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

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.

[1]  Guocheng Du,et al.  Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production. , 2014, Metabolic engineering.

[2]  J. Singer,et al.  Water-soluble poly-(L-glutamic acid)-Gly-camptothecin conjugates enhance camptothecin stability and efficacy in vivo. , 2001, Journal of controlled release : official journal of the Controlled Release Society.

[3]  M. Kunioka,et al.  Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms , 1997, Applied Microbiology and Biotechnology.

[4]  A. Margaritis,et al.  Poly(glutamic Acid) for Biomedical Applications , 2001, Critical reviews in biotechnology.

[5]  H. Misono,et al.  Biochemistry and molecular genetics of poly-γ-glutamate synthesis , 2002, Applied Microbiology and Biotechnology.

[6]  S. Foster,et al.  Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. , 2000, Microbiology.

[7]  R. Losick,et al.  A major protein component of the Bacillus subtilis biofilm matrix , 2006, Molecular microbiology.

[8]  Chao Yang,et al.  Complete Genome Sequence of Bacillus amyloliquefaciens LL3, Which Exhibits Glutamic Acid-Independent Production of Poly-γ-Glutamic Acid , 2011, Journal of bacteriology.

[9]  J. Wall,et al.  Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency , 2009, Applied and Environmental Microbiology.

[10]  I. Shih,et al.  The production of poly-(gamma-glutamic acid) from microorganisms and its various applications. , 2001, Bioresource technology.

[11]  Xueping Guo,et al.  Improved poly-gamma-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. , 2010, Bioresource technology.

[12]  G. Stark,et al.  Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Skaracis,et al.  Optimization of xanthan gum production by Xanthomonas campestris grown in molasses , 2003 .

[14]  J. Qiao,et al.  Depressed Biofilm Production in Bacillus amyloliquefaciens C06 Causes γ-Polyglutamic Acid (γ-PGA) Overproduction , 2010, Current Microbiology.

[15]  Jane-Yii Wu,et al.  Levan production using Bacillus subtilis natto cells immobilized on alginate , 2010 .

[16]  M. Ogura,et al.  Bacillus subtilis Response Regulator DegU Is a Direct Activator of pgsB Transcription Involved in γ-Poly-glutamic Acid Synthesis , 2009, Bioscience, biotechnology, and biochemistry.

[17]  B. Bassler,et al.  Regulation of Uptake and Processing of the Quorum-Sensing Autoinducer AI-2 in Escherichia coli , 2005, Journal of bacteriology.

[18]  M. I. Gusdal,et al.  The Production of , 1979 .

[19]  Chao Yang,et al.  Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3 , 2015, Journal of Industrial Microbiology & Biotechnology.

[20]  Hong Xu,et al.  Economical production of poly(γ-glutamic acid) using untreated cane molasses and monosodium glutamate waste liquor by Bacillus subtilis NX-2. , 2012, Bioresource technology.

[21]  Shufang Wang,et al.  Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. , 2011, Bioresource technology.

[22]  A. Galizzi,et al.  Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis , 2013, Biotechnology and bioengineering.

[23]  A. R. Kulkarni,et al.  Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. , 2006, Biomaterials.

[24]  M. Surette,et al.  Quorum sensing in Escherichia coli and Salmonella typhimurium. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  K. Devine,et al.  The essential YycFG two‐component system controls cell wall metabolism in Bacillus subtilis , 2007, Molecular microbiology.

[26]  J. Sekiguchi,et al.  Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto). , 2011, The Journal of general and applied microbiology.

[27]  Chao Yang,et al.  Curing the Plasmid pMC1 from the Poly (γ-glutamic Acid) Producing Bacillus amyloliquefaciens LL3 Strain Using Plasmid Incompatibility , 2013, Applied Biochemistry and Biotechnology.

[28]  Jun Feng,et al.  Chromosome integration of the Vitreoscilla hemoglobin gene (vgb) mediated by temperature-sensitive plasmid enhances γ-PGA production in Bacillus amyloliquefaciens. , 2013, FEMS microbiology letters.

[29]  V. Yang,et al.  Controlled release of clot-dissolving tissue-type plasminogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. , 2001, Journal of controlled release : official journal of the Controlled Release Society.

[30]  K. Gunka,et al.  Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation , 2012, Molecular microbiology.

[31]  I. Mandic-Mulec,et al.  Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms , 2013, PloS one.

[32]  Satoru Miyano,et al.  Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species , 2005, PLoS Comput. Biol..

[33]  B. Palsson,et al.  Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data* , 2007, Journal of Biological Chemistry.

[34]  T. Ohmachi,et al.  Glutamic Acid Independent Production of Poly(γ-glutamic acid) by Bacillus subtilis TAM-4 , 1996 .

[35]  A. Sonenshein,et al.  Role and Regulation of Bacillus subtilisGlutamate Dehydrogenase Genes , 1998, Journal of bacteriology.

[36]  A. Fouet,et al.  Poly‐gamma‐glutamate in bacteria , 2006, Molecular microbiology.

[37]  Chun Xing Li.  Poly(L-glutamic acid)--anticancer drug conjugates. , 2002, Advanced drug delivery reviews.

[38]  G. Beldman,et al.  Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation , 1994, Molecular microbiology.

[39]  M. Surette,et al.  Regulation of autoinducer production in Salmonella typhimurium , 1999, Molecular microbiology.

[40]  M. Kunioka,et al.  Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335. , 1992, Bioscience, biotechnology, and biochemistry.

[41]  J. Park,et al.  Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs , 2013, Nature Biotechnology.

[42]  Dieter Jahn,et al.  Fermentative Metabolism of Bacillus subtilis: Physiology and Regulation of Gene Expression , 2000, Journal of bacteriology.

[43]  J. Stülke,et al.  Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression , 2007, Molecular microbiology.

[44]  Y. Beyatlı,et al.  Characterization and production of the exopolysaccharide (EPS) from Pseudomonas aeruginosa G1 and Pseudomonas putida G12 strains , 2008 .

[45]  O. Kuipers,et al.  Regulation of Glutamine and Glutamate Metabolism by GlnR and GlnA in Streptococcus pneumoniae* , 2006, Journal of Biological Chemistry.

[46]  Yajun Yan,et al.  Conversion of proteins into biofuels by engineering nitrogen flux , 2011, Nature Biotechnology.

[47]  Bastien Chevreux,et al.  The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains , 2008, Journal of bacteriology.

[48]  J. Stülke,et al.  A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene , 2011, Journal of bacteriology.

[49]  M. Marahiel,et al.  Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu , 1991, Journal of bacteriology.

[50]  P. Youngman,et al.  Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. , 1992, Biochimie.

[51]  Jane-Yii Wu,et al.  Production and characterization of curdlan by Agrobacterium sp. , 2009 .

[52]  F. Bolivar,et al.  Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism , 2007, Applied and Environmental Microbiology.

[53]  F. Smith,et al.  COLORIMETRIC METHOD FOR DETER-MINATION OF SUGAR AND RELATED SUBSTANCE , 1956 .

[54]  Chao Yang,et al.  Metabolic engineering of Bacillus amyloliquefaciens for poly-gamma-glutamic acid (γ-PGA) overproduction , 2014, Microbial biotechnology.