, Bowryococeus, Botrsosphacrella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bum Feria, Bumilleriopsis, Caloneis, Calorie, Campytodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centrtractus, Cenironella, erat, Chactocerns, Chaetochlors, Chaeto ‘morpha, Chaetonella, Chaetonema, Chaetopelts, Chaeto- hora, Chaetosphaeridium, Chamaesiphon, Chara, Charac- fooktors, Characiopsis, Characium, Charales, Chilomonas, Chiainomonas, Chlansdoblepharis, Chlamsdocapsa, 0 o 2 Chlamydomonas, Chiamydomonopsis, _ Chlamydomsa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlo- ella, Chiorobotrys, Chlordbrachis, Chiorockyrium, Cloro- ‘caccum, Chlorogioca, Chloroglocopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromopheton, Chromutina, Chroococcidiopsis, Chroococeus, Chroodact om, Chroomonas, Chroothece, Chrysamoeha, Chrysapss, Chrysidiastrum, Chrysocapse, Chrssocapsella, Chrysocha. ete, Chrssochromulina, Chrysococeus, Chrysocrinus, Chr solepidomonas, Chrysolylos, Chrysonebula, Chrysophyta, Chrysopysis, Chrysasaccus, Chrysophacrella, Chrysos. tephanosphaera, Clodophora, Clastidium, Closteriopss, Clasterium, Coccomysa, Coccancis, Coelastella, Coelas. ‘rum, Coelosphacriam, Coenochloris, Coenococcus, Coeno- ‘ystis, Colacium, Coleochaete, Colldictyon, Compsogonop- sis, Compsopogom Conjugatophsta, Conochact, Coronasirum, Cosmariun, Cosmioneis, Cosmocladium: Crateriportla, Craticula, Crinaliu, Crucigenia, Cracig eniella, Cryptoaulas, Crypiomonas, Cryptophsta, Cteno- hora, Cyanodicivon, Cyanonephron, Cyanophora, Cyano- phyta, Cyanothece," Cyanothomonas, Cyclonesis, lostephanos, Cyeloella, Cylindracapsa, Cylindrocy sts, Ghlindrospermum, Cylindrheca, Cymatopleura, Cymbla, Gymbellontsschia, Cystodinium Dactslococeopsis, Debarya, Demicula, Dermatochrssis, Dermocarpa, Der ‘mocarpella, Desmatractum, Desnidum, Desmococcus, Des ‘monema, Desmosiphon, Diaeanthos, Diacronema, Diades- ‘nis, Diatoma, » Diatomella, Dicellul, Dichothris, Dichotomococeus, Dieranachaete, Diciyoohloris, Dictyo caccus, Dictvosphaerium, Didymocysts, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococeus, Dinobryon Dinococcus, Diplochlors, Diploneis, Diplostauron, Distr ‘nella, Docidium, Draparnaldia, Dunalilla, Dssmorphoc- ‘cous, Feballocysts, Flakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entociadia, Entomoneis, Entophysalis, Bpichnsis, Epipysis, Epithemia, Eremosphacra, Evastrop” sis, Euastrum, Bucapsis, Eucocconels, Eudorina, Euelen, Euglenophsta, Eunotia, Eustigmatophyta, Eutreptia, Falla. ia, Fischerelia, Pragiaria, Frogilaiforma, Franceia, Frus ‘alia, Curilla, Geminella, Genicularia, Glaucocysti, Glaw- cophyta, Glenadiniopsis, Glenodinium, Gloeocapsa, Glocochaete, Gloeoohnsis, Gloeococeus, Gloeoesstis, Glocodendvon, Glocomonas. Gloeoplax, Gloeothece, Glove” tila, Glosotrchia, Gloiadictvon, Golenkinia, Golenkiniopss, Gomontia, Gomphocymbella, Gomphonema, Gomphes: hacria, Gonatozygon, Gongrosia, Gongrosira, Goniochlo- 1s, Gonium, Gonvostonsm, Gramulochloris, Gramulocys topsis, Groenbladia, Grmnodinuum, Grmnozys, Gyrosigma, Haematococens, Hajniomonas, Hallassia, Ham. ‘matoidea, Hannaea, Hantzschia, Hapalasiphon, Haplotae- nium, Haptophyta, Haslea, Hemidiaiun, Hemitoma, Herib ‘audiela, Heteromastix,Heterothris, Hibberdia, Hildenbrandia, Hille, Holopedium, Homocothris, Homan. thonema, Hormotila, Hyalobrackion, Hyalocardim, Hyalo- discus, Hyalogonium, Hyalotheca, Hydrianun, Hydrococ- cus, Hydrocoleum, Hrdrocoryne, Hydrodictyon, Hydrosera, Hydrarus, Hella, Hymenomonas Isthnochloron, Johannes. aptistia, Juraviella, Karavevia, Kathablepharis, Katod- inuum, Kephyrion, Keratococcus, Kirchnerilla, Klebsor ‘midium, Kolbesia, Koliella, Komarekia, Korshikoviela, Kraskella,Lagerheimia Lagynion, Lamprothamnivan, Lema nea, Lepocincts, Leptosira, Lobococeus, Lobocysts, Lobomonas, Lusicola, Iyngbya, Malleochlovs, Mallomonas, Mantoniella, Marszoniella, Martyana, Mastgocoles, Gas. togloia, Melosira, Merismopedia, Mesostigma, Mesotae- trum, Micractnium, Micrasterias, Microchaete, Microco-US 7,968,321 BI 25 feus, Microcystis, Microglena, Micromonas, Microspora Microthamnion. Mischococcus, Monochrysis, Monodus Monomastic, Monoraphidium, Monosiroma, Mougeota, Mougeotiopsis, Mvochlois, Miromecia, Msxosarcina, Nae~ ‘geliella, Nonnochloris, Nautococcus, Navicula, Neglectlla, Neidium, Nephroclamys, Nephrocytium, — Nephrodilla, ‘Nephroselmis, Nerrium, Nella, Nitellopss, Nizschia, Nadu aria, Nostoe, Ochromonas, Ovdogonium, Oligochaeto hora, Onvehonema, Oocantium, Ooeysts, Opephora, Ophiocytium, Orthoscira, Oscllatoria, Oxyneis, Pachycla- delta, Palmelta, aimodictyon, Pnaorina, Panmus, Paralia, Pascherina, Paulschulsia, Pdiastrum, Pedinella,Pedinomo- nas, Pedinopera, Pelagodictvon, Penium, Peranema, Peri ddiniopsis, Peridinium, Peronia, Petrone, Phacotus, Phacus, Phacaster, Phacodermatium, Phaeophta, Phacosphacra, Phaeothanion, Phormidium, Phycopelis,Phylariocklors, Phyllocardium, Phylomitas, Pinnularia, Pitophora, Placo eis, Planctonema, Planktosphaeria, Planothidium, Plee tonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleuro- 2 ‘lado, Pleurodiscus, Pleurosigma, Pleurosira, Preurataenium, Pocillomonas, Podohedra, Polyblepharides, Polvehactophora, Polvedrella, Polvedriopsis, Polygonio- ‘chioris,Plvepidomonas, Pol taenia, Polvtoma, Polvtomella, Porphyridium, Posteriachromonas, Prasinochlovis, Prasin. ‘ocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochloro- thr, Protoderma, Prowsiphon, Provasoliella, Promnesium, Psammodictvon, Psammothidium, Pseudanabaena, Pseue roclonium, — Psuedocarteria, Pseudochate, Pseudoch- ‘aricium, Psendococcomysa, Psewedodictyosphaerium, Pseudolephyrion, Pseudoncobyrsa, —Pseudoquadrigula, Pseudosphaerocsstis, Pseudostaurastrum, Pseudostauro~ sira, Pscudotetrastrum, Pteromanas, Punctasiruata, Pyran- ‘ohlams, Pyramimonas, Pyrrophyta, Quadrichloris, Quad- ricoceus, — Quadrigula, Radiococcus, — Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophta, Poimeria, Rhabdoderma, Rhabdomonas, Rhizoclonivm, Rhodomonas, Rhodophsta, Rhoicosphenia, Rhopelodia, Rivalaria, Rosemingiella, Rossithidiun, Roya, Seenedes- ‘mus, Scherfelia, Schizochlamydella, Schizachlamys, Sohi- imeris, Schizothris, Schroederia, Scolioneis, Scotilla, ‘Scotellopsis, Scourfeldia, Sestonema, Selenastrum, Sele~ nnochlors,Sellaphora, Semiorbis, Sderocelis, Diderocystop- sis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, ‘Sleleronema, Sorasirum, Spermatozopsis, Sphaereloesstis, ‘Sphaerellopis, Sphaerodinium, Sphaeroplea, Sphacrozo sma, Spiniferomonas, Splrogira, Spiotaenia, Spirulina ‘Spondylomoram, Spondslosinm, Sporottras, Spumella, ‘Staurastrum, Stauerodesmus Stauroneis, Staurosira, Stauro- sirella, Stenopterobia, Stephanocostis, Stephanodiseus, ‘Stephanoporos, Stephanosphaera, Sichococcus, Stichog- Toca, Stigeaclonium, Stigonema, Stipitococeus, Stokesilla, ‘Strombononas, Stylochrysalis, Stylodinium, Styloyxis, St losphaeridium, Surirella,Sykidion, Symploca, Synechocoe- ‘us, Synechoeysis, Syuedra, Synochromonas,Synura, abel: aria, Tabularia, Teltingia, Temmogametum, Tetmemoras, Tetracklorelia, Tetracyetus, Tetradesmus, Tetraedrielta, Tet- racdron, Tetraselis, Tetraspora, Tetrasirum, Thalassiosira, Thamniochacte, Thorakoohloris, Thorea, Tolypella,Tolypo- thrix, Trackelomonas, Trachydiseus, Treboua, Tremtepho- lia, Treubaria, Tribonema, Trichodesmivm, Trichodiscus, Trochiscia, Tyblionela, Uothrix, Uroglena, Uronema, Uro- solenia, Urospora, Uva, Vacuolaria, aucheria, Volvo, Vol~ vauling, Westella, Woloszynskia, Xanthidium, Xanthophyta, Kenococeus, Zygnema, Zygnemopsis, ad Zygonium. 0 o 26 Green non sulfur bacteria include but are ot Timited tothe {allowing genera: Chlorflesus, Chloronema, Oscillochlors, Heliothrie, Herpewsiphon, Rosefles, and Thermomicrs” ium. Groen sulfur baeteria include but ate not limited 10 the following genera: CBorobium, Clathrochlors, and Prosth- ecochlors. Purple sulfur bacteria include but re not limited to the following genera: Allochromatium, Chromatin, Halockro- ‘matin, Isochromatium, Marichromativn, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococeus, and Thio- eysts Purple non-sulfur bacteria include but are not Kimi othe following genera: Phacospirillun, Rhodobaca, Rhodobacter, Rhoslomicrobium, Rhodopile, Rhodopseudomonas, Rhodot. hhaiassium, Rhodospirillum, Rodovibri, and Roscospirs. Aerobie chemoithotrophie bacteria ‘include but are not limited to nitrifying bacteria such as Nirobacteraceae sp. Nitrobactor sp., Nitospina sp., Ninococeus sp. Niirspira sp., Nitrosomonas sp. Nitrosococcus sp. Nitrosospira sp. Nitrosolobus sp, Nitrosovibro sp; coloress sulfur bacteria suchas, Thiovulum sp. Thiobacllussp., Thiomicrospira sp. Thiosphaera sp. Thermodhvix sp: obligately chem= olithotrophie hydrogen bacteria such as Hsdrogenobacter sp fon and manganese-oxidizing and/or depositing bacteria suchas Sideracoceus sp. ax magnetotactie bacteria such as Aquaspirilum sp. “Archaeobaeeria include but are not limited to methano- genic archacobacteria such as Merhanobacterium sp. Metha- hobrevibacter sp, Methanothermus sp. Methanococcus SP Methanomicrobiun sp., Methanospirilun sp, Methanoge- nium sp., Methanasarcina sp., Methanofobus sp. Methano- thrix sp, Methanococeoides sp... Methanoplanus sp. extremely thermophilic Sulfar-Metabolizers suchas Thermo- proteus sp, Pyrodictium sp.. Sulfolobus sp., Acidianus sp. ‘and other microorganisms sueh as, Bacillus subils, Sacha romyees cerevisiae, Siepiomyces sp, Ralstonia sp. Rkodo- ‘coccus sp., Corynebacteria sp. Brevibacteria sp., Myeobac- teria sp., and oleaginous yeast HyperPhotosynthetic conversion requires extensive ‘genetic modification; thus, in prefered embodiments the parental photoaufotrophic organism canbe transformed with ‘exogenous DNA, Prefetred organisms for HyperPhotosynthetie conversion include: Arabidopsis thaliana, Panicum virgatwn, Miscant- ‘ius giganeus, and Zea mays (plants), Botryocoecus bral Chlamydomonas reinhardtit od Dunaliela salina (alga). Symechacocens sp PCC 7002, Spnechococens sp, PCC 794; Synechocystis sp. PCC 6803, and. Thermosynechococcus ‘lomgatus BP-1 (eyanobscteria), Chlorobium tepidum (green Sulfur bacteria), Chioroflewus awanticus (grees non-sullar bacteria), Chromatium tepidum and Chromarium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rkodo- Dacter capsulatus, and Rhodopseudomonas palusris (pumple ‘non-sulfi bacteria). ‘Yetother stitableorganisms include synthetic cells oral produced by synthetic zenomes as described in Venter etl US Pat, Pub. No. 200710264688, and cell-like systems or etic eels a deserbed in Glass et al. US Pat, Pub, No, 200710260862. Sill,ther suitable organisms include mieroorganisms tht can be engineered to fix carbon dioxide bacteria such as Escherichia col, Acetobacter ace, Bacillus subtilis, yest ‘nd fungi such as Clastridium fiungdahli, Clostidivon ther- ‘mocellum, Penicillium chrysogenum, Pichia pastors, Sac- charomyces cerevisiae, Sohizasaccharomces - pombe, Pseudomonas fluorescens, o¢ Zymomonas mobilis:US 7,968,321 BI 27 A common theme in selecting or engineering a suitable ‘organism is autotrophic fixation of carbon, sueh as CO, (0 products. This would cover photosynthesis and methanozen- tesis. Acetogenesis, encompassing the three types of CO, ‘xation; Calvin eyele, setyl CoA pathway and reductive ‘TCA pathway i also covered. The eapability to use earbon indvetion with antibiotics and the addition of 0.02% of ‘octanoie acd, the culture is contined at 25°C, from 40) hours, After that, 3 ml of acetyl actate sade to the whole ‘culture and mixed several ties. The acetyl acetate phase is analyzed by GCIMS. Fatty Esters Biodiesels and Waxes) Hostcels ae engineered to produce fatty esters (bidtiesels ‘and waxes) from acyl-CoA and alcohols. In some exam the aleohols are provided inthe fermentation media and in ‘other examples the host calls can provide the alcohol as ‘described herein, Sinictaally, fatty aid esters have an A and ‘aB side, the A side of the ester i used to describe the carbon ‘chain coatrbsted hy the alechol, andthe B side ofthe esters used to deseribe the carbon chain contributed by the aeyl- ‘CoA. Fither chain canbe saturated or unsaturated, branched ‘oF unbranched, In some embodiments, the engineered host ‘ells produce fatty aleohols or short chain aleobols. In alter- native embodiments, the host cll is engineered produce specific acyl-CoA molecules. Asused herein fatty acid esters are esters derived from a fatty aey-thioester and an alcoho, ‘wherein the A sce and the B side of the ester can vary in Jength independently. Generally, the A side ofthe ester sat least 1,2.3, 4.5, 6,7, or carbons in length, while the B side ‘of the esteris 8, 10,12, 14, 16, 18, 20, 22, 24, oF 26 carbons jn length. The A side and the B side ean be steuight chain oF branched, saturated or unsaturated Tnereased expression of ane or more wax synthases (FC 23:1.75) leads 0 the production of fatty ester, ineluding waxes from acyl-CoA and aleohols (Example 17). As used herein, waxes are long chain fatty acid esters, wherein the A side and the B side ofthe ester can vary in length indepen- ‘dently. Generally, the A side ofthe ester is atleast 8,10, 12, 14, 16,18, 20,2224 or26 carbons in length. Similarly theB side ofthe ester isat leat 8,10, 12, 14, 16, 18, 20,22, 24, oF 26 carbons in length. The A side andthe B side can be mono dd, t-unsaturated, Wax synthase peptides are capable of ‘catalyzing the conversion of an aeyl-thioester to fatty esters and some wax synthase peptides will catalyze other resetions Tor example, aecept shot chain acyl-CoAs and short eh leohols to produce fatty esters. Methods to identify wax synthase aetivity are provided in US. Pat. No. 7,118,896, ‘shih is herein incorporated by reference nother aspects, microorganismsare modified to produce fatty exter based biofuel by expressing neleie acids encod- nga wax ester synthase such hat is expressed soa to confer ‘upon ssid microorganism the ability to synthesize a saturated, ‘unsaturated, or branched faty ester, In some embodiments, a the wax ester synthesis proteins inelude, but are not limited to: fatty acid elongases, acyl-CoA reductases, acyltansferases ‘or wax synthases ity aey] translerases,diacyilyeerol acyl- transferases, ayl-co wax aleohol aeyltransferases,bftne- sional Wax esersynthase/acyl-CoA diacylglycerol aeylrans- {erase selected from a multienzyme complex from Simmondsa chinensis, Acinetobacter sp. stain ADPI (fot merly Acinetobacter calcoaceticus ADP1), Pseudomonas ‘aeruginosa, Fundibacter jadensis, Arabidopsis thalane, oF Alkaligenes euirophus. ia one embodiment, the fatty aid ‘longases, ayl-Co reductases or wan syathases are from a smultieazyme complex from lkaligenes eutrophus and other ‘organisms known in the Iterature to produce wax and fatty ‘acid esters. Adlitonal nceie acids encoding wax synthesis proteins useful in fatty ester production include, but are not Timited to, Mortierella alpina (for example ATCC 32222), Crtococeus curvatns, (also refered to a8 Apiotricum curva. tum), Aleanivorax jadensis (Tor example TYT-DSM I2TI8-AICC 700884), Acinetobacter sp. HON, (for example ATCC 14987) and. Rhodococcus opacus (for ‘example PD630, DSMZ. 4193), Fatty esters of various length are pretuced, for example the fatty ester product sa saturated or unsaturated fatty exter product having a carbon atom content between 24 and 46 tatbon atoms; 24 and 32 carbon atoms or 14 and 20 arbons. In another embodiment the fatty ester is the methyl ester fC 18:1; ethyl ester of C 16:1; methyl ester of C 16:1; or acta decyl ester of octavo, Tn another embodiment, the wax ester synthase from Acinetobacter sp. ADPL at locus AAOTT391 (described in Kalschever and Seinbuchel, 1. Biol. Chem. 278:8075-8082 2003, herein incoeporated by reference) is used. In another ‘example the wax exer synthase from Sinmondsia chinensis, at locus AADS8041 is used Optionally a wax ester exporter such as a member ofthe EAT family is used wo facilitate the release of waxes or esters ‘nto the extracellular environment, One example of a wax fester exporter that can be used is fatty acid (long chain) transport protein CGTAO-DA, isoform from D melano- _gaster, at loeus NP_524723, “Asdlesribed herein, the B side is contributed by a fatty acid produced from de novo synthesis in the host organism, In Some instances where the host is edditionally engincered to ‘make alcobols, including fatty aleobols, the A side is aso produced by the host omzanism. In yet other examples the A ‘id ean be provide in the medium, As described herein, by selecting the desired thioestease genes the B sie, and when fatty alcohols are being made the A side, can be designed to be ‘have certain earbon chain characterises. These charaters- tics include points of unsaturation, branching, and desired carbon chin lengths. Exemplary methods of making long chain faty acid esters, where the A and B sideare produced by the production hos are provided. When both the. and 2 side are contributed by the production host and they are produced using fatty aid biosynthetic pathway intermediates {hey will have similar carbon chain characteristics. For example, at least 50%, 60%, 70%, or 80% of the fatty acid esters prodiaced will hve A sides and B sides that vary by 6, 4,02 carbons in length. The A side and the B side wil also splay similar branching and saturation level Tnone embodiment, wax esters are produced by engineer. ing Synechococcus sp. PCC 7002 to express a fatty aleohol forming aeyl-CoA reductase, thioesterise, and a Wax syn hase, Ths, the production host produces both the A and the 1B side ofthe ester and thestruture ofboth sides is influenced by the expression of the thicesterase gene 4. baviyi ADPL (Germed WSadpI, secessions AAOI7391, EC: 23.175) TheUS 7,968,321 BI 43 host is transformed and selected in LB plates supplemented with anibiotcs such as kanamycin, carbencilin or specti- rhomycin, Transformants are inoculated ia 8 and cultured ia shaker a a suitable temperature. When the eultures reach @ preferred OD, analiquotis transferred into flasks. The eulture fs then placed into conical tubes and the cells are spun dose. The cell pellet is then mixed with ethyl acotate. The ethyl acetate extract is analyzed with GC/MS. Inaddition to producing fatty alcohols for contribution to the A side, the host can produce other short chain alcohols such as ethanol, propanol, isopropanol isobutanol, and butanol for incomporation on the side using techniques Well known in the art. For example, butanol can be made by the host organism. To ereate butanol-producing cells, host cells ‘canbe furer engineered 1 express. ato} (acetyl-CoA acetyl transferase) ftom E. coli K12,P-hydroxybutyryl-CoA dey- drogenase from Buisrivibrio fibrisobvens, crotonase from, Clostridium beierinci, butyryl Co8 dehydrogenase from Clostridium beierincki, CoA-aeylating aldehytle dehy dro- penase (ALDH) from Cladosporium fidvum, and adhE 2 ‘encoding analdehyide-aleohol dehydrogenase of Clostridium ‘acetobutylicum ia aa. expression vector. Similarly, ethanol ‘ean be produced in a production host using the methods taught by Kalseheuer ef al., Microbiology 152:2520-2536, 2006, which is herein incorporated by reference. The centane number (CN), viscosity, melting point, and heat of combustion for various fatty acid estes have been ‘charscterized infor example, Knothe, Fuel Processing Tech nology’ 86:1059-1070, 2005, whichis herein incorporated by reference, Using the teachings provided herein alos cell ean be engincered to produce any one of the fatty ack! esters described in the Knothe, Fuel Processing Technology ‘61059-1070, 2008, AcyI-ACP, Aey-CoA to Hydrocarbon rious microorganisms are known to produce hydrocar- bons, suchas alkanes, olefins, and soprenoids, Many ofthese hydrocarbons are derived from fatty acid biosynthesis. The produetion of these hydrocarbons can be controled by con- trolling the genes assninted with fatty acid biosynthesis in the native hosts of some microorganisms. For example, hydrocarbon biosynthesis in the algne Botryocaceus braun ‘occurs throvgh the decarbonylation of fty aldehydes. The fatty aldehydes are produced by the reduction of fatty acyl — thioester by fatty acyl-CoA reductase. Thus, the structure of the fina alkanes ean be controlled by engineering B. braun to express specific genes, such as thioesterases, which control the chain length ofthe fatty acids hing channeled into alkane biosynthesis. Expressing the enzymes that result in branched ‘chain fatty acd biosyathesis in B. braunié will result in the production of branched chain alkanes. Introduction of genes alfecting the production of desaturation of fatty acids will result in the production of olefins. Further combinations of these genes ean provide ether conirol over de final structure ‘ofthe hydrocarbons produced. To produce higher level ofthe native or engineered hydrocarbons, the genes involved in the biosynthesis of fatty acids and their precursors or the degra- ation o other products can be expressed, overexpressed, oF attenuated. Each ofthese approaches ean be applied to the production of alkanes in engineered microonyanisms such as Vibrio fursissi MM and its funetional homologues, which produce alkanes trough the reduction of fatty alcohols. Fach ‘of these approaches can alsa be applied to the production of the olefins produced by many strains of Micrococcus feuieus, Senotrophomonas maltophilia, Jeogalicoccus (ATCC84S6), and related microorganisms. These mieroor- ‘ganisms produce long chain intemal olefins that are derived from the head to head condensation of fatty acid precursors 0 o 44 Controlling the structure and evel of the fat acid precursors ‘using the methods described herein wil result information of olefins of differen chain length, branching, and level ofsatu- Examples 9, 10, 11 and 19 provide several alternatives in ‘engineering microorganism to produce hydrocarbons sch asalkane and octane ydracarbons can also he produced using evolved oxide reductases for the reduction of primary alcohols. Primary fatty sloobols are known ta be used to produce alkanes ia imjeroorganisms such as Fbviofivmissi M1 (Myong-Ok, J. Bacterial, 187: 1426-1429, 2005). An NADIE dependent oxidoreductase isthe responsible catalyst, Synthetic NAD (P)l¥ dependent oxidoreductases ean be produced through the use of evolutionary engineering and be expressed in prodne- tion hosts to produce fatty acid derivatives. One of ordi sill inthe at will appreciate that the process of “evolving” @ {itty aloobol reductase to have the desired activity is well known (Kolkman and Stemmer Nat Biotechnol 19:423-8, 2001, Ness etal, Adv Protein Chem. SS:261-92, 2000, Min. ‘holland Stemmer Cure Opin Chom Biol. 3:284-90, 1999, ‘Huisman and Gray Curr Opin Biotechnol August 13:352.8, 2002, and sce U'S. patent publication 2006/0195947), Iihrary of NAD(P)H dependent oxidoreductases is generated by standard methods, such as ecrorprone PCR, site-specific random mutagenesis, site speifi saturation mutaeness, or site directed specific mutagenesis. Additionally, 2 hibrary ean be created through the “shuiling”™ of natuelly occurring NADAP)H dependent oxidoreductase encoding sequences ‘The library is expressed ina suitable host, such as F. col Individual colonies expressing a different memher of the oxidoreductase library is then analyzed frit expression of fn oxido/reductase that ean estalyze the reduction of a fatty alcool. For example, each cell can beassayed asa whole cell bioconversion a cell extract, 2 permeabilized cell ora puri- tied enzyme, Fatty alcohol reductases are identified by the ‘monitoring the faty alcohol dependent oxidation of NAD (@)H spectrophotometrically or huorometreally. Production ff alkanes is monitored by GC/MS, TLC, or other methods. ‘An oxidoreductase identified i this manne is used to pro duce alkanes, alkenes, and related branched hydrocarbons This is achieved either in viteo or in vivo. The later is achieved by expressing the evolved fatty alcobol reductase gene in an organism that produces fly aleobols, suel as those described herein. The faty alcohols aetas substrates for the alcohol reductase which would produce alkanes. Other oxidoreductases ean be also engineered to catalyze tis reae- ‘ion, stich as those that use molecular hydrogen, ghitathione, ADIT, or other reductive coenzy es Increased Fatty Avid Production Tnttaduction of heterologous nucleic acid sequences involved in a biosynthetic pathway for the production of lhydrocarbons can be done stably or transiently into various host cols using techniques wellknown in heart, for example, electroporation, calcium phosphate precipitation, DEAE: {doxiran-mediated transfection, liposome-mediated transfee- ‘ion, conjugation and transduction, For stable transformation, DNA sequence can futher inchudea selectable marker, such as, antibotie resistance, for example resistance to neomycin, ‘etracycline, chloramphenicol, Kanamycin and genes that complement auxotrophic deficiencies, Suitable expression contol sequences for use in prokary~ otic host cells include, but are not limited to, promoters capable of recognizing the T4, T3, Sp and 17 polymerases, the PR and P, promoters of bacterioplage lambda, the tp cA, heat shock, and lacZ promoters of F. col, the alpha- ase and the signia-specfic promoters of B, subtilis, theUS 7,968,321 BI 45 pronioters of the bacteriophages of Bacillus, Sieptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of the beta-lactamase gene of PBR322, andthe CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryatie promoters are eviewed by Glick, 1. Ind. Micro- biol. 12277, 1987; Watson et al, MOLECULAR BIOLOGY (OF THE GENES 4th Bd, Benjamin Cummins (1987); and Sambrook et al, supe. 'Non-imiting examples of suitable eukaryotic promoters for use within an eukaryotic host are viral in origin and Include the promoter of the mouse metallothionein I gene (Hamer eta. J. Ml. Appl. Gen. 1:273, 1982): the TK pro- moter of Herpes vinis (McKnight, Cell 31:335, 1982) the SV40 early promoter (enoist etal, Nature (London) 290: 304, 1981); the Rous sareomia virus promoter; he eytomega- lovirus promoter (Foecking et al, Gene 45:101, 1980); the ‘yeast gald gene promoter (Johnston, et al, PNAS (USA) 06971, 1982: Silver, et al, PNAS (USA) 81:5951, 1984) and the IgG promoter (Orland et al, PNAS (USA) 86:383: 1989), Tnsomeexamplesa genetically modified host cll isgenet cally modified with a heterologous DNA sequence encoding biosymteticpatay gene product thats operably linked to ‘ constitutive promoter. Suitable constitutive promoters are known inthe at and inelude, constitutive adenovirus major Jate promoter, a constitutive MPSV promoter, and a constitu- tive CMV promoter. Suitable constitutive promoters appli ‘able for Synechococcus sp. PCC 7002 include for example, Puael, P-EM?, Paph? and PaadA ‘The microbial host cell canbe genetically mositied witha heterologous acleic aid sequence encoding biosynthetic pathway gene product that is operably linked to an inducible promoter Inducible promoters are well known in the at. Suitable inducible promoters inelude, but are not limited 10 promoters that are aflected by proteins, metabolites, or ‘chemicals, These include: a bovine leukemia vius promoter, ‘metallothionein promoter, a dexamethasone-inducible MMTV promote, a SV40 promoter, MRP poll promoter, ttreyelinc-inducible CMV promoter (such as the human Jmmetiate-early CMV promoter) as wellas those from the trp and lac operons. ‘When a host ellis genetically modified with heterologous nucleic acid sequences encoding two or more proteins involved in a biosynthesis pathway to produce extbon-based product of interest, the nueleic acid sequences can be driven by a single promoter on a single vector or at east one pro- moter on separate expression Vectors. TInsome embodiments, he ineacelular concentration (@, the concentration othe intermediate in the genetically mex fiedhost cll) ofthe biosynthetic pathway intermediate can be increase to further boost the yield ofthe final product. For ‘example, by increasing the intracellular amount ofa substrate (ez, primary substrate) for an enzyme that i active in the biosynthetic pathway, and the like Insome examples the fatty acid orintermediteisproduced in the eytoplasm of the cell The eytoplasmie concentration ‘ean be increased in a number of ways, including, but 201 Jimited to, binding ofthe fatty seid 10 coenzyme A to form an ‘acyl-CoA thioester. Additionally, the concentration of aeyl- ‘CoAsean be increased by increasing he biosynthesis of CoA, inthe cell, such as by overexpressing penes associated with ppantthenate biosynthesis (panDD) or knocking out the genes associated with glutathione biosynthesis (glutathione syn- thas), ‘Carbon Chain Modifications TIG. provides a description ofthe various genes that an be modulated to alter the structure ofthe fatty acid derivative 0 o 46 product and the encoded enzymes thatcan be used alone or Combination to make various fatty acids and hydrocarbons. ‘The products can be produced such that they contain branch points, levels of saturation, and carbon chain length, thus, ‘making these products desirable starting materials fr use in ‘many applications, Provided are various carbon-hosed prod vets of interest produced by the microorganisms. FIG. Tals lists enzymes that are directly involved in the symtliesis of carbon-based prodets, including waxes, fatty acidesters and/or fatty alcobols. To increase he production of wanes/ftty acid esters, and ltt alcohols, one ormore ofthe enzymes canbe over expressedor mutated to reduce Feedback inhibition, Additionally, enzymes that metabolize the inter ediates to make nonfatty-acid based products (side reae- tions) can be functionally deleted or attenuated to increase the ‘Tux of carbon through the fay acid biosynthetic pathway. The Examples provided herein describe how to engineer ceazyines in the respective pathways of host organisms to yield engineered organisms that produce carbon-based products of Tn other examples, the expression of exongenous FAS. 2enes originating from different species or engineered vari. fants can be introduced into the host cell ty result in the biosynthesis of fatty acid metabolites structurally different (ia length, branching, degree of unsaturation, ete) as that of the native hos. These heterologous gene products can be aso chosen or engineered so that they are unaflected by the natu- ral complex regulatory mechanisms in the host cell and, therefore, function in a manner that is more controllable for the peoduction of the desired commercial product. For ‘example the FAS enzymes from Bacillus subtilis, Saccharo- Inyces cerevisiae, Sireptomrces spp. Ralstonia, Rhodocac- ‘us, Corynebacteria, Brevibacteria, Mycobacteria, lea ‘ou’ yeast, and the like ean be expressed inthe production bast A skilled artisan will appreciate that when a production host is engineered to produce a fatty acid from the fatty acid biosynthetic pathway’ that contains a specitie level of nsat- ‘ration, bmpching, or carbon chain length, the resulting engi- ered fatty acid ean be used in the production of fay acid erivatives. Hence, fatty acid derivatives generated from the production host ean display the characteristics ofthe engi- peered fatty acid. For example, a production host ean be ‘engineered to make branched, shor hia faty acids, Then, ‘using the teachings provided herein relating to Fatty’ leohol produetion (ie, including alcohol-forming enzymes such as PAR), the production host produees branched, short chain fat alcohols, Similarly, a hydrocarbon ean be produced by engineering a production host o produce a fatty acid having a defined level of branching, unsaturation, and/or earbon chain length, thus, producing a homogenous hydrocarbon population. Moreover, whea an unsaturated alcohol, fatty acid ester or hydrocarbon is desired, the fatty acid biosyn- thetie pathway ean be engineered to produce low levels of seturated famty acids and an addtional desaturase ean be expressed to lessen the saturated product production Satration none aspect, hosts are engineered to prodioce unsaturated {att acids by overexpressing fabB, or by prowing the host at Jow temperatures (for example less than 37° C.) FabB has preference to cis-8'decenoyl-ACP and result in unsaturated fatty acid production in. coli. Overexpression of FabRt results in the production of a significant percentage of unsat- urate fatty acids (de Mendoza et al, J. Biol. Chom., 258: 2098-101, 1983), These unsaturated Fatty acids can then be ‘used as intermediates in hosts that are engineered to produce fatty acids, such as fatty alcohols, esters, waxes, olefins,US 7,968,321 BI 47 alkanes, and the ike. Ove of ening skill in the at will appreciate ha by aeauating {aDA, oF over-expresing [ab an expressing specific ihesteases (Jserbel. Delos), tomsatrated fatty eid derivatives having @ desired carbon, ‘hain length ean be peed. Altematvey the repressor of fatty” acid biorymthesis, FabR (Genbank "secession NP_418398), can be deleted, which will also result in increased unsaturated alt aid produto in Feo (Zhang etal, J Biol Chem 277 pp 18858, 2002 ). Further increase dn umaturated fty acid i achieved by overexpression of FabM (Qras-2, cis-decenoyl-ACP isomerase, Genbank accession DAAOSSO1) and contlled expression of FabK (ans-2-enoylACP_ redvetase Il, Genbank cession NP_357969) from Sieptocaccuspmetmonie (Market al J. Biol. Chem. 277: 44800, 2002), while deleting Feo Fab 1 (rans-2-enaylACP reductase, Genbank accesso NP_415804). Additionally to increase the perentage of umalurtedfaty acid esters, the microorganism ea also ‘overexpress fADB (encoding PrketoveyIACP synthase 1, ‘ecesions: BAAISIS0, EC:2..141), Sf encoding 8 sup. pressor offbA, Accession: AACA4390) and ans and ans {both encoding seeG null mutant suppressors, aka. cold shock proteins, Accession: ABDINO7.1, ANCTA0T6.1) ‘overexpressed. Insome examples. the endogenous fbl pene ‘en be attenvated, ts, increasing the percentage of pal toleate(C 16-1) produced Fat acids can be produced that contain branch points, ‘excl moieties, aud combinations threo, using the teach: ings provided brsin (Example 1). ‘By inserting and expressing one or more exogenous uci acid seguenees,mizoorganinms that natal pro= Ace straight ftty acids (SEAS) can be engineered 10 be ‘capable of fixing carbon gioxide and producing hrsnched ‘hin at cid (FAS) For example, a host sve a Ecol naturally produces staiht fatty acid (FAS). The host can ‘so engineered to capture light a described in, eg. PCT! US20081075899, filed Sep. 10, 2008, or in PCTUS2008) ‘083056, ile Nov. 10,2008, and several genes ean be intro- duced and expressed that provide branched precursors (Dk ‘opeton) and alow inition of fatty aid biosynthesis frm branched precuros (fb). Additionally the organism ean expres genes for the elongation of brFAs (eg. ACP. Tabh). ‘Adin oralteratvely. te coresponding Ecol genes that normally leo FAs and Would compete with he neo: Aled genes (Fab, Fa) canbe delved. “The branche aeylCoAs 2-methy-butiry CoA, isov- lerl-Co. and sbuturl-CoA are the precursors ob. Ta most brFA-conaining mieroorzanisms they are synthesized Jntwo steps (escribe in dtl below) from branched amino acids (sleucine, leacine and valine) [Kadena, Microbiol ‘ev. 55: pp. 288, (1991]-A mcroonanismcan be engnesred toprodutebAs,ortooveproducebrF As, by recombinantly ‘expressing or overexpressing one or more oF tie enzymes thoxe to steps. In some instances the protion host may have an endogenos enzyme tat ean accomplish ane sepia ‘which case only enzymes involved in the second stop need be recombinantly expressed. The fist step in forming branche fatty aids isthe pro- uetion of the corresponding arketo acids by a branchied- ‘hain amino acid aminotransferase. coli has soch an ewsine, VIVE (EC 264.42; Genbank accession YP_ 026247). In some examples, a heterologous Branched chain amino aed aminatansforase may not he expressed However Boll Iie orany other branched-chain amino acid aminoansferase. eg VE from Lactococcus lactis (Gen tank accession AAT4406), IVE from Pseudomonas pula (Genbank accession NP_T4S648) or IME ron Sinton 0 o 48 cescoelicofor (Genbank secession NP_629657) ean be over expressed in 2 host microorganism, should the host’ ami rotransterse reaction tun out to be rate limiting. ‘The sccond step, the oxidative decarboxylation of the ‘cketoaeids fo the eoresponding branched-chain acyl-CoA, js catalyzed by branched-chain ceketa acid dehydrogenase complexes (bk EC 1.2.4.4) [Denoya etal. Bacteriol 177 pp. 3804, (1995)], which consist of Elau (decarboxylase) 12 (dihydrotipoy! transaeylase) and E3 (dihydrolipoyldehy= drogenase) subunits. These subunits ae similar to pyruvate and a-Ketoglutarate dehydrogenase complexes, FIG. 1 lists potential bk genes from several microorganisms tha can be expressed in a production host to provide branehed- (60,000 snd forms intracelular granules. Gyengen in synthe- sized in viv via 2 pathway originating from glucose I-phos- phate. Its hydrolysis can proceed through phosphorylation to hucose phosphates; vit the internal cleavage of polymer 10 ‘allodestrins; vi the successive exo-cleavae to mallose; oF via the concerted hydrolysis of polymer and maltodextrins t0 maltose and glucose TIncerain aspects, various routesto engineer metabolism 0 produce glucose biosynthetically are desribed, For example, rlyeogen synthesis can be interupted, and glucose-1-phos- hate of glacose-6-phosphate can be desphosphorylated 52 Glucose phosphate could be dephosphorylated intracellularly via a cloned or endogenons phosphatase o hexokinase and transported out ofthe cell via cloned or endogenous fai {ating cartier. Alternatively, the glucose phosphate could be 5 wansported and dephosphorylaied externally. The glucose phosphate could also be used directly as a fermentation sub- ste Tnaxditionto the above, another mechanism is described 10 pradice glucose biosynthetialy. In certain embodiments, the present invention provides for cloned genes for glyeogen hydrolyzing enzymes to hydrolyze glycogen to glocose and! ‘oF maltose and transport maltose and glucose from the eel Preferred enzymes are set forth below in Table 2. Glucose is transported out by glucose/hexose transporte. This lterna- tiveallows the eel to accumulate glycogen naturally but adds enzyme activities to continaouslyretim it to maltose or gh ‘ase units which ean he collected as a fermentable prodct There are a number of potential enzyme candidates for alyoogen hydrolysis, Eazymes are limited in their mecha- nisms For hydrolysisofthe 4-and 1,6-bonds ofthe glyeogen polymer and complete hydrolysis requires an ensemble of fenzymes. acamylases perform an endo-attack on large poly mers of glycogen and hydrolysis results in formation of shorter average DP 13, polymers which are attacked in an cexo-fashion by glucoamylase to result in glueose product. Neither of the aforementioned enzymes will atack at the 1.6-branches. Therefore, pulluanases and other amylo- alyoosidases, which in nature perform this hydrolysis, are ‘used to completely hydrolyze glycogen to glucose. An alter ative isa f-amylane which performs exo-allack onthe lange polymer ends and results in release of maltose units. Addi tionally, there are a number of possibilities for enzymatic dephosphorylation of glucose-s-phosphate including alka- line oF acid phosphatases and kinases, The following enzymes listed in Table 2 below have activities specific 10 sugar or sug polymer dephosphorylatioa, TABLE 2 Ene or he of eos Pramas anys Tan Sena SERIE See Sete ca SSSA costae et Soca ese Percnuameenaiee Satanic Seen Seer aaa Smaiumacenaes iaktocegiosiee peanUS 7,968,321 BI 53 TABLE -continved 34 Tze rls of we ney Nave Comes No. Fastin Iylo-o-iegheniive — EC32139 debansing canner ipdmipsof {12 apheDgloont nh kaos Ingyeogen pesos int extn powphonlae kine EC27IL19 TAP paphoyiaets 2ADP + Poslonlses Posonlae Bez —{lealseDeeonytn$ phosphates {ips Dpowoein be Dice bebe “TrunsporvEmflux Gene Products ‘A number of transport mechanisms are possible, Most bacterial cells have vectorial aetive transporters io move gli- ‘cose oF malose into the cell. To accumulate sugars, these mechanisms rely on energy eoupling in the form of ATP, 2 proton motive force or gradients of other motecular species, ‘eg. phosphate Plant chloroplasts have active mechanisms (0 facilitate efflux of glucose and maltose ro the plant oF algal ‘ytoplasm. Accordingly, in certain embodiments, building transporters into the inner membrane may involve targeting > ‘and assembly, and vectorait ofthe energy coupling mecha nism versus solute flux. For instance, maltose efox pump from chloroplast for maltose transport: MEX1: glucose per- meases, low and high Km, glocose:H symporer, glucose! frvetose permease, general sugar Hs antiporter for glucose teansport; and glucose 6-phosphate-Piantiporte,riose-phos- phate:phosphate antiporter forglucoses6-phosphate transport ‘are contemplated transport mechanisms of the present inen- “There are natural Chlorella algal strains Uht serete mal- tose and glucose at appreciable rites. These stains are nor- mally endosymbiotic and, remarkably, when isolated freshly from their hosts exerste almost all oftheir photosynthate as ‘extracellular monosaccharide, however, almost invariably, they Iose this ability soon after being removed, ‘A few Chloreta strains can be grown as axenic cultures (-12 hr doubling time, 30°C. and still secrete appreciable fractions (5-40%) of ther photosyathate almost entirely as ther glucose or mallose on autrient starvation media akinto _lycogen production upon nitrogen starvation). These exere= tion nites continve in the dark from intocellalar stores of photosynthate. Some of the best rates in the literature are ‘described by Fischer etal. 179:251-256 (1989); and Beeshig= rac etal, dd Space Res. 14:79-88 (1994), In ceriain embodiments, the above mentioned rates of sugar production are maintained or, more preferably, ‘exceeded, Operating ats biomass density of 15 g/l (~OD 50), implies a volumetric productivity of -0.08°15-0.75 sugar! he “The fermentation prodictsaccordingtn the above aspect of the invention aresugars, which are exported into the media.as a resut of carbon fixation during photosynthesis. The sugars ‘canbe reabsorbed later and ferment directly separated, oF Uullized by co-cultured organism, This apprvach has several ‘advantages, Fist, the toal amount of sigars the cell a handle is aot limited by maximum intracellular eoncentea- tions because the end-product is exported to dhe medi, See= ‘ond, by removing the sugars from the cell, the equilibria of ‘carbon fixation reactions are pushed towards creating more sugar. Third, during photosynthesis, theres no need to push 0 carbon flow toward glycolysis, Fourth the sugar are poten- ‘ally les toxic than the fermentation products tht would be relly produces ‘Accordingly the invention provides cells which produce metabolic sugars, ©. glicose, through photosynthesis using Tight, water and!CO,, subsequently converting the soars into carbon-based products of interest in an elicent, sustainable yield. In certain embodiments, the photosynthetic organisms ‘re genetically modified to produce photosynthetic products suel as glucose at amounts greater than 1 mg, 100 mg, SOO sig. g.5 2,102,208, 252.302, 35, 40g, $08, 100g. 120 {8.08 150 g per liter of fermentation medium. The invention also provides engineered photosynthetic ‘organisms that produce other sugars such as sucrose, xylose, pentose, rhamnose, and arabinose according tothe same prin. ciples, Using such sugars as its primary carbon source, the ‘organism ean ferment the sugar and produce carbon-based products of interest, eg. bioftels such as ethanol (see, ez Ho et al, Appl Environ Microbiol, 68:1852-1850 (1098). describing use of glucose and xylose For the producing eth ‘ol from celalosie biomass). Consolidated Photo-Fermentation "The above aspect of the invention is an altemative to iretly producing final carbon-based product of interest asa result of photosynthesis Inthisapproach, carbon-based prod. vets oFinterst would be produced by leveraging other organ- jsms that are more amenable to making sy one particular product while culturing the photosynthetic orzanism for its ‘carbon source. Consequently, fermentation and production of carbon-based products of interest ean aceur separately from carbon source production in a photobioreactor. none aspect, the methods of producing such carbon-based products of interest include two steps. The is-step inelndes ‘using photosynthetic organisms to convert carbon dioxide to photosynthetic products such as glucose. The second-step is ‘ousethie photosynthetic prtusts.as carbon source for ells that produce carbou-hased products of interest. In one embodiment, the two-stage approach comprises a photo- bioreactor comprising photosynihetie cells; a second reactor comprising cells capable of fermentation; wherein the pho- tosynthetic eels provides a carbon source suc as glucose for cellscapableot fermentation to prauce a carbon-based prod ‘et of interest, The second reactor may comprise more than ‘one type of microorganism. The resulting earbons-based products of interest are subsequently separated andor col- Teeted Preferably, the two-seps are combined into a single-step process whereby the engineered photosynthetic organisms conver light and CO, direetly into glucose and such organ- ‘sms are capable of prthicing a variety of carbon-based pro ‘ets of interes.US 7,968,321 BI 55 The present invention also provides methods and compo sitions for sustained glucose production in photosyalbctic ‘organisms wherein these or other organisms that use the sug rs are cultured using light, water andO, forse as acarbon source o prodhice carbon-based products of interest, In such ‘embodiments, the host cells are capable of accreting the sg as, such as glucose feom within the cello the entre media jn continuous or fed-batch i a bioreactor, Certain changes in culture conditions of photosynthetic host cells, eg. cyanobacteria Jor the production of sugarsean be optimized for growth, For example, conditions are opti- mized for light intensity, light exposure, time of expose, 30% efficieney. Tn same examples where the final product s released fom the cell, a continuous process cant be employed. In this approach, a reactor with organisms produeing fatty acid derivatives can be assembled in multiple ways. In one ‘example, a portion of the media is removed and allowed 10 separate Paty acid derivatives are separated from the agne- us layer, which will in tur, be etumed tothe fermentation chauber. Tnatother example, the fermentation chamber wll enclose a fermestation thats undergoing a continoous reduction. ln this instance, a stable reductive environment would be ere- ated. The electron balance would be maintained the release of oxygen, Efforts to augment the NADVI and NADPYE balance can also fille in stabilizing the electzon balance. ‘The availability of intracellular NADPH can be also cehanced by engineering the production host to express an NADH:NADPH transhydrogenase. The expression of one or ‘more NADH:NADPH transhydrogenase converts the NADHT produced in glycolysis to NADPH which enhances the pro- ‘duction of fat acid derivatives, Fr lage-scale product production, the engineered micro organisms are grown in 10 L, 100 Lor larger batches, fer ‘mented and induced to express desired products based on the specific genes encoded in plasmids as appropriate. Cell ar boring engineered nucleic aids to over-exprestoratlensate ene products are incubated rom 3 500 mi. seed culture for TOL fermentations (51 for 100 Lfermentations)in LB med (glycerol fre) at 37°C. shaken at >200 rpm until eltares reacted a desired OD (iypically 16 hours) incubated with ‘kanamycin, ampicillin or the lke. Media is teated with con- ‘invously supplemented to maintain a 28 mM sodium prop- rionate ata suitable pl of about 8.0 toactvate the engineered zene systems for production as well as t0 slop cellular proliferation. Media is continuously supplemented with car- bon dioxide. Aliquots of wo more than 10% of the total cell volume are removed each hour and allowed to sit unaggitated 0 a8 to allow the hydrocarbon product to rise othe surface And undergo spontaneous phase separation. The hydrocar- thon component is then collected and the aqueous phase returned tothe reaction chamber. The reaction chamber is ‘operated continuously. For wax ester production, subsequesUS 7,968,321 BI 37 to isolation, the wax esters are washed briefly ia 1 M HCL 10 split the ester bond, and returned to pll 7 with extensive Washing with distilled water. Production and Release of Fatty Alcohol from Production Host “Also disclosed herein is system for continuously produc ing and exporting hydrocarbons out of recombinant host ‘microorganisms via transport protein, Many transport and ‘eltlx proteins serve to excrete a large variety of compounds tnd can be evalved to be selective fora paniular type fatty ‘acid, Thus in some embodiments an exogenous nneleic acid sequence encoding an ABC transporter will he functionally ‘expressed by the recombinant host microorganism, so thatthe microorganism exports the fatty acid into the culturemedium. Inne example, the ABC transporter is an ABC transporter from Caenorhabditis elegans; Arabidopsis thalania, Atkai- genes entrophus of Rhodococcus erythropolis (locus ‘AAN73268). In another example, the ABC transporter is an ABC transporter chosen from CERS (locuses Ail 51500 or AY734542), A(MRPS, AmiS? and AIPGPI. In some ‘examples, the ABC transporter is CERS. In yet another ‘example, the CERS pene is from Arabidopsis (locuses AU 1251500, AV734542, A13g71000 and At Ip$1460), The transport protein, for example, ean also be an elf. protein selected from: AerAB, ToIC and AerEF from E: coll ‘oF HILL6IB, H11619 and ULO139 from Thermosymechococ ‘us elomgatus BP-I In addition, the anspor protein can be, for example, 2 fatty acid transport protein (ATP) selected from Drosophila melanogaster Cacnorhabltis elegans, Mycobacterium tuberculosis or Saccharomyces cerevisiae or any one of the ‘mammalian FATPs. The EATDs can additionally be resyathe- ‘izad with the membranous regions reversed inonder to nvert the direction of substrate flow. Specifically, the sequences of amino acids composing the hydrophilic domains (or mem- brane domsins)of the protein canbe inverted while maintain- ing the same codons for eech particlar amino acid. The ‘dentiication ofthese regions is well known inthe ar Production hosts can alsa be selected for their endogenous ability fo release fatty acids. The elficeney of product pro> ‘duction and release into the fermentation media can be ‘expressed as 2 ratio of intracellular product to extracellular product In some examples the ratio can be $:1, 41, 3:1, 2:1, 11, 12, 13, 14,07 1:3 Procesting & Separation ‘The carbon-based preucts produced by the carbon diox= ‘de fixing organisms during fementation can be separated from the feementation media, Known techniques for separat- ing Tany- acid derivatives from aqueous media can be ‘employed. One exemplary separation process. provided: herein is a two-phase (biphasic) separation process. This process involves fermenting the genetcally-enginoored pro- ‘Guetion hosts under conditions sullicieat wo produce for ‘example, fatty acd, allowing the fatty acid to collet in an ‘organic phase and separating the organic phase from the !nqueots fermentation media, This method ean be practiced in both a bated and continuous fermentation setting. ‘Biphasic separation uses the relative immiscbility of ty ‘acid to fecilitate separation, A skied artisan will appreciate that by choosing a fermentation media and the organi phase such tha the fatty acid derivative being produced has high JogP valve, evenat very low concentrations the faty acid will separate into the organic phase inthe fermentation vessel ‘When producing fatty acids by the methods described herein, sich products Will be relatively immiscible in the {Fermentation media, as well as i the cytoplasm. Therefore, the fatty seid will collect in an organic phase either intracel 0 o 58 Iulaely or extrcellolarly. The colletion of the produets in an ‘onganic phase will lessen the impact ofthe fatty acid deriva tive on cellular function and allows the production host +0 pradce more produc. “The fatty alcohols fatty acid esters, waxes, and hydrocar- bons prodhced as described herein allow forthe prodietion of homogeneous compouncls with respect to other eomponnds wherein a least 50%, 60%, 70%, 80%, UO%e, oF 95% of the {atty alcohols, fatty acid esters, waxes and hydrocarbons pr duced have carbon chain lengths that vary by less than 4 carbons, of Hess than 2 earboas. These compounds can also be produced so that they have a relatively uniform degree of Ssturation with respect to other compounds, for example at Teast 50%, 60%, 70%, 80%, 90%, oF 95% of the fatty aleo- hols, fatty acid esters, hydrocarbons and waxes are miono-, iso i-unsaturated, Pathways Associated with Production of Isoprenoids There are two known biosynthetic pathways that synthe- size isopentenyl pyrophosphate (*IPP") and is isomer, dim- cethylally pyrophosphate “DMAPP").Eukaryotes other than plants use the mevalonste-dependent (°MEV") isoprendid pathway exclusively to convert acayl-coenzyime A (acetyl. CoA") to IPP, which is subsequently isomerized to DMAP. Prokaryotes, with some exceptions use the mevalonate-inde- pendent or deoxyxylulose S duced 2-10x the amount ofeach ethyl ester than JOC723, but Milli-Q water. 0.5% (vv) ethanol wasaddeditothecultures to _ethy myristate was only producedin low quantities of | mail replace loss due to evaporation every 48 hours. At68 and 236 ores forall these cultures. No ethyl esters were ound inthe hours, S mland’ ml of culture were removed from each Mask extracts of ICC879 or JCC7SO, indicating tat the stain can- Jor ethyl ester analysis, respectively. The OD;,9S reached by not make eth] esters naturally and expression of only tesA is the cultures is given in Table 13, ‘not enough to confer production of ehyl esters (FIG. 20, TABLE 13, aah ced ee) ‘Theculturealiquots were pelleted usinga Sorvall RC6 Plas TABLE 14 superspeed centrifuge (Thermo Electron Corp) and a F138-—|§ ——— $$$ T4XSOCY rotor (5000 rpm for 10 min). The spend media __ASUCTispesive tl es fun! inthe cl pele care oF eee 7a superaataat was removed and the cells were resuspended in 135 a inl of Mil-Q water. The cells were pelted agin sing & Ciba Cl6Deta! CLA C9. benchtop centrifuge, the supematant discarded and the cell Sample ever ct oer tet pelltwarstondat~80°C:ttlanalyzed forthepresnceof KCTanGsn om ast ogg st ete. jimmie 8 ea feommaeh 093 ors Desction and quantification ofethyesersinstinsiCellIeeuaysiseh sky ahaa pellets were thawed and 1 ml aliquots of acetone (Acros ICS 72 Organics 326570010) containing 100mg, butylated 4s hydoxytolvene (Sigma-Aldrich B1378) and 50 mg/l ethyl” Methy1 Ester Production Cultring conditions: One colony valerate (Fluka 30784) were added, The cell pellets were of JCC8D3 (Table 1) was inoculated into lO mls of A+ media nixed with the acetone using a Pasteur pipetes and vortexed containing $0 jim speetinomyecin and 1% ethanol (v). twiee for 10 seconds (otal extraction time of 1-2 min). The Thisculre was incubated for 3 days in a bubble tube at 37° suspensions were centrifuged for 5 min o pellet debris, and 0 C.spangedat approximately 1-2 bubbles of1%COssirevery the supematants were removed with Pasteur pipette and 2 seconds in light (40-50 E/m2's PAR, measured with & subjected to analysis with a gas chromatograph using flame .1-250A light meter (L1-COR). Theeulture was innoculated ionization detection (GCIFID). {no two Masks toa final volume of 20.5 ml and OD 008 in A+ media containing 200 pg/ml spectinomyein and 0.5 ss mM IPTG with ether 0 5% methanol or 0.5% ethanol (iv), ‘These cultures were incubated in a shaking ieubator at 150 autosampler was tse 9 det he tiles One of ym gc Cur 24, fiance ih (7-130 ‘ach sample was injected into the GC inlet (slit $1, pres~ 2/5 PAR, measured with a LI-2S0A light meter (LI- sure: 20 psi, pulse ime: 03 min, purge ime: 0.2 min, purge COR)) forthe days. Wate loss through evaporation Was flow: 1S ml/min) and an inlet temperature of 280° C- The replaced with the addition of sterile Milli-Q water. 5 ml of column was a HP-SMS (Agilent, 30 mx0.25 mmx0.25 wm) these cultures (OD; aq-5-6) were analyzed forthe presenceot ‘and the carrier gas was helium at a flow of 1.0 ml/min, The Yel ermetiyl tay GC oven temperature program was 50° C., hold one minute: ‘Detection of ethyl- or methyl-esters: Cell pellets were 10°imin inrease o 280° C.; hold ten minutes. The GC/MS thawed. and 1 ml aliquots of acetone (Acros Organics interface was 290" C.,and the MS range monitored was 250 «8. 326870010) containing 100 mT. butylated hydroxytoluene 600 amo, Ethyl myristate retention ime (et) 17.8min], ethyl (Sigm-Aldrich 13 1378) and 80 marl ethyl valerate (Fluka Palmitate (a 198 min) and ethyl stearate: 21.6 min) were 30784) were added, The cell pellets were mixed with the An Agilent 7890A GCIFID equipped with a 7683 seriesUS 7,968,321 BI 85 ‘acetone using a Pasteur pipettes and vortexed twice For 10 seconds (otal extraction Gime of 1-2 mia), The suspensions were centrifuged for § min to pellet debris, and the superna- tants were removed with Pasteur pipettes and subjected to ‘analysis with a gas chromatograph using mass special detec tion (GC/MS), ‘An Agilent 7890A GCISOTSC ELMS equipped with «| 7683 series autosampler was used 10 measure the ethyl esters. ‘One ul of each sample was injected into the GC inlet using pulsed splitess injection (pressure: 20 psi, pulse ime: 0.3 ‘min, purge time: 0.2 min, purge ow: 1S ml min) and an inlet temperature of 280° C. The column was a HP-SMS (Agilent, 30 0.25 mmx0.25 jm) and the carrier gus was helium at 3 ‘low of mL/min, The GC oven temperate peogzam was 50°C. hold one minute: 10"/aininerease to 280" Cold ten minutes, The GC/MS interface was 290°C, andthe MS range ‘monitored was 25 to 600 amy. Compotinds indicated hy peaks present intoal jon chromatograms were identified by ‘matching experimentally determined mass spectra associated with he peaks with mass spectral matehes found by searching jn NIST O8 MS database, ‘The evlture of ICC803 incubated with ethanol contained ‘ethyl palmitate [retention time (et): 18.5 min], ethyl beptade ‘canoate (rt: 19.4 min), ethyl oleate (#: 20.1 min) and ethyl stearate (re 20.3 min) No ethyl esters were detected in the strain incubated with methanol. Instead, methyl palmitate 178 min), methyl heptadecanoate (#: [8.8 ain) and methyl stearate were found (FIG. 21). This strain apparently has the ‘capability to make both methy/ aad ethyl estes depending oa leohol used, The wax synthase gene used in this strain is known to havea very broad substrate specificity (Stéveken ot al. 2005; Kalschever etal. 20060; Kalschever et al. 2006b), ‘and therefore JCCSOS could uilize a wide variety of aleahols to produce varons fatty acid esters. REFERENCES ‘Cho, H. and Cronan, J.B, 1993, Escherichia col thioeserase |, molecular cloning and sequencing ofthe structural pene and identification a a periplasmic enzyme. The Journal of Biological Chemistry 268: 9238-9245. Kalschever, R.. Stoling, T. and Steinbichel, A. 2006, ‘Mierodiesel: Escherichia col engineered for fuel produc tion. Microbiology 152: 2529-2536. Kalschever, R., Stoveken, T., Luftman, H., Malkus, U, ‘Reichel, Rand Steinblchel, A, 2006b, Neutral lipid bio synthesis in engineered Escherichia col: jajoba cl-like wax esters and fatty acid butyl esters. Applied and Evie ronmental Mirobioloay 72: 1373-1379. Kameda, K. and Nunn, W.D. 1981. Purification and charae- terization of the acyl Coenzyme A. synthetase from Escherichia col, The Journal of Biological Chemisty 256: 5702-5707, Stiveken, -, Kalscheuer, R, Malkus, U., Reichelt, R- and ‘Steinbichel, A. 2005. The wax ester synthaselacy] coen- ‘zyme A: diaeylaycerol acyltransferase from Acineto~ acter sp. sti ADP: characterization ofa novel type of acyliansterase, Journal of Bacteriology 187:1369-1376, Example 18 Patty Alcohol Producers Acyl-CoA reductases (BC 1.2150) convert acyl-CoA and NADPH to fatty aleohol and CoA. Examples of penes t0 ‘express include: bfar from omy mori {QSROT9}. acrl from Acinetobacter baylyi ADPL (AACAS217}; jar from 0 o 86 Simmondsia chinensis; am unspecified acyl-CoA reductase from Triticum aestivum; far from Mus musculus; mlar2 rom Mus musculus; aeeMl from Acinetobacter sp. Ml; and bar from 1. sapiens: Example 19 Engineered Microonganisms Producing Ostane ‘To produce a particular alkane such as octane, several genes as identified in FIG. 1 are introduced in a selected ‘microorganism. The enzyme acetyl-CoA:ACP transacylase E.coli fabEl) (BC 23.138) generates acetyACPHCOA from acetyl-CoA and ACP. Acelyl-CoA carboxylase (E. cal aeeBCAD) (BC 6.4.1.2) generates malonyl-CoA from acetyl CoA,ATPand CO2. Malonyl-CoA:ACP transseylase (E.coli {AbD} (EC 2.3.1.39) generates maloayl-ACP and CoA from malonyl-CoA and ACP. -ketoaeyl-ACP synthase (E. col {abB)(EC23.1.41) generates COZ and3-ketoacy ACP from acyl-ACP and maloayl-ACP. 3-Ketoacyl-ACP recactase (F. oli fabG) (EC 1.11.100) generates 3-hydroxyaeylACP from 3ketoacyl-ACP and NADPH. 3-hydroxyacylACP elydratase (E. coli RADA) (EC 4.2.1.60) generates enoyl- ACP from 3-hydroxyacylACP, Enoyl-ACP reductase (E. ‘oli abt) (EC 1.3.1. {9,10}) generates aeyl-ACP from enoyl- ACP and NADH, NADPH. AcyI-ACP hydrolase (S. cerevi- sia fall) (FC 3.1.2:14) generates fatty acid and ACP from facylACP. Several aldehyde dehydrogenase found in P ‘aeruginosa (EC 1.2.1{3.4}) generate octanal from octanoste and NADH, NADPH. Aleoliol dehydrogenase (Z. mobils adh) (BC 1.1.1-(1.2}) generates T-oetanol from aetanal and NADH, NADPH, Allane I-monooxygemase (P fluorescens alk) (BC 1.14.15.3)then generates n-octane, NAD(P)H and (02 from Loctanel, Production of s-octane confers engi- ered host cell expression of the abave enzyme activities, Example 20 Production of Ioprenoids ‘To generte a strain of cyanobacteria forthe production of Semethyl-but-3-en-1-ol and 3+methyl-but-2en-l-ol, plas- ‘mids are generated by inserting a genomic DNA fragment of Symmechococeus sp. PCC 7002 comprising the coding soquence ofthe nud gene and upstream genomic sequences ino a Vector ‘Synechococcus 7002s grown for 48 from colonies in an ‘incubate shaker fask at 30° C. at 19% CO. to at ODzyq 0 | in A" medium described in Pigaard etal, Methods Mol Biol 274:325-340 (2004). 500 of eultue is added toa test-tube ‘with 3p of 1-5 uzof DNA prepped from a Qiazea Qigprep Spin Miniprep Kit Valencia, Cali.) for each construct Cells were incubated bubbling in 1% CO, at approximately 1 bubble every 2 seconds for 4 hours. 200 ul of cells were plated on A* medium plates with 5% agarose and prown at 530° C. for two days in low light. 10 ygimL of spectinomycin ‘vas underplayed on the plates. Resistant colonies were vi ‘ble in 7-10 days. The cultures are grown overnight by shak- ingona rotary shaker. TheOD,. ofeach eultureismessured, and a sample is removed. To each removed sample, ethyl ‘acetate is added, and the sample is vortexed. portion ofthe ‘upperethyl acetate phase stirnsferredtoa clean glass vial for ‘analysis by gas chromatography-mass spectrometry. “The samples are analyzed on a GC/MS. A yl, sample is separatedon the GC usinga DB-S column (Agilent Techoolo- ics Ine, Palo Alto, Calif.) and belium carier gas. The oven eyele for each sample is 60° C. for 3 minutes, increasingUS 7,968,321 BI 87 ‘temperature at 60°C Jainute to a temperature oF 300? Cand ‘shold at 300°C. for2 minutes Thetota un time s 9minutes, Theresolved samples are analyzed by a mass-selective dete. tor along with previously measured retention time of 3-me- thyl3-buten-I-ol and 3-methyl-2-butea-I-ol mass spectra using this GC protocol ‘he 3-methy!-3-buten-I-ol and isoamyl alcobol can be blended respectively with @ California Reformulatod Gaso- Tine Blendstock for Oxygen Blending (CARBOB) to form various mixtures having an oxygen content of 2 wt%, 2.7 6083.5 wt. 9%. Similarly, I-butano, ethanol, methyl tertiary- butylether (MTBF) and ethyl tertiry-butylether (ETBE) can als be blended respectively with CARBOB to form various ‘mixtures having an oxyyzen content of 2 W1%,2.7 Wi. %or3.S ‘wt. %. The APL grovty values, research cotane aumbers, motor octane numbers, ant-kndck indexes, vapor pressure data, net heats of combustion, water tolerance data, and ‘aportiqud ratio ofthe mixtures are tested Example 21 Engineered Microorganisms Producing “Terephthalate 2adehydro-3-deoxyphosphiohepionate aldolase E.coli a1oF (EC 25,154) generates. 3deoxy-D-arabino-hepti- losonate-7-P from PEP and D-erythrose-4-P. 3.dehydro- ‘quinate synthase . coli aroB (EC 4.23.4 generates 3-dehy~ droguinate from | 3-deoxy-D-arabino-heptulosonate-7-P 3edehydroguinate dehydratase E.coli aroD (EC 4.2.1.10) fenerstes 3-dehydro-shikimate from 3-dehydroguinat Sedehydroshikimate dehydratase from Acinetobacter sp ADPI guiC (EC 42.1.2) generates protocatechuste from 3edehydro-shikimate, j-ketoadipylCoA synthase from Rhodococeus sp. RHAL peaP (FC 23.1.174) generates -ke- toadipyl-CoA and CoA from acetyl-CoA and suecinyl-CoA. Beketoadipate CoA-transferase from Pseudomonas putida Peal (BC2.8.3.6) generates f-ketoadipateand suecinyl-CoA Jrom P-Ketoadipyl-CoA and succinate. 3-oxoadipate enol- Jactone hydrolase Rhodococcus sp. RHAI peal. EC 3.11.24 enerates B-ketoudipate enol lactone from f-Ketoadipate ‘Fcarboxymuconolactone decarboxylase Riadacorcus sp HAL peal. (FC 4.11.44) generates y-carboxy-muconolac- tone fm ketoadipate enol lactone and C2. y-carboxy Ciseissmuconate eyeloisomerase Rhodococcus sp. RHAL peaB (EC $5.12) generates. fcarboxy-cis.cis-muconate from y-carboxy-muconolactone. Protocatechuate 3.4-ciony- genase from Rhodococcus sp. RHAI peaGH (BC 1.13.11.3) enerates protocatechuate Irom f-carboxy-cis.cis-muconat. Protoeatechuate1,2-cs-dihydrodiol dehydrogenase Rliodo- ‘eoceus sp. RHAT tpaC (EC 13.1.n) generates DDT from protocatechnate, CO2 and NADPH. Terephthalate I2-diony- nase Rhodococeus sp. RHAI tpaAB (BC 1-14.12.15) con- verts DDI to terephthalate, NADH and 02. Example 22 Engineered Microorganisms Producing 1,3-Propanediot ‘The enyyme sn-glycerol-+-P dehydmgenase 5. cerevisiae dar (BC 11.1. (8,94}) generates sn-glycero|3-P from diy ‘droxyacetone-Pand (NADH, NADPH}. sn-glyeerol-3-phos- phatase S. cerevisiae gpp2 (HC 3.13.21) generates glycerol fiom sn-plycero-3-P. glycerol debiydratase K. pneumonia (BC 26.1.17) generates a-ketopluarateand N-suecinylLL- 2.6ediaminopimelate from L-ghatamate and Nesuccinyl-2- nino-6-ketopimelate. Nesuccinyl-Ldiaminopimelate des- 0 o 92 vecinylase E. colf dapls (EC 35.118) penerates LL. Gaminopimelate and succinate from N-suocinyl-L.L-2, iaminopimelate. Diaminopimelate epimerase F. coli dap (EC 5.1.1.7) generates meso-diaminopimelate from LL-- ‘aminopimilate, Diaminopimelate decarboxylase E.coli Iys (©C 4.11.20) generates L-lysine and CO2 from meso-diam popimelat. Alternatively, in fiew of dapD (BC 23.1.117), stg (EC 26.117), dap (EC 3.5.1.18); LL-diaminopimelate ami notransterase 4. thaliana Atég33680 (EC 2.6.1.83)is used t0 generste L-diaminopimelate and L-glutamate from tet rhydrodipicolinte and «t-Ketoglutarate. Homocitate syn- thaseS. cerevisiae lys21 (EC23.3.14) generates homocieate and CoA from acelyI-CoA and c-Ketoglutrate, Homoaconi- tase S cerevisiae lysd, lyst (EC 42.136) generates hhomoisocitate from homocitrate and homo-cis-sconitate Homoisocitrate dehydrogenase S. cerevisiae Iys12, Iysl1, |ys10 (EC 1.1. 1,87) generates 2-oxoadipateandCO2+NADH {rom homoisoeitrate. 2amincadipate transaminase S.cerev- siae AROS (EC 26.139) generates L-2-aminoadipate and ‘cketoglutarate from 2-oxcadipate and [-glutamate. 2-ami oadipate reductase S. cerevisiae lys2, IysS (EC 1.2.1.1) ‘generis [-2-aiinoadipate @semiadehyde from L-2-ani roadipate and NAD(P)H. Aminoadipate semialdehyde- alutamate reductase S, cerevisiae lys9,lys13 (EC 1.5.1 10) generates N6-(L-1,3-Dicarboxypropy))-L-lysine and NADP ‘rom L-glitamateand L-2-aminoadipate 6-semialdehydeand NADPH. Iysine-2-onoglutarate reductase S. cerevisiae lysl (EC 1.5.1.7) generates L-lysine and acketoglutarate and [NADH from N6-(L-1,3-dicarboxypropy)}--lysine Example 34 Engineered Microorganisms Producing Serine Phosphoglyeerate dehydrogenase E.coli ser (EC 1.1.98) generates 3-phosphonooxypyrwate and NADH rom 3+P-D-glycerate. Phosphoserine transaminase F. coli sorC (BC 2.6.1.2) generates 3-phosphonooxypyruvateel- lutamate from ontho-P-L-sorinet<-ketoghiarate. Phospho- Serine phosphatase F coll serlt (EC 3.1.3.3) converts ortho- PL -serine to L-serne, Example 35 Engineered Mieroonganisms Producing Aspartate Aspartate aminotransferase B: cof aspC (BC 2.6.1.1) con- verts oxaloacetate and L-glutamate to [aspartate and a-ke- toghitarate Example 36 Engineered Microorganisms Producing Sorbitol Sorbitol ({tom PSP) Glvcose-6-phosphate isomerase B. «oli pai (EC 5.3.1.9) converts D-ftrucioses-P to Deceght- cosesb-P, Phosphoglucomutase £: coll pam (BC 5.4.2.2) converts Decplucose-6-P to Decrglucose-I-P, Glucose-I-phos- phatase F. coli agp (PC 3.13.10) converts D-c-giveose-I-P fo Dacglvcose ‘Allematively, sldose-I-epimerase FE. coli_galM (PC 5.1.3.3) converts D-glucose to D-ceglucose, Polyol dehy- drogenase 8. cerevisiae GRE3 (EC 1.1.1 21) generates D-sor- bitol from D-cglucose and NADPHUS 7,968,321 BI 93 Example 37 Engineered Microorganisms Producing Ascorbate Alpha-D-glucose-6-phosphate Ketolisomerase thaliana PGH (BC 53.1.9) generates f-D-ructoxe-G-P rom D-acglucose-6-P. D-Mannose-6-phosphate ketol- isomerase A. thaliana dind (EC $3.18) converts B-D-fructose-6-P 10 D.mannose-6-P. D-Mannose 6-phosphate 1,6-phosphom- tase thaliana atpmm (BC 5.4.2.8) conver D-mannose-6-P to D-mannose-I-P. Mannose-[-phosphate_guanylyltrans- {erase A thaliana eyt(EC 27.7 22) converts Demannose-1-P © GDP-mansose. GDP-mannose 3 5-02) convents hydrogen peroxide to oxy- zen, Example 38 Engineered Microorganisms Producing Cephalosporin Homoeitrate synthase S. cerevisiae Iys21 (BC 233.14) ‘converts acetyl-CoA and a-ketoghitarate to homocitrate and oA. HomoaconitaseS. cerevisiae ly, Iys3 (EC 421.36) gen- ‘erates homocitrate or homo-cis-aconitate or homoisectate Homoisocitrate dehydrogenase S. cerevisiae lysI2, Iys!l, Iysl0 (EC 1.1.1.87) generates 2-onoadipate andl CO2 and NADI from homoisocitrate, 2-aminoadipate transaminase, ‘cerevisiae ato8 (BC 2.6.1.39) converts 2-oxoadipate and [L-gintamate to L-2-aminoadipate and a-ketoghsamte. Phos- phoglycerate dehydrogenase F. coll serA (KC 1.1.1:95) cone veris -P-D-glycerste to Sphosphonooxypyravate and NADH, Phosphoserine ansaminase E. coll ser (EC 2.6.1.82) converts orto-f-L-serine and a-ketoglutarate 10 3:phosphonooxypyruvate and L-ghutamate. Phosphoserine phosphatase £, olf see (EC 3.1.3.3) converts omtho-P-L- serine 10 Leserine. Serine O-acetyltransferase 4. thaliana AtSerat2; 1 (EC 2.3.1.30) converts acetyl-CoA and L-serine foCoA and Onscetyl- serine, Cysteine synthase A thaliana AUGSS880 (EC 25.147) converts O-acetyl serine to Leysieine and acetate. Acctolactate synthase B. col ilvN, vB (EC 2.2.1.6) converts pyruvate to CO2 and 2-aeetooe tate. Acetohyroxyacid isomerareductase F. col iv (PC 1.1.1.86) converts 2-aeetolactate and NADPH to 2-dihy- droxyisovalerate, Dihydroxyacid dehydratase F, coll ilvD (BC 42.19) converts 2,3ihydroxyisovalerate to 2-ketois- ‘ovaerate Valine transaminase, colilvE (EC 2.6.1.42)con- verts 24etoisovalerate and L-glutamate to ceketogltarate and L-valine. ACV synthetase 4. varibilis Ava_1613 (EC 63.2.26) convers 3 ATP. L-2-aminosdipate, L-cysteine and [valine to NL -S-amin-5-earboxypentanoyl 1 -cysteiny|- Davaline. Isopencillin-N synthase 4, varibilis Ava_S009 (BC 1.21.3.1) converts N-{L-S-amino-S-carboxypentanoy!]- Leeysteinyl-Dvaline and 02 t isopenicilin-N. Isopenicil- lin-N epimemse Mf. xanthus cofD (EC 5.11.17) converts ‘sopencillin-N to penicillin-N, Cephsalosporin biosynthesis 0 o of expundaselhydroxylase C acremonium cefEF (BC 1.142041, 1.14.11.26) converts peniillin-N, 2e-Ketoglotarate and 2 02 to deacetyleephalosporin C, 2 succinate and 2 CO2. Deace- tyloephalosporin-C acetyliansferase C: acremonium celG (EC23.1.175)then generatesCoA and cephalosporin C from acetyl-CoA and deacetyleephalosporin C. Example 39 Engineered Mieroorganisms Producing Isopentenol Iedcony-D-xylose-5 phosphate synthase col ds (PC 22:17) comers pyruvate and Delyeenehyde-P 10 Ieony-Deavilones-? nd CO2, T-ley-D-rline poopie redutisomersse Ecol dur (BC 1.1.1.267) con- tens -dooxy-DexslloseP and NADPHL 10 2-Cmetiy- DerytirioleP. 2Conetiyi-Dertrtel ¢phoxphate tyiyransrse F col ispD (BC 2.72.40) convene CTP a 2C-methyk Dery 4? 10 Astin SPP) 2C- Desythntl. Heytidine S-diphowpho)2-C-mctiy Deewthitl kinase £ ol isp (C271 148) convents AP and devine SPP} 2CmethylD-entitl to. 2-2-4 (Gide 5-PPy2-C.metg| Deel. 2-CanetiylD- cohol 24-cyeodiphoopate synthe Eco ip (EC Wou2) comers 2-P-Hestdine 5-PP)3-C-metiglD- cpihviol to 2-C-metiplDeerythrtel-24reyelo-PP and CR. sydroxy-Sometiylou-2en- yl diphosphate syn- thse Ecol isp (PC 1.1743) convert 2-Comethy-Deeryth- niok-2.eylo-P? to (E)--hydrony-.methyboi-2-e-l-ye PP ydiony 3 metiyibucd-enyl diphosphate edoctase coliipit BC 1-17.12) conven (E)-thydroxj--metis bu 2eneleyPP and NADPH to opens) PP. Ayden imetyibu-2-eny diphosphate redutve F. coll sph (EC 1117.12) comers (@)-4-hydroxy3-mtiyIbt2-en1-yLPP andl NADPH to dimethybllyPP.opentenytinhosphate ‘Sisomerse Ecol ii (EC 33.3.2) converts dimethyl PP isopentenyPP.nopenteny-PPpyroposptese con. vert openteny/-PP to openteno. bopentenol dikinase Convers nopeteny-PP to sopenteol and ATP Hydroxy ctislaltarytCoA sath 8 cerevisiae feral (HC 2.33.10) converts acey-CoA and aestoacey Con to (S)-:hydony-SmstylluaryhCaA and COA Tiydronymathyilatayl-CoA reductase Sconevsie hme (EC 1.1.1.34) converts (R)-mevalonate and CoA t (S)- SydonycmehylitryCoa and? NADPH. Mevaonate Kinase 5. crevisae engl? (BC 2.7.1,36)convers ATP ad {B)mcrlonste to (8) $--mevlonse. Phosphomealonate Kinase 8 cerisine em (EC 27.4.2) conver ATP and (Re S-Pamevalonate 10 (R)-SPP-mevalonate. Diphosphomesa- ionatedecarbowsaseS cerevsie nwa (EC 4.1133) con Wo isopenteni-PP and Example 40 Engineered Microorganisms Producing Lanosterol | ieoxy-Daxylose-5-phosphste synthase: col dx 221.7) converts pyrwate and Deglyceraldehivde--P 10 1 eoxy-Daaylulose-5-P and CO2. Ixleoxy-D-xylulose shosphate reductoisomerase B. coll dxr (EC 1.1,1.267) oon. vers I-deoxy-D-xylalose-5-P and NADPH to 2-C-methyl- Deenythritol4-P.2-C-methyl-D-erythritol phosphate cytidyltranserase F. col spD (BC 2.7.7.60) converts CTP and 2-C-methyl-D-erythrtal 4P to 4-€ytidine-S-PP)-2-C- D-erythsitl. 4-(eytdine $-diphospho)2-C-methyl- Deerythritl kinase £. col ispE (EC 2.7.1 148) converts ATPUS 7,968,321 BI 95 and. 4(eytidine-S-PP}-2-C-methyl-D-erthrtal 10 2-P-4- (eytidine” 5-PP)-2-C-methyl-Derytheitel. 2-C-methy-D- ‘eqthritol 2.4-cyclodiphosphate synthase £, cal spl (EC 461.12) coments 2-P-4(cytidine $-PP)-2-C-methyl-D- eqthritel to 2-Cmetiyl_D-erythriol-2.4eyeloPP and CMP, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate syn- thase E.coli ispG (BC 1.17.43) converts 2-C-methyl-D- ‘eqthrtol-2,4-eyclo-PP to (E)4:hydroxy-3-methyTbut-2-en- LylPPdshydroxy-3-methylbut-2-eny1 diphosphate reductase £: cof ispH (EC 1.17.1.2)convers (E)-4-hydroxy- 3.methylbut-2-en-L-yl-PP and NADPH to isopentenyl-PP. ‘4ydroxy-3-methylbut-2-enyl diphosphate reductase: col isp (EC 1.17.12) convents (F)-4-hydroxy-3-methyTbut-2-en- JAPP+NADPH-dimethylally PP. Isopenteny!-q measurements ‘forthe culture superatants were thereby converted to D-gh- cose concentrations ‘As shown in Table 17, JOCS43 and JOCS45, the replicate AglgAlkan AglgA2lacl-P,.-yihX-GLUTI-spee srns produced five times mare glucose than the contol AghgA Jan AgleA2:spec JCC342¢ strain, when all three cultures ‘wereatacomparablecell density (ODzap 13-15)-JOCS47. the AglgA=kan AglgA2=JachP,,-yihX-all-spec strain, grew significantly more slowly than the other three strains, filing to grow beyond ODzxy 9.6 during the course of the experi- ‘ment; at this OD. the culture supematant of JCCS47 had a alucose concentration comparable to that of ICC342e. TABLE 17 SSCS damn yan aru a sei a Densiy (ODr) Mau ite) TeGae Asencio ry > peste ‘Aan Agere suncaapee alan Ages Meals savin GC-MS assay: In separate growth experiment to the one described in the Enzymatic Assay section a single colony of each of ICC342¢, JCCSA3, JCCS4S, ane JCCSE7 was inoeu- Jatedinio 10 mil A+ mestum containing 75 yin! kanamycin and 75 jg/ml spectinomycin. These cultures were incubated at 37° C. for approximately three days, slowly and contin Cus bubbled wth air enriched wih 196CO, in 50 pE m= soe" PAR. Cells were washed pwice with 10US 7,968,321 BI 109 ‘containing 75 jgiml Kanamyein, 75 yglml spectinomyeis ‘and 0.5 mM IPTG, and seeded into 30 ml cultures of thesame medium at an initial OD;y9 of 0.05. These cultures were incubated ina shaking photoincubator (Infor) at 150 rpm at 37°C. for 8 days in a 2% CO, atmosphere and continuous light (100pE msec“! PAR)-Twoalilitersof culture was sampled on the final day. Cells were pelleted by centrifuga- tion, andthe culture supernatant was filtered through 30.2 jum fier to remove any remaining cells. 05 mi of filtered culture supernatant was Iyophilized overnight in preparation of “derivatization for GC-MS, Lyophilized residue was partially dissolved in 200 jl of ‘anhydrous pyridine by vigorous vortexing (© which was ‘added 1.0 lof silylting reagent (BSA*TMCS+TMSI, 3:2: 3; Supelco, Bellefonte, Pa.) Mixtures were subjected to sub- ‘stantial vortexing and then placed at 70° C. fortwo hours with ‘occasional vortexing. After enoling to room temperature, the derivatized sample wastransferred toaglass autosampler vial, in preparation for GC-MS analysis using sn Agilent 78908 ‘GC equipped with 2 5975C electon-impact MS. 1.0 i of derivatized sample was injected into the GC with a 7683, automate Higuid sampler equipped witha 10 yl syringe. The ‘GC inlet temperature was 280° C. and a split ratio of $ vas used, The capillary column was an Agilent HP-SMS (30 smy0.25 mmx0.25 um), The cartier gas was helium at a flow rateof 1.0 ml min”. The GC oven temperature program was 50°C. hold | min; 10°C. min" to 280" C., hold 10min. The ‘GC-MS interface temperature was 290° C., the mass spec trometer source temperatuee was 230° C.and the quadeupole temperature was 150" C, The mass range was 25-1000 amu, Suyar peaks present in the total ion ehromatograms were ‘enified by their reteation times by using authentic stan- dards (Signia-Aldrich) and by searching an NIST MS data- base (2008 version) As shown in Table 18, JOCS43 and ICCS45 had produced ‘over twice as much glacose than the control JCC342e stain, ‘despite the former being at lower cell density (OD-39 9.7 and 108) than the later (O39 15.1). JCCS47 produced basal amounts of glucose. FIG. 16 shows the ftal ion chromato- gram (TIC) lor CC342¢, JCCS4S, and JCCS47 inthe eten- tion time window during which TMS-derivatized a-D-ghi- ‘cose aud P+D-phicose elute, fom which the combined peak. ‘reas in Table 18 were derived, TABLE 18 “and JOCS4S, dete ttl chromnstouram peak aes for ooze sen by Go MS ane ct Deny Conined TC pe ares sin Guoype 0dr) fare and icone ‘uate ‘blaAzsee ‘etter Aeledtta SIX pee ‘patna alert SHX-CLUTse Aesatseee Abierto SiNOLUT- eee ioe Ts Ty “These GC-MS data corroborate the independent enzymatic assay data reported inthe previous section, namely that the 0 o 110 P,.¥ibX-GLUTI cassette, but aot the P,VihX-gl cassette i JCCS47, resulted insignificantly higher glucose produe- tion than observed in an isogenic control strain. Although GC-MS analysis indicted only basal levels of slucose were present inthe growth medium ofJCCS47, twas apparent that there were several other ion chromatogram peaks only present, or present with larger ares, inthis siuin and not in ICC342¢, JCCSA3, or ICCS4S. One o these peaks vas positively identified as sucrose based on authentic sta- ard analysis, as shown in Table 1 and FIG. 17. Basedon the ‘cancentetion of suerose used in the sutheatic standard analy sis, and assuming that the TIC peak area observed scales Tineaely with this known suerose concentration, it was est ‘mated thatthe JCCS47 culture medium contained appeo nately 600 mg liter sucrose, approximately 100 times that seen in ICC342c, ICCS43, or ICCS4S. No maltose was ‘observed in any ofthe four strains’ culture media, TABLE 19 matte 2361 gnocchi on sen by GEMS (ee) peakaa eauense rain Geneype ('e= 361) ect auton aneA2:spe 802 HECSS7—AlpAtshanAleA2sc Py sanasns vibe 1eC8#3 autho aUBA2HIE Py 0866 FeCS4S—AbeATshan ABEADIN, 00 SAK-GLUTL pee ‘The P,-yihX-alf cassette in JCC547 therefore resulted in significantly higher stierose production than observed in an ‘isogenic control stain. A possible reason for this is that the Git ransporer is able to mediate the export of suerose that is naturally synthesized, andl otherwise maintained, within the cell. There are no reports of Gif being able to mediate trans- port of disaccharides such as sucrose, Gi as been reportedas being able to mediate transport of glucose and, 0 2 much lesser degree, fructose. However, the notion of Gi-mediated saccharide export was supported by the GC-MS analysis of the cultre diam of JCCS47. Asmentioned above, GC-MS indicated several ion chromatogram peaks that were only present, or present with larger areas, inthis strain and not in ICCH42y, ICCSA3, of JCCSAS, Consistet with these poaks representing disaccharides, many of these peaks were char- acterized by a dominant m7 36] ion, which is diagnostic of ‘TMSlerivatized disaccharides [Molar Peel et al, Chem. Maier Sci, 45:321-327 (1997), as shown in Table 20, cause none of the authentic disaccharide standards that were used eluted atthe times indicated in the table above, ‘none of these corresponding peaks cul be identified with certainty However, given the presence of the m/z:361 ion in all its highly likely that hese peaks represent disaccharide ‘ordisoccharide-like molecules, most likely synthesized nat rally within the cellUS 7,968,321 BI a TABLE 20 112 Dich andor dicate ike moc proce Wy CCST a termined hy exten Srnin__Gesoype goa ato ast 298 Jecse2eaueatn 7 rr AeA2 see secssr Auaion oss gasses awn66s aso ‘IBA oP NK tape secses ypaistan « oo ‘IDA el SRGLUTI Se Tinsepai bcos Scan a ander Ex nple 58 Engineered Microorganisms Producing Maltose with Amylase Expressing Plasmids Construction of amylase expressing plasmids: The DNA. sequence ofthe amylase genes of Gylcine max (AMY_gm), Bacillus cereus (AMY_be), and the transporter genes fom Arabidopsis thaliana (MEX!) and Escherichia coli (Se\A) were obtained from Genbank. The codon sequences were ‘optimized for E.coli. The codon-optimized sequences and amino seid sequences for BAAS46S0 (SEQ TD NO: 12 and SEQ ID NO: 13, respectively), CAASOSSI (SEQ ID NO: 14 snd SEQ ID NO: 15, respectively), AAPOM3S0 (SEQ ID NO: I6andSEQIDNO: 17, respectively), and YP_025293 (SEQ ID NO: 18 and SEQ TD NO: 19, respectively) ate provided herein. The DNA sequence of aphll, amt2, and te promoters ‘were obtained from Genbank and codon-optimized for B, coll (SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively). The tre promoter was engineered with an Upstream lac” gene that is constitutively exprested. For ‘examples of codon optimization of genes for E coli, see ‘Chandler et al, “Characterization of Gibberellin receptor rutans of Barley (Hordeum vulgare LJ", Mol Plant 2008 1:285.94; Xue etal, Improved production of p-hydroxyin- namie aeid from tyrosine using a novel themostable pheny- Jaloninetyrosine ammonia lyase enzyme, Enzyme and Microbial Technol. 2007 42:58-64 ; and Chun etal, Electro transport pathway fora Sireproniyceseytachrome PAS0:cyt0- chrome P450 105DS-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2). J Biol Chem. 2007 282:17486-500, Those optimized genes were obtained by ‘contract synthesis from DNA 2.0 (Menlo Park, Calif). In addition, plasmids containing two 750 bp regions of homol- ‘ogy designed to remove the native glg® (pIB313) or the restriction site (pJB318) from Syrechacoceus sp. PCC 7002 ‘were obtained by contract synthesis from DNA 2.0 (Menlo Park, Cali) Using pJB315 and pIB3I8 as vectors, the structs were engineered by performing 4 soqu insertion of aaJA using Poel and 1 Asel, amylastransporter cassette using Ndel and FoR, insertion ‘ofthe promoter-cat cassette with NotT and Nae, an removal ‘oftheeat gene wing Si. All restriction and ligation enzymes ‘were obtained from New Fngland Biolabs (Ipswich, Mass.) jgnted constnicts were transformed into either NEB Sa, E.coli (igh Efficiency) (New England Biolabs: 0 oe Ipswich, Mass.) or Copy Cutter EP1400 competent £. coli (Gpicentre Biotechnologies: Madison, Wis) ‘Genetically Modified Synechococcus sp: PCC 7002 (7002! amylase transporter): The constructs as deseribed above ‘Were integrated onto the genome of Synechococcus sp. POC 7002 (Synechococcus 7002 or 7002) using the folowing protocol, Synechococcus TO02 was growa ia an incubated ‘Shaker Mask at 37°C. at 1% CO, to ua ODis9 of 1.2 in A* ‘medium described in rigaaed NU etal, Methods Mol. Bio 274:325-340 (2004). 1000 lof culture was added to ato tube with 50 pL of 2 yg of DNA digested with Xbal (New England Biolabs: Ipswich, Mass.) and ade to ces without further purification. DNA was prepped from a Qiagen (Giaprep Spin Miniprep Kit (Valencia, Calif) for each con- slruet, Cells were incubated in the dark fortwo hours at 37°C. The entre volume of cells were plated on A medium plates ‘with 1.5% agarose and grown to 37°C. ina lighted incubator (40-60 jhimais PAR, measured with a L1-250A light meter (LI-COR)) for appeoximately 24 hours. 25 ng/mL of spect ‘omyein Was underiayed on the plates, After further incuha- ‘ion, resistant colonies became visible in 5 days. One eolony from each of the 18 cultures (CC724-741) was restreaked ‘onto A* medium plates with 1.5% agarose and 50 y/ml. spectinomycin. Colonies from these plates were then inceu- Jated into S mlof A+ medi containing 25 pg/ml spectinomy= cia. This culture was incubated in a bubble tbe at 37° C. sparged at approximately 1-2 bubbles of 1% COs every 2 seconds in Tight (40-50 yE/m2/s PAR, meastred with @ 11-2504 light meter (L1-COR)). Strains containing amylase- mnsporterconsinics under constitutive expression were ar vested at OD aq ranging fom 1.080 9.44, Strains containing famylase-transporter constructs under tr expression Were incubated to OD, 4p between 3.76 and 8.15. 1 mL. of these ‘uninduced cultures Was harvested. The remaining cultures ‘were disted to OD, of I, induced with 0.05 mM IPT, and incubated for 24 hors. To harvest cells, cultures were spa or | mingtest 14800 pm, The supematant was subsequently submitted for GCIMS analysis. ‘Measurement of maltose by gas chromatography: Samples were partially dissolved in 300 uLanhydrous pyridine (Siz Aldrich; St Louis Mo.) before adding 1.0 mL of silylation reagent (BSA¥TMCS#TMSI_ 3:23. (Supeleo: Bellefonte a.) ARereachaddition, samples were subjectod to vigorous vortexing, Samples were then heated at 70°C. for to hours, cooled, transfered to autosampler vals, and measured with the GCIMS, Retention time in minutes of c-maltose and Fmaltose were identified by their mass spectra and their tention times.US 7,968,321 BI 113 {lent 890A GC/S97SCELMS equipped with a 7683 series autosampler was used to detect maltose. The GC inlet ‘ay set toa spit tio of 5.0 and te inlet temperature was st to280°C. 1 of sample was injected into HP-SMS column (Agilent, 30 mx0-25 mmx0.25 yum). The carrier gas was helium. The GC oven temperature program was 50° C., hold ‘one min; 10°/min inerease to 280° C., hold ten min. The ‘GC-MS interface was seo 290° C,, and the MS mass range was 25 ro 1000 amu, Peaks present in te total-ion chromto- grams were identified by retention time analysis and by Searching the NIST MS Search database version 2.0 (2008) ‘wih the associated mass spectra, Retention Gms in minutes ‘of cemaliose (24.73 min) and f-maltose (25.06 min) were ‘determined with authentie standards Feo Fluka and are rep resented in FIG. 18, ‘Maltose was detected in 1CC726, 729,735, and 738 (FIG. 19), Maitose vas produced only in strains in whieh expres- son ofthe amylase-transporter operon was controll by the tre promoter Example $9 Engineered Methanogenesis Pathway A host cell of interest is engineered to produce methane Preferably, a host is selected or engineered to have methano- nie properties, TARL Exyne FON. pm Tecmyincaotian 12995 Meahamorsia Syaegetse ‘erhonant fateacba tevayonetanoyerin 2 eet feensernnese inca oneetn 427 Meaning Relyleoeteraydonetsaeerin 1.8989 Meohmovarna Simao 159811 Mah ‘edonsinsopterin diss Meshinnarna Example 60 Engineered Acetogenesis Pathway [A host cell of interest is engineered to produce acetate Preferably, the host cell is selected or engineered to have soctogenie properties TABLE 22 a TONS, Euan Onan, pent) eee Be Echoes 0 o 14 TABLE 22-continued Engme TON Fann Onn, eee Tanyheinipeobiae IA) Chard actluicom ute exci Riovmetieneermbsdolte 35.49 Enoch ol 1D ‘este ‘ihecton Sfosmetpleneermbydine 1528 Exerc co seaydgeoe ‘einecton Imeblentemisdte 18.120 boas hata fete NADIPI irae Nets 1.— Cau sermoacecom Caron mers 113593 Claman Boj sedges es Hydrogen = _ Eesha col ys DCFOBE Example 61 Engineered Reductive TCA Cycle A host cell of interest is enginocred to have a reductive ‘TCA cyele, Preferably. the host cells selected or engineered to have photosynthetic properties. TABLE 22 Exgne EC Ne, Exsaple Onin aoe). ‘hegre ryahiss 1.273. Comptoir ian oXDABC cite pe 2, det dain CL Senate iyese 4212) noe cot faneAB Sie Con gue ADP. G28) Foon mee ne praetor 1221 loin ire po Seen Pyele A ost coll of interest is engineered to have a Calvin yee Preferably, the host cell is selected or engineered 10 have: shotosynthetie properties, TABLE 23 Bayne TECNa Expl Ononi, gene Dia tapheepiae—-AI9 caliphate cevomne Sistas sa Fomtnirole 4129 Penionons at conc binhepe ee 6.4218 Beco MRA {coretophophtare SLL lech ip Smpalseophapiae S137 dts ana roee peeps emierse SLL ach col pi ‘Syecalilgepiepinte | 13119 debe has GAPA ‘erseae NADI) (poortoriines hone icoeree $3.16 Exchrchia coli iA ‘ube facpine eens $131 Extercho cali ge Howlegyernie lime 2722. Extensis coll pak Psmkorblcknwe 22 y “dos ats PAKUS 7,968,321 BI 115 Example 63 116 TABLE 28-continued cee os Cao Orin “RodmomatGon suis Mattapan ste Atos cell of interest is engineered to havea HPA eyele, 5 (AMPominn) ih . Prferably. the hos cell is selected or enginoored to have Svseematcan entanse Mota sels photosynetie properties SGptormnyylcon depdopmase —Moutptne sds AD) ee to pomvatenytkie Menthe setae eepiCokoxtomiae 681.2 lonfaas wom Gaesccmande’ 218 Clore santas Example 65 “tmseme ADP soup " Engineered Limonene Production aa 11.1. OMlorerus auraniiaeas =a oe A host eel of interest is engineered to produce limonene aaa caer Preferably, the host cell is selected or engineered to have SSloy-coatyinite 42:1 ofa amano rhotosyntete properties. tn more prefered embodiments, Eotyicoatcicime 134 lbmfaurswommass ap the Calvin Cyele fy removed and the SHP/AHB Cyele i hom engineered into the host. PrplantCoAcatonise 6413. Claw amonices Irobymiomt Con pene $109.1 Chonan onc rectyimioytca ie” 4092 Corgan TABLE 26 Miipiconmine Co 283-~ Olonfans worms eee 3s Bane ior Poe Siete kintopoue 12081 Choa awemtane 28 SA Iniyslopi acceptor Serer pty to (Ror Sines ‘abon) ‘ere be 4212, ort once (49tinoen ane (6 (Srinoe Ging) manent S3%e Gees anon Siem pai 0 (Role ah Rule ehpiogiae 43 Cram once estan eur eae » Sec Style Snes MNRDHCOA 12992 Olona aeoenas ‘oplomeronte 40135 apap rr tn Chips onze ‘Serie Sosolaanse mh Gloves aorta fhoomneinds kane 2742 aloes 2P seins rob Shimon monte Seiten ee S236 makes owen Shima morsecn 35 nn -Seyistane 1M ake Sate tae Shona once Goren strc CoAT Shnfow wort ERI tn pty ne —— —— (4S)-timonene sys 423.16 (S)litmonene (piney) SDimmerc sine 32320 himonene fo iq Poviptomacaine P81 ew sample st Eto 53a Emde Pinte tine? 1712 patente Engincered 3HPGHB Cycle SGytty magic: 1743 Lydony 2g 2 top pace ttn pone ‘Aux cl of interes engineered to ine 2 HPN ** Kemeapebcwthe 442 Smeg tbat ‘eycle (Berg et al, Science 318:1782 (2007)). In more pre= — ita Rei nua 2aaase Yfoghestonisnes fered embodiment, the Cavin Cycle is removed and the SSigtneatiane TTI. jamie ois BHDIAHIB Cycle is engineered ino the host. Preferably, the ieorhtot host eel is selected oF engineered to have phovosyalhtic | -CaiskDeytinlt 27760 oe $0 ont eye disp. properties Deorhtot Loko Dopo Sobombte L267 Cin Dente TABLE 25 ‘ananaomse a ChogebophuceSghpiae 2217 TeeayDoghlows? mone Baie Oman po) Sie ae aletccmalicnee sie Mela lls Brample 66 Retanicevsniene —Menape sie Deen ead SinlipmpoiCoAddpiine —Mtabptore dle cplotCearcdne NADPH)” Meafaphcndals Cloning oft and construction of vectors for expressing pean on cuerioe Napoca fad in cyanobacteria: fad, (SEQ ID NO: 24) was generated snaipimaony-Ca pinerae Mtallsphoers sete bby PCR from Escherichia coll (Migula) stain genomic DNA. eee eee (ATCC #700926) using Phusio™ Hot Start High- Fidelity succinate semialdlye reductase Metallasphocra sedula 6 DNA Polymerase (Developed & Manufactured By Finnzymes Oy and distributed by New Fagland Biolabs, Ipswite, Mass.) aeconting to manufacturers instructions.US 7,968,321 BI 417 The forward primer (ll_FP) sein this reaction contains ' Néelrestetion site ad the reverse primer (ad RP) cone tain a 5" FeoRT resection site. Amplified DNA was subse- ‘quently purfed by gel slctrophorsis followed by agarose Purfcacton using the Qiagen gel extraction kit protocol {(iagen) Pune DNA prot and vector pS (a vetoe based on the DNA 2.0 pJ204 vector containing Nadel and coRI cloning sites) were bolh digested with Ndel (New England Biolabs; Ipswitch, Mass.) and Feo (New England Biolabs; Ipswitch, Mass.) followed by ligation overnight Using T4 gas (New England Biolabs; pswitc Mas) The Jigated comet was transformed into NEB EPIOO Copy Cater competent F: col! (Now England Biolabs Ipswitch, Mass). Colonies were subsequently grown in LB media and screened for insert by PCR using the same primers as above Theseleced construct Was Validate by sqhenceveriicati (GENEWIZ (South Phinfeld, N3.) This fad isert was cloned ito «recombination vector suitable for insertion into Synechococcus sp. 7002 contiing Sever elements An upstream homology region (UHR: st eg, SEQ ID NO: 26) and 8 downsteam homology region {Di see, e-2, SEQ ID NO:26 is presenti he all. insert allowing ecombinatoninto pAQT (pA? plasmid sequence See Geniank # C7000857), The homology regions Hank 8 multiple cloning site (mes) and a Kanamyein easete which provides resistance in both E.coli and Synechococcus 9. 7002. An Xba site present uptteam ofthe UHR an down- stream ofthe DH allows linearization ofthe fall. iseet to Tecltate transformation and integration int the host ongan- ‘Several variants arin fad under ferent strength pro- moter have been prepared. One such promoter (amt fom SCC160) is presented in SEQ ID NO: 26. Other variants at haveheen constricted elude consiucts wth fa under the control of Thermassnechococeuselongatus BP-1 promoters Such 3 12142 oF pa, “Assessment of fad. expression constrict. The fal con- sirets wil e integrated onto pAQ7 of he engineered Syn- chococts stain 1CC8OS (pPAQI laclg Pires fae Symhase-aA) sng the following protocol. The sti wil be grown fom colonisin an incubated shaker flask at 37°C. 812°%CO, 10490 ao0f Lin A" medium supplemented with 200 ml spctinoeayein, S00 pL of eltre will be added to “amierocentfuge tube with SOL of 1-5 yg of DNA prepped {rom a Qiagen Qiprep Spin Miniprep Kit (Valencia, CaliE) foreach const after digesting wih xbal (New Fagland Biolabs: pswitch, Mass) Cells wil be incubated the dark 137°C forfourhoursina shaking incubator, 200 of cells Will be plated on A” medium plates with 15% agarose and 100 aml spectinomyin and ineusted a 37°C. for24 hunder Jight. $0 yal. of kanamyein will be undelayed on the plats, and resisant colonies wll be visible in 7-10 days Expression and seerction of el ester in strains conain- ing the TadL expression consists will be measured and eompared as deseribed in, Fxample 17, herein In addi- tin, cular wll be parttioned with an organi solvent sich ssisooctaneor etl dette andthe organic solvent partition ‘ill be analyzed by GC/MS as detailed in example 17 10 ‘etct an eat esters present inthe mei, Other te niques known to those skilled in the art may also be used to mesure the amount of ei] eser eeeted into the medium, Different levels of expression andlor secretion may be ‘ested, depending onthe parieulrenvronment andlor pur pose forthe expression andlor secretion. A varity of con- ftrcts effecting @ range of expression levels is therefore ‘Scab, In aiton fo fa. rom Feo other transporters including the CERS wex transporter from l. taliona 0 o 118 (Q8CRK2; SEQ ID NO: 23) and XyIN from Pseudomonas ‘putida (Q8VMI2; SEQ 1D NO:25) wil be incorporated into ‘consineis and their enhancing eects on ethyl ester secretion vill be measure. In certain embodiments, two oF more di tinct transporters may be incorporated into a single strain vo achieve levels of biofuel sceretion that are greater than the Jevels achieved using single transporter. Inaddition, recom binant transporters may be modified or mutated to modulate (increase or decrease) the level of secretion achieved, or to alter the specificity ofthe transporter(s) such tht the tran port of additional types of molecules is increased or ‘ecreased relative to the unmodified form ofthe transporter, ‘Such modified transporters may have amino acid sequences that are atleast 80%, 85%, 90%, 95%, 96%, 07%, 98%, oF ‘90% identical tothe transporters disclosed hercin. Other pro- :moters in addition to those mentioned above may be used in this context, including ape, EMT, ARNA-Gilu,laelg-Pure, IA, hsp33,ebeL., cpeC, epeB, rps, Paphil, Pei and nia As with the transporter sequences, modifications may be ‘made to any of the promoter sequences to modulate the level of expression ofthe transporters under their contol Example 67 Enhanced Ethanol Production ‘This Example provides an illustration of how ethanol pro- ‘duction nan engineered orzanism can be improved and made ‘more efficient by modifying the expression levels andlor identity of enzyines involved in ethanol production, Speci cally, a series of pAQT Aldh targeting plasmids were con- structed containing Moorelfa alcohol dehydrogenase (od) ‘genes under control of different strength promoters. Tse plasmids were wansforme into JCC445 (see Table 5) and the resulting strains were examined for ethanol production. ‘Transformation of JCC44s5 by several different plasmids was show to enhance peduction of ethanol but only one plasm, Pol_adhAM, resulted in increased levels of ethanol accom panied by a concomitant decrease in acetaldehyde levels Strain Construction TABLE2T ain oat+ Pani paced) Teaser) TOG + 778 arin Pap aw Seeastae — Teeus) ne Algttncrcbatiane ‘aniceims) (SeQib No-2) Four ig of the plasmid DNA was digested with Xmal and sed to transform 400 pl of JCC44S (OD, 1.15) using the ‘transformation protocol deseribed herein. Te entire transfor lated onto A agar plates and incubated at 37 (C($50 uP) in ir After 24 hours the plates were underplated with kanamycin to final conceatration of $0 jgiml and incubated as above. After § days background growth was iminished and Km* colonies began to be visible, at which point the plates were transferred to 37 C (-50 jE )+1% COs, After 3 days, eo Kay" colonies from each transformation ‘were streaked onto A°#"""?! agar plates for single colony isolation. Stwaked plates were incubated for 4 days at 37°C ‘SO jE) inairand then transferedto 37 C (-SO uP} 196 CC Toran additional 3 days ‘Batch Ethanol in Flasks: single colony of one clone from the original streak plate fom each transformation was incew- lated into S ml AW™""520 both in a 16 mMx 50 mM plastie-capped culture tube and incubated inthe Inforsinew-US 7,968,321 BI 119 bator (37 C, 150 rpm, 2% CO.) ata ~60" angle. After 3 days the cultures were tansfeed info foam-plugged 125ml Erlenmeyer flasks containing 30 ml 182.18" t9 an ODzso-—0.1. The initial weight of each Bask culture was ‘determined so that sterile dl1,0 could be aided at sampling times to accownt for evaporation, Atcach sampling point, 300 lof eure was removed and the cultres Were replenished, ‘with an equal mown of fresh JB2.1 mediam. At roughly 24 hou intervals, samples were taken for growth rate and ete noliacetaldehyde detemainations. OD-,, measurements and “derived growth cures are shown FIG. 22. The levels of eta nol and acetaldehyde in the media ‘Were determined as ‘described herein al the data is presented in FIG, 28 and FIG, 24, respectively. Ascan be sen from the Figures, the levels of ethanol were highest in JCC44S4782, where additional adhAyy is ‘expressed under the contol of the lambda el promoter ‘The 120 cumulative Jevels of ethanol produced by this strain were approximately 4p/L. Atany given time point, the measured levels of ethanol were substantially higher than those mea- sted in C44 or JCC4454773, in some cases neatly twice as high. Inadltion, the same strain produced only 50% (or less) acetaldehyde compared to IC445 or IOC4454773 (express: ‘ng adh, under the control ofthe Paphil promoter). Thu this Example demonstrates not only that enhanced levels of ethanol and redveed levels of toxic intermediates can res {rom alterations to promoters controling adh expression, butalso provides a specific example ofan engineered eyano- bacteria UCC4454773) with enhanced ethanol producing capabilities, ‘All publication, patent documents and sequences referred tohereinarehereby incorporated hy reference in thei entirety forall purposes to the same exteat as if each were so indi- Vidually denoted. 00> sequmnce: 1 aestgogaas cteagattan tatcascges gtcagtgaga tecgegcgns sascacsstt 60 gattacgate geateategt gatexceggt aanggogett costgcatgs 380, gencacateg tgectgetet guacaanaae cagattacgt atatecatte tgatcagsts 240 gstegsgeag tactagctat togtagegst tscesgateg azgcagceas stctgtosey 360 egceeegegs teetanctar coaggagaag gectacaase eggctatege teacguttge 540, acsgostacg teagegtgaa tgeectgaae catgtegtes aagcesegne ctecanagtt — 660 ccteaggeee tgteteacce tgeaguccts accgogegtt attaccteet geatgectet 780 eeganesces geategeste tgatacogge etgetgcatt teacesacge actgguacae 940 segstagtea aucaaattta teoggetace coggagutac tegeggaant cetggnaccy 960 asacggctag cteutacegy catcactatg ansctgaaag aegegggttt ceaggetgan 1080 gatatogege grctgacega covageette accacteeat eectggasct cotgctster 2140 “ 589 10 wo 2US 7,968,321 BI 121 122 -continued S212: ORGRTEN Moorelz6 op. <#00» saqumice: 2 Met up Glu Thr ye He Aan Toe Ran 16 Val Arg ia Hhe Aig Ala Lye mis mr ved ye Phe Gly Val Ohy Ala Te Lye lye 126 Aap Aap ie Ala Arg Gu Pho tye Glu tye Giy Tyr Asp Arg Tie Tie Vat Tie ‘oe Giy tye Gly Ala Tye Lys Ala thr Gly Ala Typ Giu Tyr Tie val Pro Ala teu Aon Lys Aon Gin Tie Thr Tyx Tie His Tyr Aep Gin Vat ‘rg lu Phe oly Ala Arg Ala Val Leu Ala re Gly aly aly er Pro og Phe Ala Val Val Ser Tle Pro Gi tye Ala Tyr tys Pro Ala 7 ala tye tap cys He Tye pro Leu tyr Ser He agp Aap pro ala Lew et val tye tau pro Sex Agp Gin the Ala Tye Val Sex Val Aap Ala teu Aen Hie Val val Gis Ala Ala the ger tye Yal ala sex Pro Ty he fle Tle tu Ala tye Glu the val Arg teu Tle Ala arg Tye tes Pro Gin Ala teu Ser His Pro Ala Aap Leu the Ale Arg Tye Ty= tea lew Nye Als der les He Ala Gly 1g Ala he Aap Aan hy Lea Lew Mig the the Hie Ala teu Gls His Pro teu ger Ala Val tye Pro ais es Mag Hie hy Lew Oy Ley Shy Met Leu tau fro Al Val Ya bys ip He tye fro Als The fro Gla Vel Lea Aig ls Te bes Gls feo "ie Val Po Aep bau bye Gly Val Bo Oly Olu la GLU Lys Ata ALA The Ala Aap Arg Glu Arg Val bys Ala Tie Tyr Gin Aap Ala Phe123 US 7,968,321 BI -continued 124 <210> $90 1 No 2 <#00> sscumice: 3 catgecttog cogteeaces cagsetstos eetaneaggte stacoteogs eapcagegee ageaagtttg eesstgee ccoateactog cagugeates tgecggttee vecaeegos ceaguogeene ceactantae gangeetace actanaceat sgeageaagat estanecact otgaascty stecacggea eoagcestst egscgenate soctastage ostsnagane eugeetaaes sceggecagt ‘gecansetag stenagpate sttanaaacg cogetgetgea accegetose eassogatge cacaggttat weeguetgea ancgacoate cgtccege gaatgaagtt gctotccace gaegeeagtt cancanegeg osttatest concancgaa setgggetac taccoatoatUS 7,968,321 BI 125 126 -continued reeset eee ene ee ee cee ee eee ral ee eee cee eee ee egtaatccgg accaegttte ctetttceag gogcegtget ceatcctcas coagegtgty 7320 agratenges acgatgatte tgutgtgess ascogtates ctatgartcr ggesaasste 2350, cstanasage gttttanacy satgactess gaccagates tgaactatte tgeggtesta 2400 ea geascgttce tggccegegt gageegetgt setggascyg cgcsas <210> $89 19 no 4 285; OR omarion, Descespeton of AELESetal Sequence: Synthetic too saqumnce: « ‘agcogetene tgccogettt ceagtogggs ascctgtegt gecagctgea ttastgnste «0 ‘sorenacges eggoangags cggttegest attgggcace aggetggtte teettttcas 120 ‘seogtcoacy etggtttgee ceagcagges aanatcctst Etgatagtas tegacgeas 240 gatatazcat gagctarett sggtategte gtateccact accgagatat cogeaccaae 300US 7,968,321 BI 127 128 -continued cagcategen gtagunacge tgcecteatt cageattege atggtetgte ganaacegge 420 catageacte cagtegcett scegttcoge tatoggctaa atttgattge gagtasgata 480 egeguttege tggtgnceca atgegaceay atgerccaey cecagteges tavegtette 600 acantagtg caggeagett concageaat ggcatectgy teatecages gatagttaat 720 egccateges gottecacte tttccegegt ttogcs atcactg cataattogt gtegctcaag gegcactoce gttctggata 1320 ga getgttgacs 1380 atantcate oggctogtat aatgtgtggs attgegageg gatascaate tcacacagge 1440 ‘stoeaacegs egcaggance ctgccagege ateagcaata tteteaccty aatcaggata 400 caggagte cggataaaat gettgatggt egpaugtgse ateanttceg teagceagtt 600 casctetgge geategggct teccatacas gegatagatt gtegeaccts ategceegae 720 actacegege goscatetat acoeataraa atcageatee angetggaat ttaatosess — 790 cetegagte tecegtegan tatgycteat attertcett tetcaatate ategaageat — 640US 7,968,321 BI 129 130 -continued atagggate agesttacan ccaateance aattetgaae attategege goseatetat — 960 letgaceceat geogancten gaugtgnaac gecgtagese egatgstagt stggmancte 1080 voggecttte gecogugeta attagaagst gtogeectte tgaagtsgys cetgeagget 1200 egtcag cgustoratg agyogstege atteattcag gaggettaga cougtgateg ageggceget caratgtane aggaattcag 1620 vateacgeag cagaseagte gecctas vogcacatge aguctoggce ctgaccaagt oasstocatg oggacrgcte tegatcrret 1960 gescegceas ctaggezeg: cgcssggcse astgtctste 200 gaagetatag cgttggegct scam: stgaatgagg gtgtcnsste ttttggatac tgtansatga tagagactte cteaggegat 2580 vateotasat goatastaan tactgataae atcttatage tugtattata Eeetgtatte 2090 eatcerctea attctettta acaaactaga aatartgtat atacaasans teataantaa 3000 ‘gtgegengee eeceecteat teatanggtt aaateattct catatatens genaagtgne 9120 aggogesctt aaatatects acaaatgete etteectans ctecoeccat aanaaanece 2100 ‘accgnagoas gUetetacgt tabttgogge Ctancgatte ctegteaten gaacegeces 2240US 7,968,321 BI 131 132 -continued asanggcen tosstoagas gectterset tagtetgaty cetggeaste costactete | 3360, vcagetene tonsaggegs tantacggtt atconcagaa toagagesta acgcaggaan 2480 ‘gueeeecent aggetecgee ceectgacga geatcacaaa aategecget cangtengag 3600 sogtaacagg attageagag squggtatat aggeggtact acagagttet tgaastasta 3960 agagteg gtagctetts atoogse ‘canaceaceg ctagtagegg 4080 cetgatetee tetacggagt stgaegetea segogeataa ctcacgttaa 4200 aqgaretcaa teatgagett gogcoatese atcaagteag ogcaatgcte tact sass sequence: 6 cecateaage ogatgosege eghectgues gtcteeccey gegacttcet geuattects 790 ‘gecatggect acttecatga gecgauctte gtegestegs tegagecect ggaggegsue 900 cogangegen teaccaccey geygatenty gagnacgase tgetagacan getgcecacy 1020 ctgagegane tagcctas 1038 <210> $89 1 no 7US 7,968,321 BI 133 134 -continued

---

ORGAO astaticLa,exauence 580 19 no 21 #00» sagumce: 21 svcanctgeg copargaces cacctgttge tsccagests ggatgecatt staststess getotvaas ceteestets eostagaces evgectgene saagogseste eaatgteces sgeagectste sgegttattteUS 7,968,321 BI 147 -continued cateaagtee tgtcteggey egtetgeste tggetguets geatanatat cteactegea 1260, sttggeget gasogeastg egegcentta cogugtcogs getgesestt gutgcgsate 1440 ceatennscn ggatttrege ctgetgayge ausccagogt ggaceyetty ctgcanctet 1560 etcagaacea agcastaaag ggeaateage tattgccogt eteactasta asaagaaaaa 1620 agctagcaog acagstctce oquctagaaa goagacagta agegeaacye asttastata 1740 segottctg gogtcaggca gecatcggaa getatgatat ggctatacag gtcgtanate 1860 arataatatg tgussteatg ageggstaac aattroscas aggssacage atargcrets 2040, gagegattea sogegtatte egetageate gottaagaag agettecaca tgggtgsage 2360, getscatcag catsagegtg gagasateag egacgaages tesgcagagg cgetgtatce 2220 agencgaatt taccageatg tttegcaggs ggaaggttet teacceagey atacggtett 7520 cetegacgat aacgoogata stataguagy agcenstcag ctgggcatta coagtattct 2680 gotoasagat sassccasca tcosggucta ettegsgaag gegttarcct catgataage 2660 gatctggtta cesgtetgge tttgattgcy gesettogts gtstgetste cpgstecgse | 2760 vegtttogen cpgutggcag gesttgeas atttectgtt tettecgcte cctggetage 2060 ctgacggege ctgacaccce geactagets gttarganay geegeeatag cguggecage 2360 ‘seoggtttog atanagegat guatanatet agcgegugte tgtttgegtt tggtatcace 3480 avogtettea cgggcgctag satogcagea ttccagcaac tgatggatat caacgeaata 3540US 7,968,321 BI 149 -continued cagaccater ctateggtge aotguattte attetcaoga tgattgcany copegtgste 2660 cugeegtata tegcgststt taptatgnge taveutcoys tetgttagHt cptectgage 2010 ctggegaace eeetggtcan eebectgett aaageguets atgstagcee ggcettgaat 3960 cagacgttea atcatggttt stectatetg gtcttegeeg egetgageat cevanstage 4020 aegrggeatt cocaauagte atgataagaa ttoggtttte egtcevgtet tgatttecaa 4140 cagcaagegs aetacgceat gggtegstat ttgatattat guageagcaa ogatateacg 4440 gacctettgy anacttegge ttesectgga gagagegags teeteegege tgtagaagte «740 egtecaages gasgcogstt sgeagegegg ettaastoas gegttagatg cactaagsae | 5240 ‘gectgaacen aggecccaae aattcecagy gaagtegcta cestggtagg caagtttcte 5580 cttggttans tegctatgce ccogtgnces atgegct gen cogtegceye tapcaggast $760 ‘getaccanne anatgacegs gatgaunats acgaattteg gegogsetty tttagagaa 5060US 7,968,321 BI 151 -continued <210> $89 19 xo 12 [2132 OTHER IPORATION, Description of AzEifietal Sequence: synthetic polynuclestide foo» suqumice: 22 ceoguaaatg acctgegttg ggegaancaa aacggettet atgcaatcac ggetgactte 240 vecagrasta stattgucgs raattae gastacages agctgtatac tgogttogca 540 aecctgccac egrcegicay cgugcastte etgatgaaty gitscctaag catgtatage 940 egtceggaas tgaccgazes gagcagctas e=30 gestgecgas gacgctasty 140 gequpettta cectgetgey tteccaggas gtgstat sccacgateg ghgatacegt ctacatcacc ggteacegtg cggagetayg tegctgggat 1440 acegegaane getsucagac catecageaa agctggance oggteeeget gaugaccace 1620 210» 589 19 No 29 <0» saqumiee: 29 Net tye Aon Gin Phe Gin Tyr Cye Cye Tie Val Tie beu Ser Val ValUS 7,968,321 BI -continued 154 mp as mp oo Be ae mp ay ay maa bye op Met ye ay tye ay ay ne ne a ay oe oe oe tye ne ap dan ely aes tye ‘me val Aap Phe Rep Phe ‘tne Uys teu eau trp tye das Phe tye ‘the asp tye an asp val a me bysUS 7,968,321 BI -continued ep the vat ye tie the aly Aan ‘an Tyr Pro He cin teu Ala Phe Tie tye Ser Lys Rep cay 589 1 nye tye Asp, - suqumice: 2 satgncests cagsnateos cogemnots estcrsases aagargencs enatgeage scggetaces ereageagat sacgatceget ccagnccagge roceattges eeteasegse tgateegees geaanageet ceattgeges estgostate egtsatsagg ae = a ane ne ay seogecaten saggacerteg egeeeggtgt eettonagna cextgetgect rosgotactt ccastascott eategaegte cestegengte sesagtetane scantects cettgecase tacgtatets accegtetts cxceattaceUS 7,968,321 BI 157 -continued snccetgeegt treogtggct gcogsasney gacutgaagg tegatggtta & sion <210> 89 19 no 25 #00» sequmiee: 25 Net Ala thr Sex Aop Sex Aon Het Lou Low Aon Tyr Val Pro Val Tyr op Guy Val Net Val Aop Val Trp Trp Gly Tie Tie Giu teu tye a1y Pro tye Gin ays Rep Tap Arg Ala tye Reg Ser Leu Phe Gie Leu val ‘ein lu eye Oly teu The teu in Ala Hie Het Ser Phe tie ain ye eu Aap Tie Gly Glu Ser Aan His Aap Te Phe Tyr Thr Aen Arg Ser fis the Hie Gly Arg The Ala tle Glu te tyr Ser Aop Tyr Met Lye aap Hie cle val Gly Leu Gly pro aia aly clu Leu arg Tyr Pro Ser ye Pro gin Sex Gin Gly trp Gia phe Pro Arg Tle aly Glu Phe ain eye Sys Rep tye Tye Lew Lys Ala Aap Phe Lys Ala Ala Val Ala Arg 22g Oy We Pro Oly tap alse fso Aap Aap MDa Oy le Tye Ane ‘ep Val Pro Oly Ser The hy fhe fhe Lys Ser Aan ly She Bye Vel hr he tye Oy tye Phe Phe Leu The tp Spr Ser Aon Uys Le bas ve Lys Yak Wye bea Ala 220 Lye Val Ser Gy Hae ie Tap tap 2 Lye a hu Ran Me Me A 6O4 Lew hr AD8 Gy YE TYE Aon La Pro Sex Aop Ala Lye Sex Gly Pro Gin Glu Leu Val Gin Gin Val LeuUS 7,968,321 BI -continued 160 Pro Arg Tye Aop Ala The Ala Tyr Val Thr Tyr Leu Arg Leu Ser Aap Aap Leu Leu ‘em Tae Be Lye Lye Phe Val ta feye Ala Aan Pro Gin tyo Tye Aan tye Met Hie Ser Ala Pro tye Hie Pro tie Giu Vat teu Lou <210> 580 1D no 26 #00» sagumice: 26 eosarsccas eecaneeses accateggee cacancagee eeagocaaae accegetsts egateagees cegeetegtee <210> $89 19 no 27 ceatagcast ssstoetgacs sctgtomaes seagaccaat gectgaccast sctggeets cxegasegae gatagtaace eetergaace eestestags ‘seseeatage cxgsestees cam Uys Ser Ron Phe a Aap Gin Rep Tye ccagateacat cogacotegtt seancaeoat sgecogtan azagtaccas atscttegtt cetgagegte getgttcats tanagatets cceacggttac gecgtecaatUS 7,968,321 BI 161 -continued 162 [210s ORGMMISN Aransdopose thatsana #00» saqomice: 27 Net Olu Oly Wye Ma Te Ala The Sex Leu Oy GIy Aap ArG Val Lew ie Phe Pro Oye Sex Pro Arg Sex Sex ohe Val Phe The Sex Arg Leu op Phe Pro Wie ciu Aan Gin Gin Giy Aan Pro Gly Leu Gly Lys Phe tye Gis tye Gin als trp Asp Sex Top the Ala tye Phe Sex aly aly: Tye Phe Ala tye bys Arg Gla tye Gis Ala Ala Val Val Gie The ue ‘Gly Leu He val Asn cye Leu tyr tyr Me Gly ys Leu Ser Lye The ‘val trp oln tau trp Glu Aep Val Tle Thr rie aly aly eu Ser val Met Ala Arg the Gly bys Leu ger Gis tye Gly Val Arg Phe Val aly: Ser Gin Met top The Aon Phe Leu Aan fro Asp Aan fe Lys aly Lew Ser Ser Tle The Met Leu Leu Sex Met Met Gly Aen Giy Leu Mee 116 Val Aen oye the Sar Gin Sar ehe bhe Val Ala Ala the Te Gly Le Iie Ser trp Te Gty beu Ala teu Tzp Arg Aop Ala Val Ala Tyr GlyUS 7,968,321 BI 163 -continued 164 <210> $89 19 no 28 [2132 OTHER TPoRATION: Description of Artificial Sequence: synthetic -<#00> sequmiee: 29 avgatetaga teatguogat ggeacgeogt atgeacgata tetacgcege sttratacta setegegaag tegstaogca gccgttctag attagtttge tetacacgat tancgegate aaactgtten tectctatts cetgatgges attsgtaata cgctgetste tgegtttase eatcactact tgacctegat sacgtgegge attctactaa cgagcotage aaacaccast atgcogcage tgttepegct ageasgeaag tatgetgata seagcasgey tgsgatgatt cogecegaaa acgccctgag satgeanggt ggctggraag attctaatgt teptatgcta cetsastoce gtatagesct gatgucgtts casctgttes atgctgectt sattagcatt) ‘togegguen ttugtetact gtugttccag gacctgstge cgustcate twgtgeegc te cabttogace ggtgicatce tggeggtgt gatccagast ‘gegsttgcee agagetggys teactteges gtgtactagy Ctattgcgst tetttccots grtgegstge eetgusgge ganggttaag gatgestas too» secumice: 29 Met He trp Me tat the mot Ala Ang Ang Wet Aon Gy Vol tye Ala 2a Hho Met Leu Val Ala Phe Hat Mot Ghy Val Ala Oly Ale Leu con tye Leu Te Te Phe cye Cye Leu Met Ala Tle Gly Aen Ala Leu Leu.US 7,968,321 BI 165 166 -continued fxg Gi Tye Aa Rap Aan Sox ALA Arg GIM Val Val Moe Phe Sor Sor aL Mee Ag Ala On Ser ABA Tey Yad Te Gy Pro Pro La ‘ia Bho Met Leu Aig Ue Aen BYE GLY Be THE Va Wet Phe sex 126 ‘hr tou Not Tep ThE Cys Aon ThE HOE TYE Hie Tie Aop Hot Pro Low ye tye Val tye Reg tye aly tye Reg Arg Mat Mer Val Te Ala val Phe Ala Val ays Tap Val Ze Ala val Ze Ser vs too» secumice: 20 <210> 989 19 No 21 > RGRIEN: Aetiticlat SequenceUS 7,968,321 BI 167 -continued 168 too» sauumiee: 21 ‘gtacegtgca ettectegtt ceactagatt ggageceaaa tateatcaga gtactgcttt 120 cvectaagat gocatettga gananattte actettcons ggagttgatt tagtatagge 240 560 10 wo 22 <#00» suqomice: 22 soacgaacag geasttegeg tattaagegs caggatagtt tetctettea ceagtasaae 120 guictogsta stagegogea ttgogcooag ogccatctga tegttgycaa coageatege 360 agtoggsacy atgecetcat teageatctg eatggtttge tgaas aatsatactg tegatgggtg tetggteaga gacetea: cegtengcee geragtegtt gtgscacgsg gttgggaaty tastteagct cogscatsge 960 sacggtctgs tasgagscac egycatacts tgegacateg tatauegtta ctggttteat 1080 agtgcaccaat gettctggey teuggeages atcggaaget gtgstataye tgtgceggte 1260, ‘geoeegacat cataacggtt ctggcassta tctgaaatg agctgttgae a: 920 SEDs ORGUISN: Aransdepete thalsana #00» seuumiee: 23US 7,968,321 BI 169 -continued 170 nu Te xy Ag Gay Ala Tye Leu Ale Esp OLL Aap Lau TRE Val Vad ‘aay Ser Gly Lye Ser Thr Lou Geu Aap Ser Lau Ala Gly Arg Lew Ata fg tow Aap Tye Gly bow Val ALA Tyx Val Thr Gin GIu Aep 226 beu Met oiy The Lew The Val Arg Glu thr Te Thr Tyr Ser Ala His bow ‘ly Thr Tle Tie ola Leu Oly Leu Gin Aap cya Ala Aap Arg Val The ‘ctu pro thr ser Gly Leu Acp ser Ala ser Ala Phe Phe Val rie ain feu teu Sex Sex ahy alu The Val tyr Phe Ghy Glu Ser Lys Phe Ala Val Gia Phe Phe Ale Gly Ala Gly Phe Pro Cys Pro Lys Lys Arg Aen fro Ser dap tie he tes tag tye He hon Ser Aep fhe Aep The Val fer Aap Pro Lew Wet Aan ew Ala Thr fer Gha te law Ala Ang Law ‘val lu aon Tyr Arg Arg ser val tyr ala Uys Ser ala Lye ser arg ‘rg fer Me Vol Aan Met Gye Reg Rep The Gly Tyr Tyr Tep Ser Reg Hig Yel fe tye he Vek Valder Phe ye Yah ly The Be Phe Bye ‘ep Vol Ghy ke Sex Tyr Mh Ser THe baw AA Aig Val Ser Ope Gly chy Me Te ts Giy Phe Met ThE She Het Ser THe Gly Gly Be Bro er the He Glu Gis Met bys Val the tyx tys Glu Arg teu sex oly,US 7,968,321 BI 1m -continued 172 ve Rye ly Wal ser Val Tye Te Te Ser an tye Val Sor Ser the Pro Phe Leu Val Ala Tie Ala Ue Tle The Gly Ser Tle The Tye Aan Net Val Wye Phe Rag Peo Gly Val Sex ke tp Ala Phe Bho Cys Leu op tou Pro tye Val Phe Trp Arg Tyx Pro Tie Ser Phe Hot Ser Tyr ly ser trp Ala Tle ain aly Ala yr Lys Aan Aap Phe Leu aly Lew ‘Val Tie Aan tye Te phe Oly Yal Gin Val Thr His ser ys Trp Trp ep Lou dor Ala Te Val Low Te eu Val Oye Tyr Arg Te Leu Phe ie Gin Ala tye Arg The Mat Lys Sex Leu Lys Lys Arg Pro Sex Phe too» secumice: 24 Met Ser Gln tye he teu Phe the Lys Ger Ale es Als Ve} Als Val Val Aen fle ger Ghy the ger ero ger Gly Arg Sex Lew Lys Ala Aap ‘Aen Tle Ala Pro The Ala Tep Val Pro Aan Met iio Phe Val ALA Pro "ea op Gln Phe Gly Tep Ghy Ala Sex Te Thr Sex Aen 2y= Oly bey Ala the Glu Phe Aen Aep the Tyr Ala Gly Gly Ser Val Gly olyUS 7,968,321 BI 173 -continued 174 The Thr Aap Law G14 The Wet Bam Un om tau Ser hy Ale TYE Rog Leu han a Ala Tup Set Bho Ghy Leu Oly Me Aan Ria Val TYE Ata dog Ala ye Te Ol Rag Phe Ra hy Aap Le chy Gn Lay VOL Ala teu Aon Giy Aan Gin Trp Giy Phe Giy Trp Aon Ala Gry The Leu Tyr ‘ctu tou Asp Lye Aon Aon Aug Tyr Ala Cou Thr Tyr Arg Ser Glu Val hye Tle Aap Phe Lys Gly Aan Bye sex sex Aap Leu Aan Arg Ala Phe -Aen Aon Tyr Gly teu Pro Fle pro Thr Ala The Gly Gly Ala The in Ser Gly Tyr teu The teu Aen tes Pro Glu Mer Trp Glu Val ser ay: Tye Aan Arg Val Aop Pro Gin Trp Ala The His Tyr Ser Leu Ala Tyr ep Thr Leu Phe Gin tye tie Glu aly ohe tye agp ala tyr arg le ‘Ala Leu Gly Thr The Tye Tyr yr Aap Aap Aan Trp Thr Phe Arg The Ser Tle pro Aap Gin Aap Arg Phe Top teu Ser Ala Ghy The The Tye [Ala Phe Aun Lye Aep Ala Ser Val Aap Val Gly Val Ser Tyr Met Hie Shy On fer Yak tye He hen i Oby Bro ye Gin Phe hu see Oe <210> $89 19 No 25 too» saqumiee. 25 Net tye Me tye Aen baw Pro Aan Lye Val te Ser Gly Ala Tie tea. hy Me Gly Ala The Sor Atg Sex Met Oly Oly The Raa Val ALA ke 1 Wal Gly Peo Ala Ser Wt Nat Val Ban Peo ALA THe Hee Op LaUS 7,968,321 BI 175 -continued 176 ip He hy Ria The Ram Peo OLN Me Gy In Hs Va See See Se ‘Aap Hie Ser tan han Aig Oly fs0 Tye Val AA Pro Ik he Ala Tye "ie Mie Wye Val Ser Aon Tep The the Oly A GIy Val Bho ALA Gln ep Val Giy Gly Lys Gly Tyr Ala Ala Gly Ala Aep Thr Gly bow Gi ‘Glu Wot Ser Ser Gly Val Asp Gly Trp Gly Tyr Ser Ala Arg tou Gly tos Lou tyr tye Val Ala Pro Thr The Aon Val Gly Val Sor Tye Met Phe tye Sex Hie Mat Aan Asp Leu Lye Gly tye Gly the val he Ala, ‘val Thr Aap tye ep teu fle Ala Phe Aap Yal ser Arg Yel Phe Trp lye Rep Ala Lew Lys Rep Tle tye Lew Gly Phe Ala Ser Gly Net Gly Guy Tye Meg His Ala Thr Gin fro the ton top Gls Oy Ue Les Aaa leu He ro Al Yel teu gin top te Ala er Les Ghy Phe Ser tye Cis Lau Ser We See Gly Reg She Rap Ra Ala Tye Sor ka ALA She 89 19 no 26177 US 7,968,321 BI -continued

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ORGAO astaticLa,exauence #00» saqomice: 26 ‘geoaceacag gotganagee aactasacet atanagars ‘canatcrata ceatggtct geteeggtce cegtgetge eeegtaceas cetteagteg cgttantats cececengae seaggactet esttgstgtt cangtetaaa gaceegcege ceagaagace estagee gstocoate ‘gangacagee veteatatge gstsaazeot cegoeeectte sngnguegett ccagutcctge acostcagas egactaaca sangettecee sacgtanata cezsctetote cxtgaceattUS 7,968,321 BI 179 -continued cteogatere taateggagy cabetgeete egttratege aaaanceana aazattgtte 7290 casstettea cagsctatts agectsoogt oataaataat tegcoartta ctagtteres 2340, atanccces atanatgett caataatatt gaasuagges gugtargate gancaagatg 2460, nctgactat tagctgtage setgcageay Uitteegtet gtetgeneay astegtecas 2580 ceetgettat gaaaacogae stgtocgges cactgazogs actacagsae gaagogucce | 2640 aagcogatoa ogattggsts stactagata aagttccgaa teaguatcta ctaageagce | 2760 sagacecage aacttgeceg rttgaccate ager: crostataga agcaggtcts attgateagy acgacctaga tanagagcac caggacctes 2940 saacgeatgg ogacgectat stgocanaca ctatgataga aaacygccge ttetctgatt 3060 ceategacts tguceptstg agtgtagets atagetates ggatategce ctegctacce 7320 gogstattge agaaguactg agtastosat gazctgnceg tetcctasts ctatacgsta 7380 cestotggeg ateteggtge satagscagt cacagszacs gtagecscaa sacsaagaat gecgaggaca tagtctetta guganacges egtgaccees taggeatagt tangeagatt 3540 210» $89 19 No 27 sequmiee: 27 ‘gotggaggea aaccatats 1” 580 10 10 26 SRG. nett saquence OTHER TPORNATION: Deecription of ArELficlal Sequence: SyntheticUS 7,968,321 BI 181 182 -continued Shoo> saqumice, 28 ctegagtegs atee 580 10 10 29 GROIN Acetic soquence scams: 29 ‘What is claim is 1, 4 method for the biogenic produetion of ethanol, co prising: culturing aa engineered eysnobscteiu in cultore medium inthe presence of light and!CO,, wherein ssideyano- bacterium comprises a recombinant pyruvate decarboxylase ‘gene and atleast one recombinant sleohol dehydrogenase ‘zene, wherein sad recombinant pyruvate decarboxylase gene ‘and at least one recombinant alcohol dehydrogenase pene belong to distinet operons wherein the expression of iid recombiniant alcohol dehydrogenase gene is increased rela- tive to the expression of said recombinant pynvate decar- boxylase gene, and wherein the amount of ethanol released ino said cultore medium by said engineered organism is ‘equal to or greater than 10 mg/L, °2. The method of claim 1, wherein sad engineered eyano- bacterium comprises a frst eopy and a second copy of said ecombinant algohol dehydrogenase gene, and wherein said first and second copies of said recombinant aleohol dehydno- tenase gene belong to distinet operons. 3. The method of claim 2, wherein said recombinant pyr vate decarboxylase gene and said second eopy of said recom- binant aleobol dehydrogenase gene are part of the same ‘operon 4 Themethod of elaim 2, wherein the expression ofa east ‘one copy of said aleohol dehydrogenase gene is operably linked toa lamba cl promoter 8. The method of claim 2, wherein said first and second copies of sid rocombinant alcoho dehytdrogenase genes are encoded by distinct plasmids. ‘6. The method of elsim 1, wherein the activity of aleoho! dehydrogenase encoded by ssid recombinant alcohol dehy- tdrogenase is varied by controlling the level of a cofactor required by alcohol dehydrogenase "7. The method of claim I, wherein the measured level of acetaldehyde released ito said culture medium by ssid engi- ‘ered organism is less than 7 mg/L after 10 days of culture 8. The method of claim f, wherein the measured level of thal released into said euleure medium by sad engineered ‘organism i atleast 1750 mgs 9. The method of claim 1, wherein the measured concen- ‘cation (mg/ml) of ethanol released into said culture medinm by said engineered organism is atleast 100 fld higher than the measured level of acetaldehyde in said culture mestum, 10. The method of claim 1, wherein said cyanobacterium is thermophilic eyanobacterium, 11, The method of elaim 1, wherein said recombinant aleoio! dehydrogenase gene encodes an alcohol dehydroge- ‘nase which is from Moorella species,