Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering

A Bacillus amyloliquefaciens strain with enhanced gamma-PGA production was constructed by metabolically engineering its gamma-PGA synthesis-related metabolic networks: by-products synthesis, gamma-PGA degradation, glutamate precursor synthesis, gamma-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased gamma-PGA purity and slight increase of gamma-PGA titer from 3.8 to 4.15 g/L. The gamma-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased gamma-PGA production from 4.15 to 9.18 g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11strain showed comparable gamma-PGA titer to NK-E10 (from 9.18 to 9.54 g/L). In addition, we overexpressed the pgsBCA genes (encoding gamma-PGA synthetase) in NK-Ell strain; however, the overexpression of these genes led to a decrease in gamma-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest gamma-PGA titer (11.04 g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final gamma-PGA titer of 20.3 g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced gamma-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of gamma-PGA or of other valuable metabolites. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.