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

doi:10.13523/j.cb.2009029

China Biotechnology

2021, Vol. 41

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

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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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

Received: 17 September 2020 Published: 09 February 2021

ZTFLH:

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Corresponding Authors: Guo-qiang XU E-mail: xuguoqiang@jiangnan.edu.cn

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	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

Cite this article:

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.

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

Table 1 Strains and plasmids

Table 2 Primer sequences used in this study

Fig.1 Fermentation profile of C. glutamicum F343-pZM1-capBCA

Fig.2 Stereochemical composition of γ-PGA produced by C. glutamicum F343-pZM1-capBCA

Fig.3 The effect of adding D-Glu exogenously on the D/L monomer ratio of γ-PGA

Fig.4 PCR confirmation of recombinant strain C. glutamicum F343 pZMI-capBCA-R0/RH/RL-racE M:DNA marker;1:capBCA-R0-racE; 2: capBCA-RH-racE; 3: capBCA-RL-racE

Fig.5 The effect of regulating the expression of glutamate racemase on the fermentation characteristics of γ-PGA based on the RBS calculator (a)Biomass (b)Residual glucose (c)L-glutamic acid (d)γ-PGA

Fig.6 The effect of RBS regulating the expression level of glutamate racemase on the D/L monomer ratio of γ-PGA (a) and enzyme activity of RacE (b)

Fig.7 PCR confirmation of recombinant strain C. glutamicumF343 pZMI-capBCA- e/11/12/16-racE M:DNA marker;1:capBCA-e-racE; 2: capBCA-11-racE; 3: capBCA-12-racE;4: capBCA-16-racE

Fig.8 The effect of regulating the expression of glutamate racemase on the fermentation characteristics of γ-PGA based on constitutive promoters (a)Biomass (b)Residual glucose (c)L-glutamic acid (d)γ-PGA

Fig.9 The effect of RBS regulating the expression level of glutamate racemase on the D/L monomer ratio of γ-PGA (a) and transcriptional level of RacE (b)


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