PRODUCTION 1

Proposal title

Microbial Hyaluronic Acid Production

Winter 2018

1
Product General Information:

Product market:

The global hyaluronic acid market was valued at USD 7.2 billion in 2016 and is expected to
reach a value of USD 15.4 billion by 2025.

The market is growing rapidly due to increase in application of HA based cosmetic, HA coated
cardiovascular implants, such as stents and vascular grafts and viscosupplements for pain
reduction in growing population of obese and aging patients.

Hyaluronic acid characteristics

Hyaluronic acid (HA) is a non-immunogenic, biodegradable and non-adhesive, non-sulfated

glycosaminoglycan, which composed of disaccharides blocks of β4-glucuronic acid (GlcUA)-β3-
N-acetylglucosamine (GlcNAc). A hyaluronic acid molecule may contain more than 10000
disaccharide blocks. The molecule is significantly rigid, and high molecular weight molecules
have the overall spherical shapes.
HA exists in many parts of vertebrates and human body such as skin, joint, eye, or umbilical
cord with different physiologic roles. For instance, HA prevents skin drying and keeps the
resilience and elasticity of the skin.
This molecule has high moisture retention capability, is biocompatible and viscoelastic. HA
binds to a large amount of water (1gram HA holds up about 6 liter H 2O) forms a highly viscous
gel. The gel like properties of HA makes it a shock-absorber for the surrounding tissues and a
lubricant in joints. These properties make HA an important ingredient of medical and cosmetic
products with high commercial value.  It is worth noting that the application of HA mostly
depends on its molecular weight, a parameter that determines HA properties.
HA could be obtained from animal (rooster comb, bovine vitreous) and microbial sources.
The animal based HA production encounters a rising concern due to ethic issues and risk of viral
infection. In addition, during purification steps due to difficulties in isolation of high molecular
weight molecule from its proteoglycan complexes the molecular weight of the polymer is
decreased.

2
On the other hand, microbial production of hyaluronic acid has arisen as safer source for HA
production. Recently bacterial hyaluronic acid is industrially produced from Streptococcus
zooepidemicus and Streptococcus equi.

Applications

Since hyaluronic acid is a naturally occurring polymer in human body, and more importantly it
has non-immunogenic, non-reproductive or developmental toxicant, non-genotoxin
characteristics, its applications in clinical and cosmetics in different forms such as injection has
been reported as safe and free of significant adverse effects. Due to having such a safety
characteristics, it has wide range of applications which are briefly presented below and
summarized in appendix 1&2.

Ophthalmological application:

HA is extremely beneficial in anterior or posterior segment eye surgery; cataract extraction,

with or without intraocular lens implantation, kertoplasty and vitreous – retinal surgery. It
protects exposed eye tissue during surgery.

The exogenous HA has no negative effects on post-surgical intraocular pressure and does not
initiate any inflammatory sequelae in the intraocular environment.

Biodegradation property makes HA injection possible into eye; and also it is very similar to
natural tears. Therefore, hyaluronic acid based products have received the approval to be used
as drugs or surgical soft instruments.

Hyaluronan based products are widely used both in the general and local therapy of eye
diseases. Several HA based eye products with amino acids, antioxidants and other compounds
have been developed.

Osteoarthritis

Synovial fluids of osteoarthritic tissues shows 2-3 times lower rate of HA biosynthesis and lower
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HA molecular weight than that of healthy synovial fluids.

Intra-articular injection of HA leads to pain syndrome reduction and improvement of joints

activities. Furthermore injection of HA helps to recover physiological properties of synovial
fluids by suppressing inflammatory and catabolic and activate anabolic reactions.

Wound healing

Hyaluronic acid has central role in wound healing and tissue regeneration. Therefore,
Hyaluronic acid is used for the treatment of wounds such as abrasions, burns, metabolic ulcers.

Cosmetics

Reduced HA production and changes in HA metabolism are among the main causes of skin
aging. In this case injection of exogenous HA helps the skin to obtain its moisture and normal
elasticity. HA has market value of hundred millions dollars as dermal filer.

Immunology

HA is used to reduce development of the herpes simplex infection. To have the antimicrobial
effects of HA within cells or intercellular space, HA is conjugated with hydrophilic polymers to
be able to pass through cell membrane.

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Economic justification of the project

This project has two phases. The final cost of each phase has been presented separately.

Phase 1:

Full development of the semi-developed strain is defined as phase 1.

⮚The bacterial strain produces 4-5g/l hyaluronic acid. For further enhancement,
hyaluronidase gene must be deleted.
⮚Hemolysin phenotype should be omitted.
⮚Colony selection

Required space for the phase 1: 20 m2, basic molecular genetic laboratory

No. of required technician: 0-1 (will be done by me)

Estimated time: 6-9 months, provided that all the materials are on bench.

Total cost: 224986.9$, Tax and shipping , Depreciation ,technician, and

overhead costs were not included

Required devices:

The main devices are listed below. The cat. No. of the devices are also provided. If there is
suitable device in lab, it will be used.

Name # $ Cat.No.

Fermenter 5 lit 1 40000

High capacity centrifuge 25000

Electroporator 1 3500 1652100,

GenePulser®/MicroPulser™
Electroporation Cuvettes,
0.1 cm gap

Incubator shaker 1 2000 Heidolph Unimax 1010

DT 543-12319-0
Incubator Shaker
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 Platform w/ High Hood
(2342
Incubator (stationary, 1 1000 EW-03613-30
37°C)

Microfuge 1 1000 392244 

Refrigerated centrifuge 1 12000

Refrigerated centrifuge 15000

PCR machine 1 3000

Electrophoresis 1 1000
apparatus

Hotplate (vial) 1 3000

Ice maker 1 9900 89166-532

Water bath 1 1100 VWR Catalog # 89032-220 26L

Digital Water Bath

Gel doc 1 5000

UV transluminator 1 6000 Z692743-1EA

Chemical hood 1 12000

Autoclave 1 3000

Balance 1 3400 01-912-402

pH meter 1 5612 01-910-926

Spectrophotometer 1 7000

Light microscope 1 1000

Freezer -20C 1 5000

Refrigerator 4C 1 5000

Freezer -70C 1 10000

Pipetor 1000,100,10ul 1 of 3000

eac
h

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 Stirrer/hot plate 1 2000

Microwave 1 1000

Bunsen burner 1 200

Rack (working, falcon, 1 100

vial)

Racks (storing in -70, -20) 1 50

Boxes 1 50

steel tray with lid for 1 50

liquid waste

Total 186962

List of main required chemicals

The main chemicals are listed below. The Cat. No are also provided. Tax and shipping cost are
not included.

Name # Cat.No/ comment Unit price $

Primers Appendix Appendix 2 500 500

2

Plasmid 1 500 500

JM109, bacterial 1 49761 65 65

strain

E.Coli DH5α, 1 100 100

Monarch® Plasmid 2 T1010S 83.00 166

Miniprep Kit 

Monarch® PCR & 2 T1030S 102.43 204.86

DNA Cleanup Kit (5
μg)

Taq enzyme 2 M0495S 82.43 164.86

T4 ligase 1 M0202S 64 64
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hindIII-hf 1 R3104S 60 60

BamH1-hf 1 R3136S 61.72 61.72

ECORI-hf 1 R3101S 60 60

BglII 1 R0144S 71 71

Q5® High-Fidelity 2 M0491S 102 204

DNA Polymerase

Deoxynucleotide 2 N0447S 82.73 165.46

(dNTP) Solution Mix

Ladder 1Kb 2 N0550S 63.03 126.06

Q5® High-Fidelity 2X 1 M0492S 162.83 162.83

Master Mix

Kanamycin 1 Sigma- 60615-5G 140.15 140.15

Erythromycin 1 Sigma-E5389-1G 39.33 39.33

Spectinomycin 1 Sigma-4014-5G 206.84 206.84

Ampicillin 1 Sigma-A9393 79.12 79.12

Chloramphenicol 1 Sigma- C1919-5G 65.22 65.22

BSA fraction V 1 Neofroxx- 82.74 82.74

1126GR050 

N-Acetylglucosamine 1 MA00834-1kg 85 85

Hyaluronidase 1 H3506-1G 291.7 291.7

hyaluronic acid 1-2 1 YH05852-500g 350 350

MD

Electroporation 1 1652089 200 200

Cuvettes, 0.1 cm gap

Ispropylalcohol 5 Sigma-I9516- 104.89 524.45

500ML

meta- 1 Sigma- 262250- 78.1 78.1

hydroxydiphenyl 5G

Glycine 1 Sigma-50046-50G 29.50 29.5

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piperazine-1,2-bis[2- 1 Sigma- P6757- 51.09 51.09
ethanesulfonic acid 25G

immersion oil 1 EMD Millipore- 29.61 29.61

1046990100

LB broth 1 L3397-1KG 158.24 158.24

Glycerol 1 G5516-1L 196.67 196.67

Sucrose 1 84100-1KG 58.21 58.21

Yeast extract 1 07533-500G 162.76 162.76

Agar 1 A5306-250G 420.46 420.46

Blood agar 1 1108860500 172.93 172.93

Tris 1 10708976001 181.97 181.97

NaCl 1 S3014-1KG 114.16 114.16

Na2-EDTA 1 E5134-1KG 273.53 273.53

NaOH 1 S8045-1KG 131.11 131.11

HCl 1 258148-2.5L 114.16 114.16

TSB broth 1 T8907-1KG 231.71 231.71

microscope slides 10 CLS294775X25- 24.64 246.4

72EA

boric acid 1 B6768-1KG 83.53 83.53

Na2hpo4 1 795410-1KG 196.67 196.67

nah2po4 1 RDD007-1KG 117.55 117.55

filter membrane for 1 Millipore 260.40 260.40

drop dialysis VSWP02500

KH2po4 1 795488-1KG 112.24 112.24

Falcon 50ml 1/500pcs Dnase/Rnase free 150 150

Falcon 15ml 1/500pcs Dnase/Rnase free 150 150

Vial 1.5ml 1/500pcs Dnase/Rnase free 150 150

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Vial 0.5ml 1/500pcs Dnase/Rnase free 150 150

Vial 0.25ml 1/500pcs Dnase/Rnase free 150 150

Cryovial 2ml, 1/500pcs SIAL0659-500EA 378.64 378.64

external threaded

non adhesive 1/500pcs CLS430591- 440.13 440.13

petridishes 500EA

2-propanol 3 59304-1L-F 135.63 406.89

Ethanol 1 1134LT2P5  220.18 220.18

Agarose 1 1057GR500 508.06 508.06

SYBR® Green I 1 S9430-.5ML 499.58 499.58

SDS 1 L3771-500G 549.31 549.31

tip 1000 filtered 1/1000pc Dnase/Rnase free 100 100

s

tip 100 filtered 1/1000pc Dnase/Rnase free 100 100

s

tip 10 filtered 1/1000pc Dnase/Rnase free 100 100

s

tip 1000 2/1000pc Dnase/Rnase free 100 100

s

tip 100 2/1000pc Dnase/Rnase free 100 100

s

tip 10 2/1000pc Dnase/Rnase free 100 100

s

Inoculating loop 5ul 20 20

H2O2 1 H1009-500ML 147 147

Chlroform 1 1024312500 96.1 96.1

sodium acetate 1 W302406-10KG-K 192.15 192.15

Zipkeep 3 - 10 30

syringe filter 0.2u 2 - 220 440

sterile, PES, 25mm

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syringe 10ml 1 pack - 100 100

Todd Hewitt broth 1 T1438-100G 99.46 99.46

BDDE 1 220892-50G 170.67 170.67

BCA kit 1 QPBCA-1KT 457.76 457.76

stericup filter   1 212.5 212.5

S2GPU05RE

Dispensing Pressure 1 XX6700P10 1560 1560

Vessel, 10 L

1(12x) 150 150

Sartolab® P20 Plus
Pressure Filter
18068----------D, GF
0.2 µm
Polyethersulfone

Tubing for 1 set 150 150

pressurized filtration

Magnet 10 50 50

Parafilm 1 P7793-1EA 50 50

forceps 2 100 100

Charcoal 1 161551-5KG 357 357

autoclave tape 1 50 50

autoclave bag 4 50 50

plate 96 well- 1 150 150

nonsterile

DMSO 1 C6164-50ML 331 331

disodium 1 1063060250 93.81 93.81

tetraburate

Total 17571.58

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Phase 2:

Pilot production of hyaluronic acid

⮚50 lit fermentation set up

Required space for the phase 1: 40 m2,

No. of required technician: 2

Estimated time: 3-6 months, provided that all the materials are on bench.

Total cost: Tax and shipping, Depreciation, technician salary, and overhead costs were not included

Required devices:

The main devices are listed below. The Cat. No of the devices are also provided. If there is
suitable device in lab, it will be used.

Fermenter 100 lit 90000

Tubular centrifuge 100000 claryfing type (solid-liquid, pH4-10, 500-

2000cp, 20-30lit/h flow rate,

Diafiltration housing

Filtration housing

Carbon filtration housing

Spry dryer

Vacuum pumps

Freeze dryer

Mixing vessel

Portable CIP

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 Portable viscometer

Water filtration system

Autoclave

List of main required chemicals

The main chemicals are listed below. The Cat. No are also provided. Tax and shipping cost are
not included.

NaCl 40kg

NaOH 45kg

K2HPO4 5kg

MnSO4 5kg

CaCO3 10kg

Casein pancratic hydrolyzed 25kg

TSB broth 10kg

Glucose 40kg

Yeast Extract 15kg

sodium fumarate 1kg

sodium oxalate 1kg

sodium citrate 1kg

Carbazol 1kg

sodium deoxycholate 1kg

h2so4 specific gravity 1.84 10lit

N-acetylglucosamin 1kg

acetic acid 5lit

Sucrose 40kg

sodium acetate 45kg

mgso4 5kg

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Ispropylalcohol 500lit

calcium chloride 45kg

cuso4 5kg

Filter1 1000

Filter2 1000

Filter3 1000

Filter4 1000

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Appendix 1

Cosmetic Product

Shampoos Hair coloring products Beard softeners

Lotions, oils, powders, and Dyes and colors Men’s talcum
creams Tints Pre-shave lotions
Bath products Rinses Shaving creams
Oils, tablets, and salts Color sprays Shaving soaps
Soaps and detergents Lighteners with color Skin care products
Bubble baths Bleaches Skin cleansing creams,
Capsules Makeup Blushers lotions, liquids, and pads
Eye makeup Face powders Depilatories
Eyebrow pencils Foundations Face and neck creams,
Eyeliners Leg and body paints lotions, powders, and
Eye shadow Lipsticks sprays
Eye lotions Makeup bases Body and hand creams,
Mascara Rouges lotions, powders, and
Fragrance products Makeup fixatives sprays Foot powders and
Perfumes Nail care products sprays
Powders Basecoats and undercoats Moisturizers
Sachets Cuticle softeners Night creams, lotions,
Non-coloring hair care Creams and lotions powders, and sprays
products Nail extenders Paste masks/mud Skin
Conditioners Nail polishes and enamels fresheners
Sprays/aerosol fixatives Nail polish and enamel Suntan products Suntan
Straighteners removers gels, creams, liquids, and
Permanent waves Personal hygiene products sprays
Rinses Underarm deodorants Indoor tanning
Shampoos Feminine deodorants preparations
Tonics, dressings, etc Shaving products
Wave sets Aftershave lotions

Appendix 2

Non cosmetic applications

MEDICAL DEVICES

•Ophthalmic surgery • Intra-articular injections

(viscosupplementation)
• Topical preparations for wound and burns
• Eye drops for dry eye (eg. artificial tears) healing
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•Intradermal injections for aesthetic • Anti-adhesive wound healing products
correction
•Bandages with exudates absorbing
• Anti-adhesive and shape keeping products capacity
for thoracic and abdominal surgery

• Cartilage (joint) and tendon disorders • Wound and burns healing preparations

REMEDIES/DRUGS

•anti-cancer preparations •Topical anti-inflammatory and analgesics

preparations
• Wound and burns healing preparations
• Cartilage (joint) and tendon disorders
•Preparations for reduction of scar
formation •Nutraceuticals supporting the treatment of
skeleton or prevention of skeleton disorders
• Eye drops
• Preparations for liver regeneration

TISSUE ENGINEERING

• Artificial skin grafts for plastic and • Artificial bone grafts

reconstruction surgery
•adipose tissue replacement (mammary
• Artificial skin for test of cosmetic raw replacement)
material
• Support for growing of soft tissue grafts
• Replacement of injured cartilage, artificial
• Aorta and large venous grafts industry
cartilage grafts

APPLICATION IN BIOTECHNOLOGY AND MOLECULAR BIOLOGY

• Component of special cell culture medium • supporting material for gene transfer

OTHER BIOLOGICAL APPLICATION

• Sperm separation according to their • Medium for in vitro ovum fertilization

mobility

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Appendix 2. list of required primers

Name sequence quantit

y

lyase-F1 5' GTCAATTTCAGGCAATCACA 3' 50nMol

lyase-R1 5' TCACTTGGCTTATCACCAG 3' 50nMol

lyase-F2 5' CTGGTGATAAGCCAAGTGAT 3' 50nMol

lyase-R2 5' TGAGCGAAATTGTCTAGGAT 3' 50nMol

lyase-F3 5' AGCCTTTCTTTACCCTTATGTCA 3' 50nMol

lyase-R3 5' AAGATAAAACATGCCATCTCC 3' 50nMol

lyase-F4 5' TAAGACTGGCTGACGCATT 3' 50nMol

lyase-R4 5' TGGTCATCTAAGATCGTCCAA 3' 50nMol

lyase-F5 5' TGTCTACCCAGCAATCGT 3' 50nMol

lyase-R5 5' TACTAATGTGCTTGAATGGCTA 3' 50nMol

stplS-F1 5' GCACTCTATGTCACAAACGA 3' 50nMol

stplS-R1 5' GCCCTTGTTAAAGTGACCA 3' 50nMol

stplS-F2 5' CCTTTCTGGAAAAATAATGACA 3' 50nMol

stplS-R2 5' TGATAACGATAAGTGATTGGT 3' 50nMol

stplS-F3 5' CTAACAAGAGAAGAAGCACGA 3' 50nMol

stplS-R3 5' TGATTTCCATATTTACGTGTG 3' 50nMol

hyd-F1 5' AAGCCTCTCAGTTAATCAGCC 3' 50nMol

hyd-R1 5' TAAAATGAGCCACCCAGTCC 3' 50nMol

hyd-F2 5'GGCTTTTATGGCACTCCTT 3' 50nMol

hyd-R2 5' GGATTAGACACAATTCCCCTG 3' 50nMol

hyd-F3 5' CATTTGCCAATCGCTCT 3' 50nMol

hyd-R3 5' ATAGGAATTGTGCCCCA 3' 50nMol

hem-F1 5' TCTTTCCCAGTCGAGCTA 3' 50nMol

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hem-R1 5' TCCTTGACGGTTTGACA 3' 50nMol

hem-F2 5' AAGTCTTGACGAGGCTAC 3' 50nMol

hem-R2 5' TGGCAATAACCTGTAAAGCA 3' 50nMol

hem-F3 5’ GCTTTACAGGTTATTGCCAT 50nMol

hem-R3 5’ TTGACACAAGCCATCGT 50nMol

SodAF 5’ CAGCATTCCTGCTGACATTCGTCAGG 50nMol

SodAR 5’ CTGACCAGCCTTATTCACAACCAGCC 50nMol

M13/pUC Reverse 5’ AGCGGATAACAATTTCACACAGG 50nMol

M13/pUC Forward 5’ CCCAGTCACGACGTTGTAAAACG 50nMol

18