Journal Pre-proof

Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways

enables high-level production of miltiradiene in yeast

Tianyuan Hu, Jiawei Zhou, Yuru Tong, Ping Su, Xinlin Li, Yuan Liu, Nan Liu, Xiaoyi
Wu, Yifeng Zhang, Jiadian Wang, Linhui Gao, Lichan Tu, Yun Lu, Zhouqian Jiang,
Yongjin J. Zhou, Wei Gao, Luqi Huang
PII: S1096-7176(20)30066-5
DOI: https://doi.org/10.1016/j.ymben.2020.03.011
Reference: YMBEN 1656

To appear in: Metabolic Engineering

Received Date: 15 October 2019

Revised Date: 25 February 2020
Accepted Date: 29 March 2020

Please cite this article as: Hu, T., Zhou, J., Tong, Y., Su, P., Li, X., Liu, Y., Liu, N., Wu, X., Zhang,
Y., Wang, J., Gao, L., Tu, L., Lu, Y., Jiang, Z., Zhou, Y.J., Gao, W., Huang, L., Engineering chimeric
diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene
in yeast, Metabolic Engineering (2020), doi: https://doi.org/10.1016/j.ymben.2020.03.011.

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Author statement

Tianyuan Hu: Conceptualization, Investigation, Formal analysis, Data curation, Methodology,

Writing - original draft, Writing - reviewing & editing.

Jiawei Zhou: Investigation, Formal analysis, Writing - reviewing & editing.

Yuru Tong: Investigation, Validation.

Ping Su: Supervision, Writing - reviewing & editing.

Xinlin Li: Investigation.

Yuan Liu: Investigation.

Nan Liu: Data curation.

Xiaoyi Wu: Formal analysis, Writing - Reviewing and Editing.

Yifeng Zhang: Formal analysis.

Jiadian Wang: Investigation.

Linhui Gao: Data curation.

Lichan Tu: Visualization.

Yun Lu: Validation.

Zhouqian Jiang: Writing- Reviewing and Editing.

Yongjin J. Zhou: Methodology, Writing- Reviewing and Editing.

Wei Gao*: Conceptualization, Project administration, Resources, Validation, Supervision,

Writing- Reviewing and Editing.

Luqi Huang: Project administration, Resources, Supervision.

 1 Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables

2 high-level production of miltiradiene in yeast

3 Tianyuan Hu1,2 , Jiawei Zhou1, Yuru Tong2, Ping Su6, Xinlin Li1, Yuan Liu1, Nan Liu1, Xiaoyi Wu1,

4 Yifeng Zhang1, Jiadian Wang1, Linhui Gao4, Lichan Tu1, Yun Lu1, Zhouqian Jiang1, Yongjin J.

5 Zhou4, Wei Gao1, 2, 5 ,*, Luqi Huang3

7 1 School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, China

8 2 School of Pharmaceutical Sciences, Capital Medical University, Beijing, 100069, China

9 3 State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese

10 Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China

11 4 Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,

12 Dalian, 116023, China

13 5 Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing,

14 100069, China

15 6 Department of Chemistry, the Scripps Research Institute, Jupiter, Florida, 33458, USA

*
16 Corresponding author: Wei Gao. Jieping Building 381, Capital Medical University, Beijing,

17 100069, China. Tel: +86-10-83916572. E-mail: weigao@ccmu.edu.cn

18

1
19 Abstract

20 Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene

21 compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid

22 and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal

23 compounds. In this study, comprehensive engineering strategies were applied to construct a

24 high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1

25 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for

26 their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II

27 diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I

28 di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP

29 to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein

30 modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L

31 bioreactor. This work offers an efficient and green process for the production of the important

32 intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the

33 biotechnological production of valuable natural diterpenes.

34 Keywords：
：miltiradiene, geranylgeranyl diphosphate, diterpene synthase, metabolic engineering

35

36

2
37 1. Introduction

38 Many diterpenes have valuable pharmaceutical or biological activity, but their structure makes

39 them challenging to be synthesized. According to their main scaffold, diterpenes can be divided into

40 the clerodane, labdane, pimarane, abietane, and kaurane types (Christianson, 2017; Gao et al., 2012;

41 Jia et al., 2016). All these diterpenes are synthesized from (E,E,E)-geranylgeranyl diphosphate

42 (GGPP), which is derived from isopentenyl diphosphate (IPP) and its isomer dimethylallyl

43 diphosphate (DMAPP) produced via the mevalonate (MVA) pathway or the methylerythritol

44 phosphate (MEP) pathway (Fig. 1) (Ignea et al., 2015). Many of the abietane-type diterpenes are of

45 particular interest and value. This includes triptolide, a bioactive compound isolated from

46 Tripterygium wilfordii, the derivatives of which have been evaluated in phase I and II clinical trials,

47 as well as tanshinones, a group of active diterpenoids found in the Chinese medicinal herb danshen

48 (Salvia miltiorrhiza), which exhibit diverse pharmacological activities, including cardioprotective,

49 antioxidant and antitumor effects (Corson and Crews, 2007; Feng et al., 2019; Jiang et al., 2019;

50 Noel et al., 2019).

51 In spite of the many promising activities of these diterpenes, their low abundance in natural plant

52 tissues and the tedious and inefficient extraction processes severely limit their further study and

53 application. By contrast, biotechnological production using modified microorganisms is eco-friendly

54 and efficient, already providing sustainable and reliable supplies of many natural compounds, such

55 as artemisinic acid and taxadiene (Ajikumar et al., 2010; Paddon et al., 2013). It is essential in

56 enhancing the supply of their common precursors for high-level production of these diterpenes (Chen

57 et al., 2020). Miltiradiene is widely present in medicinal plants and is an important intermediate in

58 the biosynthesis of many abietane diterpenes, such as triptolide, tanshinone, carnosic acid, carnosol
3
59 and rubesanolides A-D (Bozic et al., 2015; Bruckner et al., 2014; Gao et al., 2009; Hansen et al.,

60 2017; Jin et al., 2017; Pateraki et al., 2014; Su et al., 2018; Sugai et al., 2011; Zerbe et al., 2014). In

61 our previous study, modular pathway engineering of diterpene synthases and key enzymes of the

62 MVA pathway in Saccharomyces cerevisiae led to a miltiradiene titer of 365 mg L-1 in a 15-L

63 bioreactor (Zhou et al., 2012). Then, the production of miltiradiene was further improved to 488 mg

64 L-1 in fed-batch cultivation by increasing the carbon flux toward FPP and GGPP through

65 plasmid-based overexpression of the key genes involved in the GGPP biosynthetic pathway (Dai et

66 al., 2012). However, the titer was still insufficient for industrial production.

67 In the biosynthetic pathway of miltiradiene, class II diterpene synthases, which contain a

68 characteristic DXDD motif, initiate the cyclization of GGPP to (+)-copalyl diphosphate ((+)-CPP),

69 after which the class I diterpene synthases containing a DDXXD motif cyclize and rearrange the

70 (+)-CPP to miltiradiene (Fig. 1). In recent years, more diterpene synthases that catalyze miltiradiene

71 biosynthesis have been identified in different plants, but we are not aware of any publications

72 comparing their ability to produce miltiradiene and the effects of combining different class I and II

73 diterpene synthases (Bozic et al., 2015; Bruckner et al., 2014; Gao et al., 2009; Hansen et al., 2017;

74 Inabuy et al., 2017; Jin et al., 2017; Pateraki et al., 2014; Pelot et al., 2017; Su et al., 2018; Sugai et

75 al., 2011; Zerbe et al., 2014; Zhang et al., 2018). Using recombinant expression of class I and II

76 diterpene synthases from different species, Jia et al. (2016) identified 13 previously unknown

77 diterpene products and achieved remarkably high yields with some of the enzyme combinations. In

78 addition, protein modification of key enzymes, including translational fusion and truncation of transit

79 peptides at the N-terminus of the enzymes, is also an effective strategy for increasing the production

80 of terpenes (Ajikumar et al., 2010; Chen et al., 2016; Zhou et al., 2012). Jiang et al. (2017) improved

4
81 the geraniol production from trace amounts to 524 mg L-1 by manipulating the key enzymes geraniol

82 synthase (GES) and farnesyl diphosphate synthase (Erg20). It is conceivable that an integrated

83 application of strategies including metabolic pathway optimization of host cells, non-native enzyme

84 combinations, and engineering of diterpene synthases, could lead to a major breakthrough in

85 miltiradiene production.

86 Here, we further improved the production of miltiradiene by increasing the carbon flux toward

87 GGPP in S. cerevisiae and optimizing the gene module of miltiradiene synthases. The final strain

88 with an optimized pathway and efficient miltiradiene synthase module reached a miltiradiene titer of

89 3.5 g L-1 in a 5-L bioreactor. Our work offers a good example for terpene production in microbial

90 cell factories and lays a foundation for the biosynthesis of other valuable natural diterpenes.

91
5
 92 Fig. 1. The biosynthetic pathway of GGPP and miltiradiene in S. cerevisiae. The genes in pink

93 ellipses were overexpressed in BY-T20.

94 2. Materials and Methods

95 2.1. Strains, plasmids and culture media

96 The initial strain used in this study was BY-T20 (MATα trp1Δ0 leu2Δ0 ura3Δ0,

97 trp1::HIS3-PPGK1-BTS1/ERG20-TADH1-PTDH3-SaGGPS-TTPI1-PTEF1-tHMG1-TCYC1, a schematic of

98 strain construction is shown in Fig. S1), which is ultimately derived from S. cerevisiae S288C

99 (Giaever et al., 2002; Su et al., 2018). E. coli Trans1-T1 (TransGen Biotech, Beijing, China) was

100 used for plasmid amplification. The gRNA expression vector p426-SNR52p-gRNA.CAN1.Y-SUP4t

101 (ref. no. 43803) and the Cas9 expression vector p414-TEF1p-Cas9-CYC1t (43802) were purchased

102 from Addgene (http://www.addgene.org/) (DiCarlo et al., 2013). The vector used for the construction

103 of miltiradiene synthases was pESC-LEU (Cat. no. 217452; Agilent Technologies, USA). All the

104 strains and plasmids used in this study are respectively listed in Table S1 and S2. Synthetic dropout

105 (SD) medium was purchased from FunGenome Company (China), and was supplemented with 2%

106 glucose, minus the auxotrophy factors complemented by the propagated plasmids. Yeast extract

107 peptone dextrose (YPD) medium was composed of 1% yeast extract (OXOID, England), 2% peptone

108 (OXOID, England) and 2% glucose.

109 2.2. Construction of gRNA plasmids and preparation of ds-oligos

110 Specific gRNA sequences targeting YJL064w, YPL062w and ROX1 were obtained using an

111 open-source tool http://yeastriction.tnw.tudelft.nl which is specialized for the identification of

112 suitable Cas9 target sites in S. cerevisiae strains (Mans et al., 2015). Target sequences with highest

6
113 scores without any 100% identity to other genomic loci were selected. The gRNA sequence targeting

114 the EGR9 promoter was described in a previous study (Jakounas et al., 2015). All gRNA target

115 sequences used in this study are listed in Table S3. For the assembly of single gRNAs, two reversed

116 AarI recognition sites (8NGCAGGTGNNNNCACCTGCN4) were inserted upstream of the gRNA

117 scaffold by restriction-free cloning based on the p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid using

118 the primer pair pU01-F/R, and the product was re-circularized by ligation. Next, 24 nt gRNA oligos

119 consisting of a 20 nt target sequence and 4 nt 5’ overhangs were synthesized. Equal volumes of 100

120 μM solutions of oligos F and R were mixed and annealed resulting in a double-stranded insert with

121 overhangs at both ends (Xing et al., 2014). Then, plasmids expressing single gRNAs were

122 constructed by inserting the double-stranded oligos into the AarI recognition site using Golden Gate

123 assembly (Fig. S2A) (Engler et al., 2014; Lee et al., 2015). Similar to the construction of the

124 single-gRNA plasmids, plasmids expressing multiple gRNAs were efficiently constructed by first

125 inserting two reversed AarI recognition sites into the plasmid with a single gRNA expression cassette,

126 and then amplifying different gRNA fragments using unique primers with different overhangs which

127 were inserted into the AarI cloning site according to the needs of the experiment by Golden Gate

128 assembly (Fig. S2B). The details of the reaction conditions and the primers can be found in the

129 legend of Fig. S2 and Table S4.

130 The 120 bp double-stranded oligos (ds-oligos) for YJL064w, YPL062w and ROX1 knockout were

131 obtained by annealing pairs of complementary single-stranded 120 nt oligos. The repair fragments

132 for EGR9 knockdown and UPC2.1 knock-in were amplified by PCR using synthetic DNA sequences

133 as templates. All ds-oligos were purified and then stored at -20°C for further use, and their sequences

134 are listed in Table S5.

7
135 2.3. Genome editing in S. cerevisiae using the CRISPR/Cas9 system

136 To obtain a higher genome editing efficiency, the Cas9 expression vector

137 P414-TRP1-TEF1p-Cas9-CYC1t was first introduced into BY-T20 using the lithium acetate

138 transformation method according to the directions of the Frozen-EZ Yeast Transformation II Kit

139 (Gietz and Schiestl, 2007). The strain with the Cas9 expression plasmid was named BY-HZ09 and

140 used for the following manipulations. A total of 1 μg of the gRNA expression plasmid was mixed

141 with 1 nM of each corresponding ds-oligo and electroporated into BY-HZ09 at 3.0 kV in a 2-mm gap

142 electroporation cuvette using a Bio-Rad MicroPulser followed by cultivation on synthetic drop-out

143 medium without tryptophan and uracil at 30°C for 2-3 days. After incubation, 5 mutant colonies

144 were selected and genomic DNA was isolated for use as template for PCR of all targeted loci. The

145 isolated genomic DNA of strain BY-T20 was used as negative control. The correct PCR bands were

146 further verified by DNA sequencing. The mutants with desired genotypes were used in the following

147 experiments after removing the gRNA and Cas9 plasmids as described in a previous study (Mans et

148 al., 2015).

149 2.4. Construction of miltiradiene synthase expression plasmids

150 The sequences of miltiradiene synthases from 7 plants were downloaded from the National

151 Center for Biotechnology Information (NCBI) database

152 (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), codon-optimized for yeast and ordered as synthetic

153 DNA. The vectors were directly constructed in the yeast strain BY-HZ16 by electroporation as

154 described above. Each miltiradiene synthase fragment had 100 bp overhangs at both ends which

155 were homologous to the linearized eukaryotic pESC-LEU vector. The class II and I di-TPSs were

8
156 inserted into the multiple cloning site 1 (MCS1) and 2 (MCS2), respectively, to be transcribed from

157 the pGAL10 and pGAL1 promoter, respectively (Fig. S3).

158 The fusion-protein expression plasmids were constructed using the same method. The class II

159 di-TPS CfTPS1 and the class I di-TPS SmKSL1 were coupled together via the widely used flexible

160 linker GGGS encoding sequence “GGT GGT GGT TCT” and the stop codons of the upstream genes

161 were removed. Additionally, four truncated variants of fusion protein SmKSL1-CfTPS1 were also

162 generated. The truncation 1 named tSmKSL1-CfTPS1 lacks the transit peptide at the N-terminus of

163 SmKSL1 (M1-C47), and the truncation 2 tSmKSL1-tCfTPS1 has a further deletion of the transit

164 peptide at the N-terminus of CfTPS1 (M1-N81). Furthermore, the β domain (N48-S256) away from

165 the active-site of SmKSL1 was removed and the variant was named as SmKSL1α-CfTPS1. Similarly,

166 the α domain (D518-A786) far from the active-site of CfTPS1 was removed and the variant was

167 named as SmKSL1α-CfTPS1βγ. All the plasmids encoding the truncated versions were constructed

168 by electroporation using BY-HZ16 as the host strain. After cultured in synthetic drop-out medium

169 without leucine (SD-Leu medium), single clones were selected and plasmids were isolated. The

170 sequence of each variant was verified by specific PCR and sequencing.

171 2.5. Homology modeling and structural analysis by computational simulation

172 Since the crystal structures of the fusion protein complexes (SmKSL1-CfTPS1 and

173 CfTPS1-SmKSL1) have not been solved, we submitted the sequences to web-based tool I-TASSER

174 (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Yang et al., 2015). To select the models,

175 I-TASSER uses the SPICKER program to cluster all the decoys based on the pair-wise structure

176 similarity, and reports five models which corresponds to the five largest structure clusters. The

177 models were visualized using the versatile molecule model rendering software, PyMOL, and the

9
178 final models of fusion protein complexes were selected by align to the protein models of SmKSL1

179 and CfTPS1 which were also predicted using I-TASSER.

180 The proteins secondary structure and chloroplast transit peptides of SmKSL1 and CfTPS1 were

181 predicted using the web-based tools http://www.compbio.dundee.ac.uk/jpred/index.html and

182 http://www.cbs.dtu.dk/services/ChloroP/ (Drozdetskiy et al., 2015; Emanuelsson et al., 1999). We

183 truncated the transit peptides and domains based on the analysis of protein secondary structure.

184 2.6. GGOH and miltiradiene fermentation in shake flasks

185 A two-phase extractive fermentation process was performed in this study. For GGOH production,

186 six individual colonies of strain BY-T20 and its mutants were first picked from the agar plates into

187 sterile culture tubes containing 5 mL YPD medium and cultivated at 30°C and 220 rpm for 24 h to

188 the exponential phase. Then, the resulting exponential culture was diluted to an initial OD600 of 0.2 in

189 5 mL of fresh YPD medium and cultivated at 30°C and 220 rpm for 12 h until an OD600 of

190 approximately 5.0. Next, aliquots were diluted to an initial OD600 of 0.05 in 10 mL medium and

191 cultivated at 30°C and 220 rpm. An extractive n-dodecane phase comprising 10 % (v/v) of the

192 culture volume was added aseptically after 10 h. The organic layer was harvested for GGOH analysis

193 at 72 h by centrifugation of the fermentation broth at 10,000 ×g for 1 min, and 10 µL of the

194 n-dodecane from each sample were transferred into 990 µL of n-hexane (dilution of 1:100) in a glass

195 GC vial. The miltiradiene fermentation process was similar to the method mentioned but the medium

196 used here was different because the miltiradiene synthases were expressed from the

197 D-galactose-induced promoters pGAL1 and pGAL10. Correspondingly, the medium used for strain

198 activation was SD-Leu with 2.0 % glucose and the production medium was replaced with SD-Leu

199 medium containing 1.8 % galactose and 0.2% glucose because a small amount of glucose can

10
200 minimize the lag phase during metabolic adaption to galactose (Rodriguez et al., 2014). The organic

201 layer was harvested for miltiradiene analysis after 144 h. Samples and standards were transferred to

202 the gas chromatograph-mass spectrometer (GC-MS) autosampler for qualitative analysis and

203 quantification.

204 2.7. Fed-batch fermentation for GGOH and miltiradiene production

205 To produce larger amounts of GGOH, strain BY-HZ16 was used for fed-batch fermentation in a

206 5-L bioreactor (Shanghai Baoxing Bio-Engineering Equipment Co., Ltd., China). Firstly, a single

207 clone was seeded into a 100 mL flask containing 20 mL YPD medium and grown at 30°C and 200

208 rpm for 24 h. The resulting exponential culture was diluted to an initial OD600 of 0.2 in 150 mL of

209 fresh YPD medium and cultivated for another 12 h until the OD600 reached approximately 5.0. The

210 seed culture was then used to inoculate a bioreactor containing 1.5 L of optimized YPD medium

211 (OYPD, 5% glucose, 1% yeast extract, 3% peptone, 0.8% KH2PO4, and 0.6% MgSO4) to an OD600

212 of approximately 0.1. The temperature was set to 30°C and the pH was maintained at 5.5 by the

213 addition of ammonium hydroxide. The dissolved oxygen (DO) concentration was kept above 30% of

214 atmospheric oxygen by adjusting the agitation (300-900 rpm) and aeration. Concentrated glucose

215 solution (80%, w/v) was fed at an exponential rate to control the glucose concentration below 1 g L-1

216 (Song et al., 2017). Additionally, 5×YP mixture (5% yeast extract, 15% peptone) was fed

217 periodically to provide adequate nutrition for cell growth. An extraction phase comprising

218 n-dodecane was added to 20% (v/v) of the medium volume at 10 h of the fermentation to start the

219 two-phase extractive fermentation. Duplicate culture aliquots were collected periodically to

220 determine cell density and production of GGOH. The miltiradiene fed-batch fermentation was

221 processed in accordance with previous studies (Tippmann et al., 2016; Yu et al., 2018; Zhou et al.,

11
222 2016). The single clone of BY-HZ70 was activated for two passages in SD-Leu medium with 20 g

223 L-1 glucose. Minimal medium containing 5 g L-1 (NH4)2SO4, 3 g L-1 KH2PO4, 0.5 g L-1 MgSO4∙7H2O,

224 60 mg L-1 uracil, 60 mg L-1 tryptophan, 20 g L-1 glucose, as well as trace metal and vitamin solutions

225 was used as the initial fermentation medium (Yu et al., 2018; Zhou et al., 2016). The temperature,

226 pH, DO, agitation and aeration were controlled as above. The carbon source was changed into

227 galactose after 6 h. A concentrated galactose solution (60 %, w/v) and 5× minimal medium were fed

228 to provide adequate carbon source and nutrition for cell growth and miltiradiene production. The

229 galactose and 5× minimal medium was initially fed with a rate that was exponentially increased to

230 maintain a constant increase of the biomass yield and miltiradiene concentration. After the biomass

231 yield stabilized, the feeding was started once the dissolved oxygen level was higher than 40%.

232 Similarly, 20 % (v/v) n-dodecane was added at 10 h and duplicate culture aliquots were collected

233 systematically to determine the cell density and production of miltiradiene and GGOH. Dry cell

234 weight measurements were performed as reported previously (Yu et al., 2018).

235 2.8. GC-MS analysis of GGOH and miltiradiene

236 For qualitative analysis of GGOH, GC-MS analysis was conducted with an Agilent 7000 gas

237 chromatograph equipped with a DB-5 MS column (15 m×0.25 mm×0.10 μm film thickness) using

238 helium as the carrier gas. The initial oven temperature was set to 100°C for 1 min followed by a

239 40°C min-1 gradient to 200°C, hold for 1 min, and 20°C min-1 gradient to 300°C, and a final 1 min

240 hold (total run time 10.5 min). The mass spectrometer was operated in the electron impact (EI) mode

241 at 70 eV in the scan range of 0–300 m/z. Mass Hunter software (Agilent, USA) was used for data

242 acquisition and processing. Quantification of GGOH was conducted in MRM mode with the same

243 GC conditions, a collision energy of 15 eV, and a solvent delay of 3 min. The dwell time was 150 ms

12
244 and the scan rate was 3.3 cycles s-1. The fragment ions were quantified using 81 m/z as the

245 quantification ion and 41 m/z along with 53 m/z as qualifiers. Calibration standard solutions for the

246 quantification were prepared using authentic GGOH (the purity is 95 %, aladdin, China) reference

247 standard (Fig. S4). GC-MS analysis of miltiradiene was conducted using the same instrument and

248 column with the following program: The initial oven temperature was set to 50°C for 2 min followed

249 by a 40°C min-1 gradient to 170°C, a 20°C min-1 gradient to 240°C, a 40°C min-1 gradient to 300°C,

250 and a final 1 min hold (total run time 11 min). The quantification conditions were similar to those of

251 GGOH using the MRM method, whereby 134 m/z was used as the quantification ion and 65 m/z

252 along with 91 m/z were used as qualifiers. The collision energy was 20 eV with 100 ms dwell time,

253 and the scan rate was 5 cycles s-1.

254 3. Results and Discussion

255 3.1. Pathway modification for GGPP overproduction in S. cerevisiae using the CRISPR/Cas9

256 system

257 GGPP is the common precursor of diterpenes and its accumulation is essential to the production

258 of downstream products. In S. cerevisiae, the 3-hydroxy-3-methyl glutaryl coenzyme A reductase

259 (HMG-R), encoded by the HMG1 gene is the major rate-limiting enzyme in the MVA pathway, and

260 overexpression of the catalytic domain of HMG1 (tHMG1) led to an improved production of

261 isoprenoids, IPP and DMAPP (Hampton et al., 1996; Polakowski et al., 1998). The isopentenyl

262 transferase Erg20 condenses IPP and DMAPP to form the linear isopentenyl diphosphate precursor,

263 farnesyl diphosphate (FPP). Next, another isopentenyl transferase, Bts1, converts FPP to GGPP.

264 Overexpression of the key genes in the GGPP pathway and modification of culture conditions are
13
265 widely used strategies to improve GGPP production (Dai et al., 2012; Song et al., 2017; Tokuhiro et

266 al., 2009). However, the metabolic system of S. cerevisiae is a complex and precisely regulated

267 network, and combinatorial design of expression cassettes could be helpful for the further

268 accumulation of GGPP. Here, BY-T20, an engineered S. cerevisiae with an expression cassette

269 harboring tHMG1, the BTS1-ERG20 fusion protein module and a special gene SaGGPS, coding

270 isopentenyl transferase from Sulfolobus acidocaldarius that can convert both DMAPP to FPP and

271 FPP to GGPP, and is therefore more efficient than the wild-type yeast was used as the host strain (Su

272 et al., 2018). More comprehensive strategies were applied to achieve a higher titer of GGPP using

273 the CRISPR/Cas9 system (DiCarlo et al., 2013; Flagfeldt et al., 2009; Mans et al., 2015; Ohnuma et

274 al., 1994). In general, GGPP is nearly undetectable in yeast, and its dephosphorylated derivative

275 (E,E,E)-geranylgeraniol (GGOH) is used as a common and direct reporter of GGPP production

276 instead (Song et al., 2017). Therefore, we measured the GGOH titer to characterize the GGPP

277 production capacity of BY-T20 and its mutants.

278 First, we downregulated the metabolic flux that towards competing pathways. Erg9 is the

279 squalene synthase of S. cerevisiae, which converts FPP to squalene. It is the first enzyme in the

280 branching pathway for triterpenoid and ergosterol biosynthesis, and its high expression consumes

281 significant amounts of FPP so that it decreases the precursor supply for GGPP production

282 (Asadollahi et al., 2008; Paramasivan and Mutturi, 2017). Considering that ERG9 is essential for

283 ergosterol biosynthesis and yeast cannot survive under aerobic conditions in its absence, in this study

284 we repressed the expression of ERG9 by knocking out the upstream activating sequence (UAS) of its

285 promoter (Fig. S5C) (Kennedy and Bard, 2001). The mutant strain BY-HZ10 showed a significant

286 improvement of the GGOH production from 40.3 to 196.4 mg L-1 in YPD medium in shake flasks

14
287 (Fig. 2A, S5 and S6). Additionally, we tried to knock out the cis-prenyltransferase gene, RER2,

288 because it can competitively convert FPP to polyprenyl compounds in S. cerevisiae. However, the

289 mutant strain without RER2 showed a very low growth rate compared with the other strains, and the

290 editing of this locus was decided against. Schenk et al. (2001) reported that yeast cells with a

291 deletion of the RER2 locus are viable, but defective in protein N-glycosylation, which suggested that

292 Rer2 plays an essential role in the normal growth of S. cerevisiae.

293 Next, we manipulated the genes and transcriptional regulators that have impact on the MVA

294 pathway. Rox1 (repressor of hypoxia) is a transcriptional regulator that can decrease the

295 accumulation of GGPP by downregulating the expression of genes involved in the ergosterol

296 biosynthesis (Denby et al., 2012; Henry et al., 2002; Montanes et al., 2011). Here, we knocked out

297 the open reading frame (ORF) of ROX1 in BY-T20 (Fig. S5B). The resulting mutant, BY-HZ11,

298 produced 1.96-fold higher GGOH than that of the initial strain (Fig. 2A). To study the combined

299 effect of ROX1 and ERG9, we constructed the strain BY-HZ12, which further improved the GGOH

300 production to 206.9 mg L-1 (Fig. 2A). It was reported that the yeast strains with deletions of the

301 distant genetic loci YPL062w and YJL064w showed good plasmid maintenance, high cell density, as

302 well as good titers of bisabolene and carotenoids at the end of production (Giaever et al., 2002;

303 Ozaydin et al., 2013). In this study, the ORFs of YPL062w and YJL064w were knocked out, resulting

304 in the single knockout strains BY-HZ13 and BY-HZ15 (Fig. 2B), which showed increased GGOH

305 levels of 54.8 and 80.8 mg L-1, respectively. Moreover, the GGOH production was significantly

306 improved to 274.7 mg L-1 in strain BY-HZ16, in which YPL062w and YJL064w were knocked out

307 simultaneously in the background of BY-HZ12 (Fig. 2A). Additionally, the uptake control

308 transcriptional regulator, Upc2, enables S. cerevisiae to take up external sterols during aerobic

15
309 cultivation, and its mutant Upc2.1 (G888D) has stronger uptake ability than the wild type. Thus,

310 Upc2.1 was often introduced into engineered yeast to produce specific terpenes of interest in earlier

311 studies (Dai et al., 2012; Lewis et al., 1988; Paddon et al., 2013; Ro et al., 2006; Vik and Rine, 2001).

312 In our work, the ORF of UPC2.1 was integrated into the YPL062w deletion site (Fig. S5C), and the

313 mutant strain BY-HZ17 had a 1.36-fold increase of GGOH titer compared with BY-HZ13. However,

314 when we integrated the UPC2.1 gene into the strain BY-HZ16, the GGOH production failed to be

315 improved further (BY-HZ18). A possible reason is that the overexpression of UPC2.1 increased the

316 metabolic burden to the strain due to multiple genetic manipulations. In view of this, we introduced a

317 site-directed mutation (G888D) into the native UPC2 gene of S. cerevisiae, but disappointingly, a

318 back mutation (D888G) was observed in the next generation of the mutant strain (Fig. S7). Leak et al.

319 (1999) found that UPC2.1 mutant yeast had increased sensitivity to metal cations and a reasonable

320 hypothesis is that UPC2.1 results in a general increase of cellular permeability, which could be

321 indicative of a plasma membrane defect. This phenomenon suggested that Upc2 plays a central role

322 in the cellular homeostasis of S. cerevisiae. Among the tested strains, the quadruple mutant

323 BY-HZ16 (ERG9p;ROX1;YJL064w;YPL062w) had the highest ability to produce GGPP.

324 In addition, medium optimization is an effective approach to improve terpene production. Zhou

325 et al. (2019) reported that an optimized YPD medium significantly improved the production of

326 friedelin, a triterpene precursor of celastrol. Therefore, we used a medium containing 5% glucose, 1%

327 yeast extract, 3% peptone, 0.8% KH2PO4, and 0.6% MgSO4 to culture the GGPP high-yield strain

328 BY-HZ16. As shown in Fig. 2B, the optimized YPD medium led to a significant increase of cell

329 density and GGOH production. The highest yield of GGOH in stain BY-HZ16 under shake-flask

330 conditions was 557.2 mg L-1, which was 61% higher than in ordinary YPD medium. The optimized

16
331 YPD medium provided more nutritional components and a suitable pH (≈5.6) for yeast growth so

332 that a higher cell density was obtained in the same volume of fermentation medium, which resulted

333 in higher GGOH production. Given this, the optimized YPD medium was used as the initial medium

334 in fed-batch fermentation of strain BY-HZ16 in a 5-L bioreactor. The GGOH production of the strain

335 reached 2.1 g L-1 and it kept increasing along with the cell density during 10 days of fermentation

336 (Fig. 2C).

337 In summary, we integrated multiple strategies including overexpression of pathway genes,

338 downregulation of competing pathways, manipulation of transcriptional factors, and optimization of

339 the medium to improve the GGPP production in S. cerevisiae. Finally, the mutant strain BY-HZ16

340 produced 2.1 g L-1 GGOH in fed-batch fermentation which provided a suitable microbial cell factory

341 for the industrial production of GGOH and laid a foundation for the the overproduction of diterpenes.

342

17
343 Fig. 2. The GGOH production of the engineered yeasts. (A) The genotypes and GGOH levels of the

344 engineered strains. (B) GGOH production of BY-HZ16 cultured in different media. (C) Fed-batch

345 fermentation of the highest GGOH-accumulating strain BY-HZ16. The data in (A) and (B) are the

346 averages of 6 biological replicates with error bars representing standard deviations. The data in (C)

347 are the averages of 3 biological replicates with error bars representing standard deviations.

348 3.2. Broad screening of diterpene synthases for high-yield miltiradiene production

349 In addition to the sufficient supply of precursors, the catalytic efficiency of enzymes is another

350 key point for high production of diterpenes. Previous studies showed that screening enzymes from

351 diverse sources can be an effective strategy to increase the productivity of heterologous pathways in

352 specific hosts (Chen et al., 2016; Ma et al., 2016). Abietane is the most widespread diterpene

353 skeleton in the family Lamiaceae (Vestri Alvarenga et al., 2001). The miltiradiene synthases,

354 including nor-copalyl diphosphate synthases (CPS) and kaurene synthases-like (KSL), are present in

355 many species of the Lamiaceae, and they share high amino acid sequence similarity with each type

356 of cyclases. Here, 11 miltiradiene synthases, functionally annotated from five representative

357 Lamiaceae plants, including Salvia miltiorrhiza (SmCPS1, SmKSL1), Coleus forskohlii (CfTPS1,

358 CfTPS3 and CfTPS4), Marrubium vulgare (MvCPS3, MvELS), Rosmarinus officinalis (RoCPS1,

359 RoKSL1 and RoKSL2), and Salvia fruticosa (SfKSL), whose complete coding sequences can be

360 obtained from the NCBI database were selected as candidates for high-yield miltiradiene synthesis

361 (Bozic et al., 2015; Bruckner et al., 2014; Gao et al., 2009; Pateraki et al., 2014; Zerbe et al., 2014).

362 Additionally, TwCPS1 and the monoterpene synthase, TwMS from the Celastraceae member

363 Tripterygium wilfordii were also characterized in terms of miltiradiene production, but they share

18
364 lower sequence similarity with the miltiradiene synthases of the Lamiaceae (Hansen et al., 2017; Su

365 et al., 2018). In addition to higher plants, a bifunctional miltiradiene synthase (SmoMDS) from the

366 lycophyte Selaginella moellendorffii was selected as the final candidate (Sugai et al., 2011). Among

367 the 14 candidate miltiradiene synthases, CfTPS1, SmCPS1, MvCPS3, RoCPS1 and TwCPS1 are class

368 II diterpene synthases that contain a catalytic “DXDD” motif for the protonation-initiated synthesis

369 of (+)-CPP. TwMS, MvELS, CfTPS3, CfTPS4, RoKSL1, SmKSL1, RoKSL2 and SfKSL are class I

370 terpene synthases that contain a conserved DDXXD motif for further cyclization of (+)-CPP to

371 miltiradiene. SmoMDS contains both active-site motifs and can directly convert GGPP to

372 miltiradiene (Fig. S8). In the following work, we screened the best combinations of class I and II

373 di-TPSs for high-yield miltiradiene by high-efficiency recombination.

374 All of the selected di-TPSs were codon-optimized and plasmids harboring random combinations

375 of class I and II di-TPSs were introduced into the GGPP high-yield strain BY-HZ16. As illustrated in

376 Fig. 3, 39 miltiradiene-producing strains were constructed and their product titers were investigated

377 by GC-MS analysis (Fig. S9). The results revealed significant differences of miltiradiene production

378 in those strains with different groups of class I and II di-TPS. Among them, strains containing

379 CfTPS1 exhibited higher miltiradiene production than those with other class II di-TPSs together with

380 class I di-TPSs from various plants. Likewise, strains containing SmKSL1 produced more

381 miltiradiene than those with other class I di-TPSs, especially when coupled with CfTPS1 (BY-HZ49).

382 The miltiradiene production of the module consisting of CfTPS1 and SmKSL1 was 105.3 mg L-1 (Fig.

383 3). By contrast, the class I TPS from T. wilfordii (TwMS), showed much lower miltiradiene

384 production with all the tested CPSs. Hansen et al. (2017) found that TwMS is a close relative of

385 mono-TPSs but has a mutant RRx8W motif, and its sequence shares low similarity with other KSLs

19
386 used in this study (Fig. S7). This may be the reason for its low catalytic efficiency in the production

387 of miltiradiene. Furthermore, we also found that the different couples of class I and II di-TPSs

388 produced different miltiradiene titers. For instance, RoKSL2 had a higher miltiradiene production

389 than RoKSL1 when coupled with CfTPS1 and TwCPS1, but it was the opposite when coupled with

390 MvCPS3 and RoCPS1. The subtle differences in their protein structures may influence the

391 cooperation between synthases. Surprisingly, the bifunctional enzyme SmoMDS produced only trace

392 amounts of miltiradiene in strain BY-HZ16, which might attribute to the enzymatic inhibition of

393 SmoMDS by high level of GGPP in BY-HZ16. These results indicated that screening enzymes from

394 different species is an effective strategy for improving the production of miltiradiene as well as other

395 terpenes, and the cooperation between different synthases can influence the efficiency of substrate

396 utilization. This is an important concept for a further enhancement of the heterologous production of

397 terpenes in the future.

398
20
399 Fig. 3. The miltiradiene production of different combinations of class I and II di-TPSs. (A) Scheme

400 of selecting the miltiradiene synthase module with the highest miltiradiene yield. (B) Miltiradiene

401 production by combinations of diterpene synthases from different plants. The data are the averages

402 of 5 biological replicates with error bars representing standard deviations.

403 3.3. Improving the miltiradiene yield by constructing fusion proteins

404 Fusion proteins generated by fusing two or more genes with a linker region are widely used to

405 enhance the catalytic capacities of enzymes and improve the utilization of substrates between

406 proteins that catalyze sequential steps in a pathway or in which protein-protein interactions are

407 required for functionality (Woolston et al., 2013). Flexible linkers are often used in fusion proteins to

408 reduce folding interference, enabling the individual proteins to retain their native activity, and either

409 separate domains spatially or allow them to interact if necessary (Chen et al., 2013; Chichili et al.,

410 2013). Zhou et al. (2012) found that the miltiradiene synthases SmCPS1 and SmKSL1 directly

411 interact in Salvia miltiorrhiza in vivo. Based on their findings and the sequence alignment of

412 miltiradiene synthases in this study, we speculated that molecular interactions between CfTPS1 and

413 SmKSL1 may exist, too. To test this hypothesis, we fused the proteins CfTPS1 and SmKSL1 in

414 different order using the flexible linker “GGGS” (Zhou et al., 2012). The strain BY-HZ68 expressing

415 the fused module CfTPS1-SmKSL1 produced 70.5 mg L-1 miltiradiene, which was lower than the

416 yield of the original separated module (strain BY-HZ49). Nevertheless, the other fused module

417 SmKSL1-CfTPS1 (strain BY-HZ69) led to 1.81-fold increase of miltiradiene production, which

418 reached 313.4 mg L-1 (Fig. 4A). This result indicated that the SmKSL1-CfTPS1 fusion is superior to

21
419 the CfTPS1-SmKSL1 fusion as well as the two proteins being expressed separately in terms of

420 miltiradiene production.

421 To illustrate the reason for the differences of miltiradiene yield, we produced 3D models of the

422 proteins using the web-based tool I-TASSER (Yang et al., 2015). The modeling showed that the

423 class II di-TPS CfTPS1 has an αβγ domain structure. The active site “DXDD” motif is located

424 between the β and γ domains at the N-terminus. The class I di-TPS SmKSL1 has an αβ domain

425 structure, and the active site is located in the α domain at the C-terminus. The schematic diagram of

426 fusion proteins (Fig. 4B) illustrates that the distance between the two active sites of fusion protein

427 SmKSL1-CfTPS1 is shorter than that of CfTPS1-SmKSL1. A closer arrangement of active sites

428 contributes to an increase of the local concentration of intermediates by reducing the spatial diffusion.

429 This may be the reason why the SmKSL1-CfTPS1 fusion module performed better in terms of

430 miltiradiene production.

431

22
432 Fig. 4. The miltiradiene production of fusion proteins of CfTPS1 and SmKSL1. (A) The miltiradiene

433 production of different fusion proteins. The data are averages of 6 biological replicates with error

434 bars representing standard deviations. (B) The domain architecture of the fusion proteins.

435 3.4. Improving miltiradiene production by tailored truncations

436 In order to obtain a higher miltiradiene titer, we engineered the fusion protein by further structure

437 optimization. It is known that many terpene synthases are targeted to plastids in plant, and after the

438 protein has been localized to the plastid, the transit peptides will be hydrolyzed (Bohlmann et al.,

439 1998). However, yeast cannot hydrolyze the peptides due to the absence of plastidic transit

440 peptidases, which may affect the catalytic activities of the proteins. Therefore, we truncated the

441 chloroplast transit peptide (M1-C47) at the N-terminus of SmKSL1, and the resulting strain

442 BY-HZ70 showed a significant increase of miltiradiene production to 550.7 mg L-1 in shake flasks

443 (Fig. 5A). Inspired by this, we further truncated the chloroplast transit peptide located at the

444 N-terminus of CfTPS1 (M1-N81), but the result went contrary to our expectations. The miltiradiene

445 production decreased to 180.5 mg L-1 (strain BY-HZ71), while the by-product GGOH showed a

446 slight increased production (Fig. 5A and 5C). It is possible that the truncation of the loop between

447 SmKSL1 and CfTPS1 decreased the flexibility of the fusion protein and increased the distance

448 between the two active sites indirectly.

449 In addition to the transit peptides, we also investigated domain-truncated variants of the fusion

450 protein SmKSL1-CfTPS1. Generally, the α domain of class II di-TPSs is considered a nonfunctional

451 vestige due to the lack of the DDXXD motif, and the β domain of class I di-TPSs is considered

452 vestigial due to the lack of the D/E-rich motif and the DXDD motif. Here, we truncated the β domain

23
453 of SmKSL1, resulting in the strain BY-HZ72. However, the resulting protein did not produced

454 miltiradiene, and (+)-copalol, the dephosphorylated derivative of (+)-CPP was detected by GC-MS

455 analysis (Fig. 5B, 5C and S10). Similarly, we further truncated the α domain of CfTPS1 but we

456 failed to detect any miltiradiene or (+)-copalol but only the by-product GGOH in the corresponding

457 strain BY-HZ73. These results demonstrate that domains that do not contain active sites can also

458 impact the catalytic function of diterpene synthases and alter the outcome of catalysis, explaining

459 why evolution retained them.

460

461
462 Fig. 5. The miltiradiene production of strains expressing truncations of the fusion protein

463 SmKSL1-CfTPS1. (A) The miltiradiene production of strains expressing the truncated variants. The

464 data are averages of 6 biological replicates with error bars representing standard deviations. (B)

24
465 GC-MS analysis of the fermentation products of strains expressing the truncated fusion proteins. (C)

466 The titer of products in strains expressing the truncated fusion proteins.

467 3.5. High-level production of miltiradiene through fed-batch fermentation

468 In an attempt to further increase the titer of miltiradiene, we cultivated the optimal engineered

469 strain BY-HZ70 in fed-batch fermentation. Minimal medium was used to promote plasmid retention

470 (Yu et al., 2018; Zhou et al., 2016). The strains were seeded into the medium to an innitial OD600 of

471 0.1. After 6 h of growth on glucose, the carbon source was replaced with galactose. The process of

472 fed-batch fermentation was controlled as described in previous studies (Tippmann et al., 2016; Yu et

473 al., 2018; Zhou et al., 2016). Time courses of dry cell weight (DCW) and consumed galactose during

474 the fermentation are shown in Fig. S11. As shown in Fig. 6, the biomass started increasing rapidly at

475 24 h, after the lag phase of metabolic adaption to galactose, and the growth subsequently remained

476 steady until the maximum OD600 of 99.3 at 132 h. Production of miltiradiene had a significant

477 positive correlation with biomass, with continuous accumulation until 180 h. The miltiradiene titer

478 reached almost 3.5 g L-1. By contrast, we detected only trace amounts of GGOH during the first two

479 days, but it accumulated slowly with the increase of biomass and reached 92.4 mg L-1 at the end of

480 the fermentation. Compared with shake flask culture, the percentage of the by-product GGOH

481 decreased significantly. Therefore, the fed-batch fermentation efficiently improved the utilization of

482 the precursor GGPP and the process has great potential for the industrial production of miltiradiene.

25
483

484 Fig. 6. Production of miltiradiene in fed-batch fermentation using the engineered strain BY-HZ70 in

485 a 5-L bioreactor. The data are averages of 3 biological replicates with error bars representing

486 standard deviations.

487 4. Conclusions

488 In this study, multiple engineering strategies were applied to increase the supply of GGPP and

489 the production of the important intermediate miltiradiene. We first constructed a series of yeast

490 strains with high GGPP production by combining several strategies based on the CRISPR/Cas9

491 system. The best strain BY-HZ16 produced 2.1 g L-1 GGOH in fed-batch fermentation. Then, we

492 took the biosynthesis of miltiradiene as an example to explore more strategies for further improving

493 the production of diterpenes. 14 miltiradiene synthases from 7 species were evaluated. We found that

494 the chimeric diterpene synthase consisting of a fusion of CfTPS1 and SmKSL1 showed the highest

495 efficiency in the conversion of GGPP to miltiradiene in yeast, after which the yield was further

496 improved by protein truncation, reaching a titer of 550.7 mg L-1 in shake flasks. Finally, this strain

497 produced a miltiradiene titer of 3.5 g L-1 in a 5-L bioreactor, which is the highest titer in heterologous

498 production reported so far. This work adopted a push and pull strategy for improving miltiradiene
26
499 production by increasing precursor supply and enhancing the catalytic capacity of diterpene

500 synthases. The strategies combined i) optimization of isoprenoid biosynthetic pathways in the

501 microbial chassis, ii) screening out the optimal module for miltiradiene production from the

502 miltiradiene synthase pool by evaluating their combinational effects, and iii) construction of the

503 chimeric diterpene synthases and truncation of miltiradiene synthases. It lays a solid foundation for

504 the pathway discovery and biosynthesis of active pharmaceutical ingredients, such as triptolide and

505 tanshinones. Moreover, the comprehensive strategies that focus on metabolic pathway optimization

506 of host cells and protein modification of key enzymes could be a good reference for the production

507 of many valuable pharmaceuticals and chemicals.

508 Acknowledgements

509 This work was financially supported by the National Natural Science Foundation of China

510 (81773830); the Support Project of High-level Teachers in Beijing Municipal Universities in the

511 Period of 13th Five–year Plan (CIT&TCD20170324); the ability establishment of sustainable use for

512 valuable Chinese medicine resources (2060302-1806-03); and National Program for Special Support

513 of Eminent Professionals.

514 Conflicts of Interest

515 The authors declare no conflicts of interest.

27
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1 Highlights

2 A suitable yeast chassis for diterpene production was obtained.

3 Extreme promiscuity of chimeric diterpene synthases improved miltiradiene yield.

4 Miltiradiene production was further enhanced by protein engineering.

5 A highest reported titer of 3.5 g L-1 miltiradiene was achieved.