不同D/L单体比γ-聚谷氨酸的合成与调控

doi:10.13523/j.cb.2009029

中国生物工程杂志

2021, Vol. 41

Issue (1): 1-11 DOI: 10.13523/j.cb.2009029

研究报告

不同D/L单体比γ-聚谷氨酸的合成与调控

朱亚鑫^1,²,段艳婷^1,²,高宇豪^1,²,王籍阅^1,²,张晓梅³,张晓娟^1,²,徐国强^1,^2,^**(

),史劲松³,许正宏^1,²

1 江南大学生物工程学院工业生物技术教育部重点实验室无锡 214122
2 江南大学粮食发酵工艺与技术国家工程实验室无锡 214122
3 江南大学药学院无锡 214122

Synthesis and Regulation of Poly-γ-glutamic Acid with Different D/L Monomer Ratios

ZHU Ya-xin^1,²,DUAN Yan-ting^1,²,GAO Yu-hao^1,²,WANG Ji-yue^1,²,ZHANG Xiao-mei³,ZHANG Xiao-juan^1,²,XU Guo-qiang^1,^2,^**(

),SHI Jin-song³,XU Zheng-hong^1,²

1 Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
2 National Engineering Laboratory for Food Fermentation Technology and Technology, Jiangnan University, Wuxi 214122, China
3 School of Pharmaceutics, Jiangnan University, Wuxi 214122, China

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摘要：

γ-聚谷氨酸(γ-PGA)是由L-谷氨酸和/或D-谷氨酸聚合而成的一种聚氨基酸,广泛应用于化妆品、医药等领域。高聚物单体的立体构型会影响产品性质和应用,因此调控γ-PGA中D-谷氨酸/L-谷氨酸单体比(D/L单体比)具有重要意义。前期以谷氨酸棒杆菌为底盘,表达来自于地衣芽孢杆菌的γ-PGA合成酶,合成以L-Glu(97.10%)为主的γ-PGA。通过外源添加不同浓度D-谷氨酸,合成了D-谷氨酸占比为15.71%~33.52%的γ-PGA。然后,在重组菌中表达来自于枯草芽孢杆菌的谷氨酸消旋酶,并使用三个不同强度RBS调控其表达水平,合成D-谷氨酸占比30.82%~34.59%的γ-PGA,但调控范围较窄。利用四个不同强度启动子调控谷氨酸消旋酶表达水平,扩大D/L单体比可调范围,合成D-Glu占比32.71%~52.53%的γ-PGA。提供一种理性调控γ-PGA的D/L单体比策略,实现了D-谷氨酸占比为2.90%~52.53%的γ-PGA的合成,为高效合成不同D/L单体比γ-PGA提供了基础。

关键词： γ-聚谷氨酸; D/L单体比; 谷氨酸消旋酶; RBS; 启动子

Abstract:

Poly-γ-glutamic acid (γ-PGA) is a kind of poly-amino acid formed by polymerization of L-glutamic acid (L-Glu) and / or D-glutamic acid (D-Glu), and is widely used in cosmetics, medicine and other fields. The stereochemical composition of its monomer often affects the properties and applications of the product. Therefore, it is of great significance to control the monomer ratio of D-Glu/L-Glu (D/L monomer ratio) in γ-PGA. In our previous study, Corynebacterium glutamicum was used as the chassis to overexpress γ-PGA synthetase from Bacillus licheniformis, and synthesize γ-PGA with L-Glu as the main component. On this basis, different concentrations of D-Glu were added exogenously to synthesize γ-PGA with D-Glu accounting for 15.71% ~ 33.52%. Then, the glutamate racemase from Bacillus subtilis was overexpressed in the recombinant bacteria, and three RBS of different strengths were used to regulate its expression level, however, and γ-PGA with D-Glu accounting for a narrow range (30.82%~34.59%) was synthesized. Subsequently, four different strength promoters were used to regulate the expression level of glutamate racemase, and γ-PGA with D-Glu accounting for 32.71%~52.53% was synthesized. A rational strategy for regulating the D/L monomer ratio of γ-PGA was proposed, and γ-PGA with a D-Glu ratio of 2.90%~52.53% was synthesized, which laid the foundation for the efficient synthesis of γ-PGA with different D/L monomer ratios.

Key words: Poly-γ-glutamic acid; D/L monomer ratio Glutamate racemase RBS Promoter

收稿日期: 2020-09-17 出版日期: 2021-02-09

ZTFLH:

Q815

基金资助: * 国家重点研发计划(2018YFA0900300);江苏省六大人才高峰项目(2015-SWYY-006)

通讯作者: 徐国强 E-mail: xuguoqiang@jiangnan.edu.cn

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	朱亚鑫
	段艳婷
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	王籍阅
	张晓梅
	张晓娟
	徐国强
	史劲松
	许正宏

引用本文:

朱亚鑫, 段艳婷, 高宇豪, 王籍阅, 张晓梅, 张晓娟, 徐国强, 史劲松, 许正宏. 不同D/L单体比γ-聚谷氨酸的合成与调控[J]. 中国生物工程杂志, 2021, 41(1): 1-11.

ZHU Ya-xin, DUAN Yan-ting, GAO Yu-hao, WANG Ji-yue, ZHANG Xiao-mei, ZHANG Xiao-juan, XU Guo-qiang, SHI Jin-song, XU Zheng-hong. Synthesis and Regulation of Poly-γ-glutamic Acid with Different D/L Monomer Ratios. China Biotechnology, 2021, 41(1): 1-11.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2009029 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I1/1

表1 菌株与质粒

表2 本研究所用引物

图1 重组菌C. glutamicum F343-pZM1-capBCA发酵特性

图2 产自C. glutamicum F343-pZM1-capBCA的γ-PGA立体构型

图3 外源添加D-Glu对γ-PGA的D/L单体比影响

图4 C. glutamicum F343 pZMI-capBCA-R0/RH/RL-racE菌落PCR验证

图5 基于RBS计算器调控谷氨酸消旋酶表达对γ-PGA发酵特性影响

图6 RBS调控谷氨酸消旋酶表达水平对γ-PGA的D/L单体比(a)及酶活(b)影响

图7 C. glutamicum F343 pZMI-capBCA-e/11/12/16-racE菌落PCR验证

图8 基于组成型启动子调控谷氨酸消旋酶表达对γ-PGA发酵特性影响

图9 组成型启动子调控谷氨酸消旋酶表达水平对γ-PGA的D/L单体比(a)及转录水平(b)影响

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