可控分子量透明质酸的生物合成研究进展

doi:10.12211/2096-8280.2024-062

合成生物学 ›› 2025, Vol. 6 ›› Issue (2): 445-460.DOI: 10.12211/2096-8280.2024-062

可控分子量透明质酸的生物合成研究进展

肖森¹^,²^,³, 胡立涛¹^,²^,³, 石智诚¹^,², 王发银¹^,², 余思婷¹^,², 堵国成²^,⁴, 陈坚²^,³^,⁴, 康振¹^,²^,³

^1.江南大学生物工程学院，糖化学与生物技术教育部重点实验室，江苏无锡 214122
^2.江南大学，未来食品科学中心，江苏无锡 214122
^3.江苏省产业技术研究院，江苏集萃未来食品技术研究所有限公司，江苏宜兴 214200
^4.江南大学生物工程学院，工业生物技术教育部重点实验室，江苏无锡 214122

收稿日期:2024-08-14 修回日期:2024-10-24 出版日期:2025-04-30 发布日期:2025-05-20
通讯作者: 康振
作者简介:肖森（2002—），男，硕士研究生。研究方向为透明质酸的生物合成与酶工程改造。 E-mail：6240201118@stu.jiangnan.edu.cn
康振（1982—），男，博士，教授，博士生导师。研究方向为食品微生物合成生物学与生物制造。E-mail：zkang@jiangnan.edu.cn
基金资助:
江南大学食品科学与资源国家重点实验室研发项目(5962060204240220);国家自然科学基金(32370066);中央高校基本科研基金(JUSRP622003);无锡市产业创新研究院先导技术预研项目(XD24006)

Research advances in biosynthesis of hyaluronic acid with controlled molecular weights

XIAO Sen¹^,²^,³, HU Litao¹^,²^,³, SHI Zhicheng¹^,², WANG Fayin¹^,², YU Siting¹^,², DU Guocheng²^,⁴, CHEN Jian²^,³^,⁴, KANG Zhen¹^,²^,³

^1.Key Laboratory of Carbohydrate Chemistry and Biotechnology，Ministry of Education，School of Biotechnology，Jiangnan University，Wuxi 214122，Jiangsu，China
^2.Science Center for Future Foods，Jiangnan University，Wuxi 214122，Jiangsu，China
^3.Institute of Future Food Technology，Jiangsu Industrial Technology Research Institute，Yixing 214200，Jiangsu，China
^4.Key Laboratory of Industrial Biotechnology，Ministry of Education，School of Biotechnology，Jiangnan University，Wuxi 214122，Jiangsu，China

Received:2024-08-14 Revised:2024-10-24 Online:2025-04-30 Published:2025-05-20
Contact: KANG Zhen

摘要/Abstract

摘要：

透明质酸（hyaluronic acid， HA）是一种在化妆品、食品和医疗领域广泛应用的天然直链酸性黏多糖。根据分子量大小，HA可分为高、中、低三类，不同分子量的HA具有不同的功能和应用场景。随着微生物发酵技术取代传统动物组织提取法，HA工业化生产取得了巨大进步。然而，天然HA合成菌株兽疫链球菌的缺点（如潜在致病性以及难以分子改造），限制了不同分子量HA的生物合成研究。近年来，随着特定分子量HA需求的不断增长，代谢工程和合成生物技术已广泛应用于HA生物合成与分子量调控。本文首先分析了中高分子量HA合成的限制性因素，重点讨论了HA前体合成途径的基因调控及竞争支路的弱化。其次，探讨了HA合酶、前体供应和发酵条件对超高分子量HA合成的影响。最后，总结了低分子量HA的制备策略，包括物理化学法、酶法和微生物直接发酵法。还针对HA的生物合成与分子量调控面临的挑战——高分子量HA分子量不够高、中高分子量HA合成能力弱和低分子量HA分子量可控性差三方面展开系统性综述，加强对HA生物合成与分子量调控策略的理解，助力实现可控分子量HA的高效生物合成。

关键词: 透明质酸, 分子量, 生物合成, 代谢工程, 合成生物学

Abstract:

Hyaluronic acid (HA), a natural linear acidic polysaccharide composed of disaccharide units of D-glucuronic acid (D-GlcA) and N-acetylglucosamine (N-GlcNAc), has been widely used in the cosmetic and medical fields. HAs with different molecular weights exhibit distinct biophysical properties. While high molecular weight HAs have stronger viscoelasticity and resistance to degradation, low molecular weight HAs demonstrate enhanced biological functions. Significant progress has been made for the industrial production of HAs, with the shift from traditional extraction from animal tissues to microbial fermentation. However, the use of the natural HA-producing species Streptococcus zooepidemicus presents challenges, such as potential pathogenicity and difficulties in molecular modifications, which limit the study on the biosynthesis of HAs with varying molecular weights. Recently, the increasing demand for specific molecular weight HAs has driven the application of metabolic engineering and synthetic biology techniques for their biosynthesis and molecular weight regulation. By identifying the key factors involved in the processes, researchers have developed various strategies to optimize the synthesis of HAs and control their molecular weights. This article first analyzes the limiting factors in the synthesis of medium and high molecular weight HAs, focusing on the genetic regulation on the synthesis pathways of HA precursors and the weakening of competitive branches. Secondly, it discusses the impact of HA synthase, precursor supply, and fermentation conditions on the synthesis of ultra-high molecular weight HAs. Finally, it summarizes the preparation strategies for low molecular weight HAs, including physical and chemical methods, enzymatic methods, and microbial direct fermentation as well. The review summarizes the latest research progress regarding challenges faced in the biosynthesis and molecular weight regulation of HAs: specifically, the insufficient molecular weight of high molecular weight HAs, the weak synthesis capability of medium molecular weight HAs, and the poor controllability of low molecular weight HAs. It provides a systematic overview on enhancing the understanding of strategies for HA biosynthesis and molecular weight regulation, aiming to facilitate the efficient biosynthesis of HAs with controlled molecular weights.

Key words: hyaluronic acid, molecular weight, biosynthesis, metabolic engineering, synthetic biology

中图分类号:

Q815

肖森, 胡立涛, 石智诚, 王发银, 余思婷, 堵国成, 陈坚, 康振. 可控分子量透明质酸的生物合成研究进展[J]. 合成生物学, 2025, 6(2): 445-460.

XIAO Sen, HU Litao, SHI Zhicheng, WANG Fayin, YU Siting, DU Guocheng, CHEN Jian, KANG Zhen. Research advances in biosynthesis of hyaluronic acid with controlled molecular weights[J]. Synthetic Biology Journal, 2025, 6(2): 445-460.

图/表 6

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工程菌株	改造策略	HA产量 /（g/L）	HA分子量 /Da	参考文献
S. zooepidemicus ATCC 39920	敲除HA裂解酶编码基因hylB	3.40	2.28×10⁶	[67]
S. zooepidemicus	过表达HAS编码基因sehasA，提高碳源浓度	6.90	2.75×10⁶	[68]
S. zooepidemicus MTCC 3523	调节溶解氧水平与N-乙酰葡萄糖胺供应水平	2.4	2.53×10⁶	[69]
S. zooepidemicus	添加铁纳米颗粒	0.435	1.48×10⁶	[66]
S. zooepidemicus ATCC 39920	紫外诱变策略，开发两阶段半连续发酵工艺	29.38	—	[10]
E. coli K12 W3110	敲除6-磷酸果糖激酶Ⅰ编码基因pfkA和葡萄糖-6-磷酸脱氢酶编码基因zwf，表达HA合成途径基因簇galU-ugd和glmS-glmM-glmU	0.02998	—	[44]
E. coli Top10	表达兽疫链球菌来源HAS编码基因sehasA和UDP-葡萄糖脱氢酶编码基因ugdA	0.19	0.35×10⁶～1.9×10⁶	[45]
E. coli JM109	表达多杀巴斯德杆菌来源HAS编码基因pmhasA和大肠杆菌K5来源UDP-葡萄糖脱氢酶编码基因ugdA	3.8	—	[48]
Lactobacillus acidophilus	表达兽疫链球菌来源HAS编码基因sehasA和UDP-葡萄糖脱氢酶编码基因ugdA	1.7	—	[70]
Lactococcus lactis CES15	PnisA启动子调控兽疫链球菌来源HAS编码基因sehasA表达	6.09	—	[52]
Streptomyces albulus	表达兽疫链球菌来源HAS编码基因sehasA和阿维米蒂利斯链霉菌来源UDP-葡萄糖脱氢酶编码基因ugdA、乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因glmU、葡萄糖-6-磷酸尿酰胺转移酶编码基因gtaB	6.2	2×10⁶	[71]
Pichia pastoris	表达非洲爪蟾来源HAS编码基因xhasA2和UDP-葡萄糖脱氢酶编码基因xhasB，毕赤酵母来源的葡萄糖-6-磷酸尿酰胺转移酶编码基因hasC，乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因hasD，磷酸葡萄糖异构酶编码基因hasE	1.7	1.2×10⁶～2.5×10⁶	[59]
B. subtilis 168	表达兽疫链球菌来源HAS编码基因sehasA，枯草芽孢杆菌来源UDP-葡萄糖脱氢酶编码基因tuaD，葡萄糖-6-磷酸尿酰胺转移酶编码基因gtaB，乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因glmU，磷酸葡萄糖胺变位酶编码基因glmM和谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因glmS；下调6-磷酸果糖激酶Ⅰ编码基因pfkA表达	3.16	1.4×10⁶～1.83×10⁶	[72]
B. subtilis BGSC 1A751	表达细菌血红蛋白编码基因vhb、C组链球菌来源HAS编码基因hasA、枯草芽孢杆菌来源UDP-葡萄糖脱氢酶编码基因tuaD	1.8	—	[58]
C. glutamicum 13032	表达酿脓链球菌来源HAS编码基因spHasA，恶臭假单胞菌来源谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因ptglmS，谷氨酸棒杆菌来源UDP-葡萄糖脱氢酶编码基因cgugdA2；敲除糖基转移酶编码基因cg0420和cg0424	34.2	3.21×10⁵	[64]
C. glutamicum 13032	表达兽疫链球菌来源HAS编码基因sehasA，谷氨酸棒杆菌来源UDP-葡萄糖脱氢酶编码基因hasB	8.3	1.3×10⁶	[61]
C. glutamicum 13032	表达兽疫链球菌来源HAS编码基因seHasA，UDP-葡萄糖脱氢酶编码基因hasB，敲除果糖1,6-二磷酸醛缩酶编码基因fba，葡萄糖-6-磷酸脱氢酶编码基因zwf，阻断乳酸和乙酸合成	28.7	2.1×10⁵	[60]

工程菌株	改造策略	HA产量 /（g/L）	HA分子量 /Da	参考文献
S. zooepidemicus ATCC 39920	敲除HA裂解酶编码基因hylB	3.40	2.28×10⁶	[67]
S. zooepidemicus	过表达HAS编码基因sehasA，提高碳源浓度	6.90	2.75×10⁶	[68]
S. zooepidemicus MTCC 3523	调节溶解氧水平与N-乙酰葡萄糖胺供应水平	2.4	2.53×10⁶	[69]
S. zooepidemicus	添加铁纳米颗粒	0.435	1.48×10⁶	[66]
S. zooepidemicus ATCC 39920	紫外诱变策略，开发两阶段半连续发酵工艺	29.38	—	[10]
E. coli K12 W3110	敲除6-磷酸果糖激酶Ⅰ编码基因pfkA和葡萄糖-6-磷酸脱氢酶编码基因zwf，表达HA合成途径基因簇galU-ugd和glmS-glmM-glmU	0.02998	—	[44]
E. coli Top10	表达兽疫链球菌来源HAS编码基因sehasA和UDP-葡萄糖脱氢酶编码基因ugdA	0.19	0.35×10⁶～1.9×10⁶	[45]
E. coli JM109	表达多杀巴斯德杆菌来源HAS编码基因pmhasA和大肠杆菌K5来源UDP-葡萄糖脱氢酶编码基因ugdA	3.8	—	[48]
Lactobacillus acidophilus	表达兽疫链球菌来源HAS编码基因sehasA和UDP-葡萄糖脱氢酶编码基因ugdA	1.7	—	[70]
Lactococcus lactis CES15	PnisA启动子调控兽疫链球菌来源HAS编码基因sehasA表达	6.09	—	[52]
Streptomyces albulus	表达兽疫链球菌来源HAS编码基因sehasA和阿维米蒂利斯链霉菌来源UDP-葡萄糖脱氢酶编码基因ugdA、乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因glmU、葡萄糖-6-磷酸尿酰胺转移酶编码基因gtaB	6.2	2×10⁶	[71]
Pichia pastoris	表达非洲爪蟾来源HAS编码基因xhasA2和UDP-葡萄糖脱氢酶编码基因xhasB，毕赤酵母来源的葡萄糖-6-磷酸尿酰胺转移酶编码基因hasC，乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因hasD，磷酸葡萄糖异构酶编码基因hasE	1.7	1.2×10⁶～2.5×10⁶	[59]
B. subtilis 168	表达兽疫链球菌来源HAS编码基因sehasA，枯草芽孢杆菌来源UDP-葡萄糖脱氢酶编码基因tuaD，葡萄糖-6-磷酸尿酰胺转移酶编码基因gtaB，乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶编码基因glmU，磷酸葡萄糖胺变位酶编码基因glmM和谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因glmS；下调6-磷酸果糖激酶Ⅰ编码基因pfkA表达	3.16	1.4×10⁶～1.83×10⁶	[72]
B. subtilis BGSC 1A751	表达细菌血红蛋白编码基因vhb、C组链球菌来源HAS编码基因hasA、枯草芽孢杆菌来源UDP-葡萄糖脱氢酶编码基因tuaD	1.8	—	[58]
C. glutamicum 13032	表达酿脓链球菌来源HAS编码基因spHasA，恶臭假单胞菌来源谷氨酰胺-果糖-6-磷酸氨基转移酶编码基因ptglmS，谷氨酸棒杆菌来源UDP-葡萄糖脱氢酶编码基因cgugdA2；敲除糖基转移酶编码基因cg0420和cg0424	34.2	3.21×10⁵	[64]
C. glutamicum 13032	表达兽疫链球菌来源HAS编码基因sehasA，谷氨酸棒杆菌来源UDP-葡萄糖脱氢酶编码基因hasB	8.3	1.3×10⁶	[61]
C. glutamicum 13032	表达兽疫链球菌来源HAS编码基因seHasA，UDP-葡萄糖脱氢酶编码基因hasB，敲除果糖1,6-二磷酸醛缩酶编码基因fba，葡萄糖-6-磷酸脱氢酶编码基因zwf，阻断乳酸和乙酸合成	28.7	2.1×10⁵	[60]

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可控分子量透明质酸的生物合成研究进展

Research advances in biosynthesis of hyaluronic acid with controlled molecular weights

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