期刊
ACS SYNTHETIC BIOLOGY
卷 9, 期 9, 页码 2450-2459出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00207
关键词
poly-gamma-glutamic acid; molecular weight; hydrolase; CRISPRi; dynamic regulation; Bacillus amyloliquefaciens
资金
- National Natural Science Foundation of China [21878152, 21776133]
- National Key Research and Development Program of China [2019YFA09005203]
- Natural Science Foundation of Jiangsu Province [BK20171010]
- Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture [XTB1804]
Poly-gamma-glutamic acid (gamma-PGA) is a decomposable polymer and has been useful in various industries. The biological functions of gamma-PGA are closely linked with its molecular weight (MW). In this study, we established an efficient method to produce variable MWs of gamma-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 gamma-PGA overproduction, in which 24.95 g/L gamma-PGA generation was attained. Second, 27.12 g/L gamma-PGA with an MW of 20-30 kDa was obtained by introducing a gamma-PGA hydrolase (pgdS) into the platform strain constructed above, which reveals a potential correlation between the expression level of pgdS and MW of gamma-PGA. Then, a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was further designed to regulate pgdS expression levels, resulting in gamma-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 gamma-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.
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