CRISPRi-Based Dynamic Regulation of Hydrolase for the Synthesis of Poly-γ-Glutamic Acid with Variable Molecular Weights.,ACS Synthetic Biology

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CRISPRi-Based Dynamic Regulation of Hydrolase for the Synthesis of Poly-γ-Glutamic Acid with Variable Molecular Weights.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-08-14 , DOI: 10.1021/acssynbio.0c00207
Yuanyuan Sha _{1,

2} , Yibin Qiu ₃ , Yifan Zhu _{1,

2} , Tao Sun _{1,

2} , Zhengshan Luo _{1,

2} , Jian Gao ₄ , Xiaohai Feng _{1,

2} , Sha Li _{1,

2} , Hong Xu _{1,

2}

Affiliation

Poly-γ-glutamic acid (γ-PGA) is a decomposable polymer and has been useful in various industries. The biological functions of γ-PGA are closely linked with its molecular weight (MW). In this study, we established an efficient method to produce variable MWs of γ-PGA from renewable biomass (Jerusalem artichoke) by Bacillus amyloliquefaciens. First, a systematic engineering strategy was proposed in B. amyloliquefaciens to construct an optimal platform for γ-PGA overproduction, in which 24.95 g/L γ-PGA generation was attained. Second, 27.12 g/L γ-PGA with an MW of 20–30 kDa was obtained by introducing a γ-PGA hydrolase (pgdS) into the platform strain constructed above, which reveals a potential correlation between the expression level of pgdS and MW of γ-PGA. Then, a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was further designed to regulate pgdS expression levels, resulting in γ-PGA with variable MWs. Finally, a combinatorial approach based on three sgRNAs with different repression efficiencies was developed to achieve the dynamic regulation of pgdS and obtain tailor-made γ-PGA production in the MW range of 50–1400 kDa in one strain. This study illustrates a promising approach for the sustainable making of biopolymers with diverse molecular weights in one strain through the controllable expression of hydrolase using the CRISPRi system.

中文翻译：

基于 CRISPRi 的水解酶动态调节，用于合成不同分子量的聚-γ-谷氨酸。

聚-γ-谷氨酸 (γ-PGA) 是一种可分解的聚合物，可用于各种行业。γ-PGA 的生物学功能与其分子量 (MW) 密切相关。在这项研究中，我们建立了一种通过解淀粉芽孢杆菌从可再生生物质（耶路撒冷朝鲜蓟）中生产可变 MW γ-PGA 的有效方法。首先，在解淀粉芽孢杆菌中提出了系统工程策略，以构建γ-PGA 过量生产的最佳平台，其中γ-PGA 生成量为24.95 g/L。其次，通过将 γ-PGA 水解酶 ( pgdS ) 引入上述构建的平台菌株中，获得了 27.12 g/L MW 为 20-30 kDa 的 γ-PGA ，这揭示了pgdS表达水平之间的潜在相关性和 γ-PGA 的 MW。然后，进一步设计成簇规则间隔短回文重复干扰（CRISPRi）系统来调节pgdS表达水平，从而产生具有可变 MW 的 γ-PGA。最后，开发了一种基于具有不同抑制效率的三种 sgRNA 的组合方法，以实现pgdS的动态调节，并在一个菌株中在 50-1400 kDa 的 MW 范围内获得定制的 γ-PGA 产量。这项研究说明了一种有前途的方法，通过使用 CRISPRi 系统可控地表达水解酶，在一个菌株中可持续制造具有不同分子量的生物聚合物。

更新日期：2020-09-20

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