透明质酸的生物合成及其代谢工程的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2021-0443

摘要/Abstract

摘要：

透明质酸（hyaluronic acid,HA）是广泛存在于生物体内的功能性糖胺高分子聚合物,在日化、医疗和食品领域应用前景广阔。随着基因工程与代谢工程等合成生物学技术的发展,人们对HA的生物合成过程和机理解析越发深入的同时,也伴随一些新的挑战来临。该综述从分子生物学角度总结了HA的关键合成酶基因及合成途径,对不同来源的HA合成酶进行了氨基酸序列比对分析,并详细描述了HA在常用微生物宿主中生产的安全性、底物不平衡性、发酵低溶氧性等瓶颈问题及其相应的合成生物学策略开发,从而归纳出了目前微生物中高产HA的有效策略,为进一步推动HA的绿色生物制造提供了重要的科学依据和理论支撑。

关键词: 透明质酸, 透明质酸合成酶, 基因工程, 代谢工程, 枯草芽孢杆菌, 谷氨酸棒状杆菌

Abstract:

Hyaluronic acid（HA）,a functional glycosamine polymer widely existing in organism,presents broad potential applications in daily-use chemicals,medicine and food. With the development of synthetic biology technologies such as genetic engineering and metabolic engineering,some new challenges are emerging while the biosynthesis process and mechanism of HA have been studied in depth. This review summarizes the key genes and pathways of HA synthase from the perspective of molecular biology,aligns and analyses the amino acid sequence of HA synthase from different sources. It also describes in detail the bottleneck issues such as the safety of HA production,substrate imbalance and low oxygen solubility in fermentation,and introduces the development of strategies on corresponding synthetic biology in commonly used microbial hosts,thereby induces the effective strategies for high-yield HA by microorganism. This review may provide an important support for further promoting the green biomanufacturing of HA.

Key words: hyaluronic acid, hyaluronic acid synthase, genetic engineering, metabolic engineering, Bacillus subtilis, Corynebacterium glutamicum

马艳琴, 邱益彬, 李莎, 徐虹. 透明质酸的生物合成及其代谢工程的研究进展[J]. 生物技术通报, 2022, 38(2): 252-262.

MA Yan-qin, QIU Yi-bin, LI Sha, XU Hong. Research Progress in the Biosynthesis and Metabolic Engineering of Hyaluronic Acid[J]. Biotechnology Bulletin, 2022, 38(2): 252-262.

图/表 6

参考文献 83

[1]	Chen WY, Abatangelo G. Functions of hyaluronan in wound repair[J]. Wound Repair Regen, 1999, 7(2):79-89. pmid: 10231509
[2]	Armstrong DC, Cooney MJ, Johns MR. Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus[J]. Appl Microbiol Biotechnol, 1997, 47(3):309-312. doi: 10.1007/s002530050932 URL
[3]	Liu L, Sun J, Xu W, et al. Modeling and optimization of microbial hyaluronic acid production by Streptococcus zooepidemicus using radial basis function neural network coupling quantum-behaved particle swarm optimization algorithm[J]. Biotechnol Prog, 2009, 25(6):1819-1825.
[4]	Izawa N, Serata M, Sone T, et al. Hyaluronic acid production by recombinant Streptococcus thermophilus[J]. J Biosci Bioeng, 2011, 111(6):665-670. doi: 10.1016/j.jbiosc.2011.02.005 URL
[5]	Liu L, Wang M, Du G, et al. Enhanced hyaluronic acid production of Streptococcus zooepidemicus by an intermittent alkaline-stress strategy[J]. Lett Appl Microbiol, 2008, 46(3):383-388. doi: 10.1111/j.1472-765X.2008.02325.x pmid: 18221275
[6]	Weigel PH. Functional characteristics and catalytic mechanisms of the bacterial hyaluronan synthases[J]. IUBMB Life, 2002, 54(4):201-211. pmid: 12512859
[7]	Itano N, Kimata K. Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase[J]. J Biol Chem, 1996, 271(17):9875-9878. doi: 10.1074/jbc.271.17.9875 pmid: 8626618
[8]	Shyjan AM, Heldin P, Butcher EC, et al. Functional cloning of the cDNA for a human hyaluronan synthase[J]. J Biol Chem, 1996, 271(38):23395-23399. doi: 10.1074/jbc.271.38.23395 pmid: 8798544
[9]	Spicer AP, Augustine ML, McDonald JA. Molecular cloning and characterization of a putative mouse hyaluronan synthase[J]. J Biol Chem, 1996, 271(38):23400-23406. doi: 10.1074/jbc.271.38.23400 pmid: 8798545
[10]	Watanabe K, Yamaguchi Y. Molecular identification of a putative human hyaluronan synthase[J]. J Biol Chem, 1996, 271(38):22945-22948. doi: 10.1074/jbc.271.38.22945 pmid: 8798477
[11]	DeAngelis PL, Jing W, Graves MV, et al. Hyaluronan synthase of Chlorella virus PBCV-1[J]. Science, 1997, 278(5344):1800-1803. pmid: 9388183
[12]	DeAngelis PL, Jing W, Drake RR, et al. Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida[J]. J Biol Chem, 1998, 273(14):8454-8458. doi: 10.1074/jbc.273.14.8454 pmid: 9525958
[13]	Tlapak-Simmons VL, Baron CA, Weigel PH. Characterization of the purified hyaluronan synthase from Streptococcus equisimilis[J]. Biochemistry, 2004, 43(28):9234-9242. pmid: 15248781
[14]	Jongsareejit B, Bhumiratana A, Morikawa M, et al. Cloning of hyaluronan synthase(sz-hos)gene from Streptococcus zooepidemicus in Escherichia coli[J]. ScienceAsia, 2007, 33(4):389-395. doi: 10.2306/scienceasia1513-1874.2007.33.389 URL
[15]	Nho SW, Hikima J, Cha IS, et al. Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis[J]. J Bacteriol, 2011, 193(13):3356-3366. doi: 10.1128/JB.00182-11 URL
[16]	Ward PN, Field TR, Ditcham WG, et al. Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynjournal genes hasA, hasB, and hasC in Streptococcus uberis[J]. Infect Immun, 2001, 69(1):392-399. pmid: 11119529
[17]	Semino CE, Specht CA, Raimondi A, et al. Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synjournal of Nod-like chitin oligosaccharides during early embryogenesis[J]. PNAS, 1996, 93(10):4548-4553. pmid: 8643441
[18]	Itano N, Sawai T, Yoshida M, et al. Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties[J]. J Biol Chem, 1999, 274(35):25085-25092. doi: 10.1074/jbc.274.35.25085 pmid: 10455188
[19]	Jeong E, Shim WY, Kim JH. Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight[J]. J Biotechnol, 2014, 185:28-36. doi: 10.1016/j.jbiotec.2014.05.018 URL
[20]	Gomes AMV, Netto JHCM, Carvalho LS, et al. Heterologous hyaluronic acid production in Kluyveromyces lactis[J]. Microorganisms, 2019, 7(9):294. doi: 10.3390/microorganisms7090294 URL
[21]	Wang Y, Hu LT, Huang H, et al. Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum[J]. Nat Commun, 2020, 11:3120. doi: 10.1038/s41467-020-16962-7 URL
[22]	朱丽雅, 吴善善, 欧阳水平, 等. 外源添加氨基酸对兽疫链球菌产透明质酸的影响[J]. 林业工程学报, 2019, 4(3):74-79.
	Zhu LY, Wu SS, Ouyang SP, et al. Effect of addition of amino acids on production of hyaluronic acid by Streptococcus zooepidemicus[J]. J For Eng, 2019, 4(3):74-79.
[23]	Lai ZW, Rahim RA, Ariff AB, et al. Biosynjournal of high molecular weight hyaluronic acid by Streptococcus zooepidemicus using oxygen vector and optimum impeller tip speed[J]. J Biosci Bioeng, 2012, 114(3):286-291. doi: 10.1016/j.jbiosc.2012.04.011 URL
[24]	Jia YN, Zhu J, Chen XF, et al. Metabolic engineering of Bacillus subtilis for the efficient biosynjournal of uniform hyaluronic acid with controlled molecular weights[J]. Bioresour Technol, 2013, 132:427-431. doi: 10.1016/j.biortech.2012.12.150 URL
[25]	Widner B, Behr R, Von Dollen S, et al. Hyaluronic acid production in Bacillus subtilis[J]. Appl Environ Microbiol, 2005, 71(7):3747-3752. doi: 10.1128/AEM.71.7.3747-3752.2005 URL
[26]	Jin P, Kang Z, Yuan P, et al. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168[J]. Metab Eng, 2016, 35:21-30. doi: 10.1016/j.ymben.2016.01.008 URL
[27]	Vellanoweth RL, Rabinowitz JC. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo[J]. Mol Microbiol, 1992, 6(9):1105-1114. pmid: 1375309
[28]	Hmar RV, Prasad SB, Jayaraman G, et al. Chromosomal integration of hyaluronic acid synjournal(has)genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis[J]. Biotechnol J, 2014, 9(12):1554-1564. doi: 10.1002/biot.201400215 URL
[29]	Sheng JZ, Ling PX, Wang FS. Constructing a recombinant hyaluronic acid biosynjournal operon and producing food-grade hyaluronic acid in Lactococcus lactis[J]. J Ind Microbiol Biotechnol, 2015, 42(2):197-206. doi: 10.1007/s10295-014-1555-8 URL
[30]	Cheng F, Gong Q, Yu H, et al. High-titer biosynjournal of hyaluronic acid by recombinant Corynebacterium glutamicum[J]. Biotechnol J, 2016, 11(4):574-584. doi: 10.1002/biot.201500404 URL
[31]	Hoffmann J, Altenbuchner J. Hyaluronic acid production with Corynebacterium glutamicum:effect of media composition on yield and molecular weight[J]. J Appl Microbiol, 2014, 117(3):663-678. doi: 10.1111/jam.12553 pmid: 24863652
[32]	Cheng FY, Luozhong SJ, Guo ZG, et al. Enhanced biosynjournal of hyaluronic acid using engineered Corynebacterium glutamicum via metabolic pathway regulation[J]. Biotechnol J, 2017, 12(10):1700191. doi: 10.1002/biot.v12.10 URL
[33]	Kim SJ, Park SY, Kin CW. A novel approach to the production of hyaluronic acid by Streptococcus zooepidemicus[J]. J Microbiol Biotechnol, 2006, 16(12):1849-1855.
[34]	Shah MV, Badle SS, Ramachandran KB. Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynjournal pathway[J]. Biochem Eng J, 2013, 80:53-60. doi: 10.1016/j.bej.2013.09.013 URL
[35]	Zhang X, Wang M, Li T, et al. Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism[J]. AMB Express, 2016, 6(1):121. doi: 10.1186/s13568-016-0296-7 URL
[36]	Pourzardosht N, Rasaee MJ. Improved yield of high molecular weight hyaluronic acid production in a stable strain of Streptococcus zooepidemicus via the elimination of the hyaluronidase-encoding gene[J]. Mol Biotechnol, 2017, 59(6):192-199. doi: 10.1007/s12033-017-0005-z pmid: 28500482
[37]	Liu J, Wang Y, Li Z, et al. Efficient production of high-molecular-weight hyaluronic acid with a two-stage fermentation[J]. RSC Adv, 2018, 8(63):36167-36171. doi: 10.1039/C8RA07349J URL
[38]	Mohan N, Tadi SRR, Pavan SS, et al. Deciphering the role of dissolved oxygen and N-acetyl glucosamine in governing higher molecular weight hyaluronic acid synjournal in Streptococcus zooepidemicus cell factory[J]. Appl Microbiol Biotechnol, 2020, 104(8):3349-3365. doi: 10.1007/s00253-020-10445-x URL
[39]	Izawa N, Hanamizu T, Sone T, et al. Effects of fermentation conditions and soybean peptide supplementation on hyaluronic acid production by Streptococcus thermophilus strain YIT 2084 in milk[J]. J Biosci Bioeng, 2010, 109(4):356-360. doi: 10.1016/j.jbiosc.2009.10.011 URL
[40]	Chen YH, Li J, Liu L, et al. Optimization of flask culture medium and conditions for hyaluronic acid production by a Streptococcus equisimilis mutant nc2168[J]. Braz J Microbiol, 2012, 43(4):1553-1561. doi: 10.1590/S1517-83822012000400040 URL
[41]	管峰, 金嘉长, 赵华国, 等. 海豚链球菌诱变发酵法制备透明质酸及其在动物皮肤修复中的应用[J]. 生物工程学报, 2016, 32(8):1104-1114.
	Guan F, Jin JC, Zhao HG, et al. Hyaluronic acid production by Streptococcus iniae and its application in rabbit skin’s regeneration[J]. Chin J Biotechnol, 2016, 32(8):1104-1114.
[42]	Im JH, Song JM, Kang JH, et al. Optimization of medium components for high-molecular-weight hyaluronic acid production by Streptococcus sp. ID9102 via a statistical approach[J]. J Ind Microbiol Biotechnol, 2009, 36(11):1337-1344. doi: 10.1007/s10295-009-0618-8 URL
[43]	Yu H, Stephanopoulos G. Metabolic engineering of Escherichia coli for biosynjournal of hyaluronic acid[J]. Metab Eng, 2008, 10(1):24-32. doi: 10.1016/j.ymben.2007.09.001 URL
[44]	Lai Z ZW, Teo C CH. Cloning and expression of hyaluronan synthase(hasA)in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture[J]. Malays J Microbiol, 2019, 15(7):575-582.
[45]	Chien LJ, Lee CK. Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin[J]. Biotechnol Prog, 2007, 23(5):1017-1022.
[46]	Li Y, Li G, Zhao X, et al. Regulation of hyaluronic acid molecular weight and titer by temperature in engineered Bacillus subtilis[J]. 3 Biotech, 2019, 9(6):225. doi: 10.1007/s13205-019-1749-x URL
[47]	Cheng F, Yu H, Stephanopoulos G. Engineering Corynebacterium glutamicum for high-titer biosynjournal of hyaluronic acid[J]. Metab Eng, 2019, 55:276-289. doi: 10.1016/j.ymben.2019.07.003 URL
[48]	Yoshimura T, Shibata N, Hamano Y, et al. Heterologous production of hyaluronic acid in an ε-poly-L-lysine producer, Streptomyces albulus[J]. Appl Environ Microbiol, 2015, 81(11):3631-3640. doi: 10.1128/AEM.00269-15 URL
[49]	Sunguroğlu C, Sezgin DE, Aytar Çelik P, et al. Higher titer hyaluronic acid production in recombinant Lactococcus lactis[J]. Prep Biochem Biotechnol, 2018, 48(8):734-742. doi: 10.1080/10826068.2018.1508036 URL
[50]	Marcellin E, Steen JA, Nielsen LK. Insight into hyaluronic acid molecular weight control[J]. Appl Microbiol Biotechnol, 2014, 98(16):6947-6956. doi: 10.1007/s00253-014-5853-x pmid: 24957250
[51]	Sonenshein AL. Control of key metabolic intersections in Bacillus subtilis[J]. Nat Rev Microbiol, 2007, 5(12):917-927. pmid: 17982469
[52]	Yan X, Yu HJ, Hong Q, et al. Cre/lox system and PCR-based genome engineering in Bacillus subtilis[J]. Appl Environ Microbiol, 2008, 74(17):5556-5562. doi: 10.1128/AEM.01156-08 URL
[53]	Kaur M, Jayaraman G. Hyaluronan production and molecular weight is enhanced in pathway-engineered strains of lactate dehydrogenase-deficient Lactococcus lactis[J]. Metab Eng Commun, 2016, 3:15-23. doi: 10.1016/j.meteno.2016.01.003 URL
[54]	Teramoto H, Inui M, Yukawa H. Transcriptional regulators of multiple genes involved in carbon metabolism in Corynebacterium glutamicum[J]. J Biotechnol, 2011, 154(2/3):114-125. doi: 10.1016/j.jbiotec.2011.01.016 URL
[55]	Liu L, Du G, Chen J, et al. Influence of hyaluronidase addition on the production of hyaluronic acid by batch culture of Streptococcuszooepidemicus[J]. Food Chem, 2008, 110(4):923-926. doi: 10.1016/j.foodchem.2008.02.082 URL
[56]	Mehmeti I, Jönsson M, Fergestad EM, et al. Transcriptome, proteome, and metabolite analyses of a lactate dehydrogenase-negative mutant of Enterococcus faecalis V583[J]. Appl Environ Microbiol, 2011, 77(7):2406-2413. doi: 10.1128/AEM.02485-10 URL
[57]	Prasad SB, Ramachandran KB, Jayaraman G. Transcription analysis of hyaluronan biosynjournal genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis[J]. Appl Microbiol Biotechnol, 2012, 94(6):1593-1607. doi: 10.1007/s00253-012-3944-0 URL
[58]	Zhang L, Huang H, Wang H, et al. Rapid evolution of hyaluronan synthase to improve hyaluronan production and molecular mass in Bacillus subtilis[J]. Biotechnol Lett, 2016, 38(12):2103-2108. doi: 10.1007/s10529-016-2193-1 URL
[59]	Westbrook AW, Ren X, Moo-Young M, et al. Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis[J]. Biotechnol Bioeng, 2018, 115(1):216-231. doi: 10.1002/bit.26459 pmid: 28941282
[60]	Westbrook AW, Moo-Young M, Chou CP. Development of a CRISPR-Cas9 tool kit for comprehensive engineering of Bacillus subtilis[J]. Appl Environ Microbiol, 2016, 82(16):4876-4895. doi: 10.1128/AEM.01159-16 URL
[61]	Zheng Y, Cheng F, Zheng B, et al. Enhancing single-cell hyaluronic acid biosynjournal by microbial morphology engineering[J]. Synth Syst Biotechnol, 2020, 5(4):316-323.
[62]	Jiang XR, Chen GQ. Morphology engineering of bacteria for bio-production[J]. Biotechnol Adv, 2016, 34(4):435-440. doi: 10.1016/j.biotechadv.2015.12.007 URL
[63]	Chubukov V, Gerosa L, Kochanowski K, et al. Coordination of microbial metabolism[J]. Nat Rev Microbiol, 2014, 12(5):327-340. doi: 10.1038/nrmicro3238 pmid: 24658329
[64]	Holtz WJ, Keasling JD. Engineering static and dynamic control of synthetic pathways[J]. Cell, 2010, 140(1):19-23. doi: 10.1016/j.cell.2009.12.029 URL
[65]	Wu MJ, Andreasson JOL, Kladwang W, et al. Automated design of diverse stand-alone riboswitches[J]. ACS Synth Biol, 2019, 8(8):1838-1846. doi: 10.1021/acssynbio.9b00142 URL
[66]	Pavicic T, Gauglitz GG, Lersch P, et al. Efficacy of cream-based novel formulations of hyaluronic acid of different molecular weights in anti-wrinkle treatment[J]. J Drugs Dermatol, 2011, 10(9):990-1000.
[67]	Termeer CC, Hennies J, Voith U, et al. Oligosaccharides of hyaluronan are potent activators of dendritic cells[J]. J Immunol, 2000, 165(4):1863-1870. pmid: 10925265
[68]	Alaniz L, Rizzo M, Malvicini M, et al. Low molecular weight hyaluronan inhibits colorectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response[J]. Cancer Lett, 2009, 278(1):9-16. doi: 10.1016/j.canlet.2008.12.029 pmid: 19185418
[69]	Stern R, Asari AA, Sugahara KN. Hyaluronan fragments:an information-rich system[J]. Eur J Cell Biol, 2006, 85(8):699-715. doi: 10.1016/j.ejcb.2006.05.009 URL
[70]	Jegasothy SM, Zabolotniaia V, Bielfeldt S. Efficacy of a new topical nano-hyaluronic acid in humans[J]. J Clin Aesthet Dermatol, 2014, 7(3):27-29.
[71]	Symonette CJ, Kaur Mann A, Tan XC, et al. Hyaluronan-phosphatidylethanolamine polymers form pericellular Coats on keratinocytes and promote basal keratinocyte proliferation[J]. Biomed Res Int, 2014, 2014:727459. doi: 10.1155/2014/727459 pmid: 25276814
[72]	Khorshidi HR, Kasraianfard A, Derakhshanfar A, et al. Evaluation of the effectiveness of sodium hyaluronate, sesame oil, honey, and silver nanoparticles in preventing postoperative surgical adhesion formation. An experimental study[J]. Acta Cirurgica Brasileira, 2017, 32(8):626-632. doi: S0102-86502017000800626 pmid: 28902938
[73]	Safdar MH, Hussain Z, Abourehab MAS, et al. New developments and clinical transition of hyaluronic acid-based nanotherapeutics for treatment of cancer:reversing multidrug resistance, tumour-specific targetability and improved anticancer efficacy[J]. Artif Cells Nanomed Biotechnol, 2018, 46(8):1967-1980.
[74]	Xu Y, Asghar S, Yang L, et al. Lactoferrin-coated polysaccharide nanoparticles based on chitosan hydrochloride/hyaluronic acid/PEG for treating brain glioma[J]. Carbohydr Polym, 2017, 157:419-428. doi: 10.1016/j.carbpol.2016.09.085 URL
[75]	Dasa V, DeKoven M, Sun K, et al. Clinical and cost outcomes from different hyaluronic acid treatments in patients with knee osteoarthritis:evidence from a US health plan claims database[J]. Drugs Context, 2016, 5:212296.
[76]	Nakamura Y, Uchiyama S, Kamimura M, et al. Bone alterations are associated with ankle osteoarthritis joint pain[J]. Sci Rep, 2016, 6:18717. doi: 10.1038/srep18717 pmid: 26776564
[77]	Tammachote N, Kanitnate S, Yakumpor T, et al. Intra-articular, single-shot hylan G-F 20 hyaluronic acid injection compared with corticosteroid in knee osteoarthritis[J]. The Journal Bone And Joint Surgery, 2016, 98(11):885-892. doi: 10.2106/JBJS.15.00544 URL
[78]	张睿聪, 孙如梦, 朴明贯. HA-GMS-INS胰岛素口服纳米给药系统研究[J]. 中国医院药学杂志, 2021, 41(2):149-153.
	Zhang RC, Sun RM, Piao MG. Oral nano drug delivery system of HA-GMS-INS insulin[J]. Chin J Hosp Pharm, 2021, 41(2):149-153.
[79]	Litwiniuk M, Krejner A, Speyrer MS, et al. Hyaluronic acid in inflammation and tissue regeneration[J]. Wounds, 2016, 28(3):78-88.
[80]	Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronic acid:a key molecule in skin aging[J]. Dermatoendocrinol, 2012, 4(3):253-258. doi: 10.4161/derm.21923 pmid: 23467280
[81]	Kawada C, Yoshida T, Yoshida H, et al. Ingested hyaluronan moisturizes dry skin[J]. Nutr J, 2014, 13:70. doi: 10.1186/1475-2891-13-70 URL
[82]	Weigel PH, Baggenstoss BA. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines[J]. Glycobiology, 2012, 22(10):1302-1310. doi: 10.1093/glycob/cws102 pmid: 22745284
[83]	Yang J, Cheng FY, Yu HM, et al. Key role of the carboxyl Terminus of hyaluronan synthase in processive synjournal and size control of hyaluronic acid polymers[J]. Biomacromolecules, 2017, 18(4):1064-1073. doi: 10.1021/acs.biomac.6b01239 pmid: 28192668

Microorganism	Strategy	HA /（g·L^-1）	Molecular weight/Da	Reference
S. zooepidemicus CKD 117	Optimizing fermentation conditions	5.4	2.9×10⁶	[33]
S. zooepidemicus WSH-24	Intermittent alkaline-stress strategy	6.5	-	[5]
S. zooepidemicus ATCC 39920	Using oxygen vector and optimum impeller tip speed	4.25	1.54×10⁷	[23]
S. zooepidemicus BCRC15414	A two-stage anoxic（static）-aerobic sequential culture	0.51	3.0×10³	[22]
S. zooepidemicus ATCC 39920	Redirection of carbon flux by addition of glycolytic inhibitors,glutamine and iodoacetate	5	3.1-3.2×10⁶	[34]
S. zooepidemicus ATCC 39920	Overexpression of scrB in S. zooepidemicus ΔfruK	5.6	-	[35]
S. zooepidemicus IBRC-M 10919 Δhyl	Removal of the hyaluronidase-encoding gene	6	3.8×10⁶	[36]
S. zooepidemicus HA-13-06	Two-stage fermentation process	4.75	2.36×10⁶	[37]
S. zooepidemicus MTCC 3523	Dissolved oxygen and N-acetyl glucosamine supply	2.4	2.53×10⁶	[38]
S. thermophilus YIT 2084	Fermentation conditions and soybean peptide supplementation	0.208	1.3×10⁵	[39]
S. thermophilus YIT 2084	Co-expressing HA synthase hasA and hasB	1.2	1×10⁶	[4]
S. equisimilis mutant nc2168	Optimization of flask culture medium and conditions	0.225	-	[40]
S. iniae	Ultraviolet mutation	0.12	3×10⁵	[41]
S. sp. ID9102	Optimization of medium components	6.94	5.9×10⁶	[42]
E. coli Top10	Co-expressing HA synthase hasA and ugd from S. equisimilis	0.19	3.5×10⁵-1.9×10⁶	[43]
E. coli BL21	Expression of hyaluronan synthase hasA from S. zooepidemicus ATCC 39920	0.532	3.46×10⁴	[44]
B. subtilis 168	Inducible expression of pmHAS,tuaD and gtaB	6.8	4.55×10⁶	[45]
	Coexpression committed genes（hasA,tuaD,gtaB,glmU,glmM,and glmS）;downregulate pfkA expression of the glycolytic pathway;integration of LHAase with the activity at 1.62×10⁶ U/mL	19.38	6.62×10³	[24]
	Engineering of cell membrane by overexpressing pgsA,clsA and reducing the expression of ftsZ	1.25	2.1×10⁶	[26]
B. subtilis WB600	Regulation of temperature using the recombinant strain carrying hasA gene from S. ubris,hasB,and hasC from B. subtilis WB600	3.65	0.39-6.94×10⁶	[46]
C. glutamicum 13032	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032	8.3	1.3×10⁶	[30]
	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032;deletion of lactate dehydrogenase	21.6	1.28×10⁶	[32]
	An additional promoter PdapB for driving hasB expression;deletion of fba,zwf deletion and knockout of lactate/acetate pathway	28.7	0.21×10⁶	[47]
	Overexpression of hyaluronan synthase spHasA,enzymes of intermediate metabolic pathways and attenuation of polysaccharide biosynthesis;Disruption of the encapsulation with the leech hyaluronidase	34.2-74.1	5.3×10⁴-3.31×10⁵	[21]
Streptomyces albulus	Overexpression of gene hasA from S. zooepidemicus,gene udgA,glmU,and gtaB genes from Streptomyces avermitilis	6.2	2×10⁶	[48]
Pichia pastoris	Overexpression of xhasA2 and xhasB genes from Xenopus laevis;hasC,hasD,and hasE genes from P. pastoris	0.8-1.7	1.2-2.5×10⁶	[19]
Lactococcus lactis CES15	Expression of hasA gene under the control of the PnisA promoter	6.09	-	[49]

Microorganism	Strategy	HA /（g·L^-1）	Molecular weight/Da	Reference
S. zooepidemicus CKD 117	Optimizing fermentation conditions	5.4	2.9×10⁶	[33]
S. zooepidemicus WSH-24	Intermittent alkaline-stress strategy	6.5	-	[5]
S. zooepidemicus ATCC 39920	Using oxygen vector and optimum impeller tip speed	4.25	1.54×10⁷	[23]
S. zooepidemicus BCRC15414	A two-stage anoxic（static）-aerobic sequential culture	0.51	3.0×10³	[22]
S. zooepidemicus ATCC 39920	Redirection of carbon flux by addition of glycolytic inhibitors,glutamine and iodoacetate	5	3.1-3.2×10⁶	[34]
S. zooepidemicus ATCC 39920	Overexpression of scrB in S. zooepidemicus ΔfruK	5.6	-	[35]
S. zooepidemicus IBRC-M 10919 Δhyl	Removal of the hyaluronidase-encoding gene	6	3.8×10⁶	[36]
S. zooepidemicus HA-13-06	Two-stage fermentation process	4.75	2.36×10⁶	[37]
S. zooepidemicus MTCC 3523	Dissolved oxygen and N-acetyl glucosamine supply	2.4	2.53×10⁶	[38]
S. thermophilus YIT 2084	Fermentation conditions and soybean peptide supplementation	0.208	1.3×10⁵	[39]
S. thermophilus YIT 2084	Co-expressing HA synthase hasA and hasB	1.2	1×10⁶	[4]
S. equisimilis mutant nc2168	Optimization of flask culture medium and conditions	0.225	-	[40]
S. iniae	Ultraviolet mutation	0.12	3×10⁵	[41]
S. sp. ID9102	Optimization of medium components	6.94	5.9×10⁶	[42]
E. coli Top10	Co-expressing HA synthase hasA and ugd from S. equisimilis	0.19	3.5×10⁵-1.9×10⁶	[43]
E. coli BL21	Expression of hyaluronan synthase hasA from S. zooepidemicus ATCC 39920	0.532	3.46×10⁴	[44]
B. subtilis 168	Inducible expression of pmHAS,tuaD and gtaB	6.8	4.55×10⁶	[45]
	Coexpression committed genes（hasA,tuaD,gtaB,glmU,glmM,and glmS）;downregulate pfkA expression of the glycolytic pathway;integration of LHAase with the activity at 1.62×10⁶ U/mL	19.38	6.62×10³	[24]
	Engineering of cell membrane by overexpressing pgsA,clsA and reducing the expression of ftsZ	1.25	2.1×10⁶	[26]
B. subtilis WB600	Regulation of temperature using the recombinant strain carrying hasA gene from S. ubris,hasB,and hasC from B. subtilis WB600	3.65	0.39-6.94×10⁶	[46]
C. glutamicum 13032	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032	8.3	1.3×10⁶	[30]
	Coexpression gene hasA from S. equisimilis,hasB from C. glutamicum ATCC13032;deletion of lactate dehydrogenase	21.6	1.28×10⁶	[32]
	An additional promoter PdapB for driving hasB expression;deletion of fba,zwf deletion and knockout of lactate/acetate pathway	28.7	0.21×10⁶	[47]
	Overexpression of hyaluronan synthase spHasA,enzymes of intermediate metabolic pathways and attenuation of polysaccharide biosynthesis;Disruption of the encapsulation with the leech hyaluronidase	34.2-74.1	5.3×10⁴-3.31×10⁵	[21]
Streptomyces albulus	Overexpression of gene hasA from S. zooepidemicus,gene udgA,glmU,and gtaB genes from Streptomyces avermitilis	6.2	2×10⁶	[48]
Pichia pastoris	Overexpression of xhasA2 and xhasB genes from Xenopus laevis;hasC,hasD,and hasE genes from P. pastoris	0.8-1.7	1.2-2.5×10⁶	[19]
Lactococcus lactis CES15	Expression of hasA gene under the control of the PnisA promoter	6.09	-	[49]

Field	Application	HA molecular weight/Da	Reference
Medicine	Treatment of inflam- matory skin diseases.	6×10⁶	[70-72]
	Anticancer and anti- proliferative	1.2×10⁵	[73-74]
	Treatment of inflam- matory joint diseases	1×10⁶	[75-77]
	Anti-diabetic	-	[78]
	Immunomodulatory	2×10⁴	[79]
Cosmetics	Anti-aging	<1×10⁴	[80]
Cosmetics	Skin-repairing	1-10×10⁴	[66]
Food	Food and health	<1×10⁴	[81]

Field	Application	HA molecular weight/Da	Reference
Medicine	Treatment of inflam- matory skin diseases.	6×10⁶	[70-72]
	Anticancer and anti- proliferative	1.2×10⁵	[73-74]
	Treatment of inflam- matory joint diseases	1×10⁶	[75-77]
	Anti-diabetic	-	[78]
	Immunomodulatory	2×10⁴	[79]
Cosmetics	Anti-aging	<1×10⁴	[80]
Cosmetics	Skin-repairing	1-10×10⁴	[66]
Food	Food and health	<1×10⁴	[81]