CRISPRi-Based Dynamic Regulation of Hydrolase for the Synthesis of Poly-γ-Glutamic Acid with Variable Molecular Weights

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.