title: Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering.
abstract: A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-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 γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15 g/L. The γ-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 γ-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-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54 g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-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 γ-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 γ-PGA titer of 20.3g/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 γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China; State Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Tianjin 300071, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Yufen
authors.initials: Y
authors.lastname: Quan
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, United States.
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Cao
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Weixia
authors.initials: W
authors.lastname: Gao
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Tianjin 300071, China. Electronic address: wangshufang@nankai.edu.cn.
authors.forename: Shufang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China. Electronic address: songcj@nankai.edu.cn.
authors.forename: Cunjiang
authors.initials: C
authors.lastname: Song
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D018441:Biofilms; D005285:Fermentation; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D011134:Polysaccharides / Q000096:biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011134:Polysaccharides; D012329:RNA, Bacterial; D034741:RNA, Small Interfering; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; D005969:Glutamate Dehydrogenase
keywords: Autoinducer AI-2; Modular pathway engineering; Poly-γ-glutamic acid; Synthetic small regulatory RNAs; γ-PGA degrading-enzymes