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Applied Microbiology and Biotechnology

, Volume 98, Issue 14, pp 6397–6407 | Cite as

Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance

  • Jun Feng
  • Weixia Gao
  • Yanyan Gu
  • Wei Zhang
  • Mingfeng Cao
  • Cunjiang SongEmail author
  • Peng Zhang
  • Min Sun
  • Chao Yang
  • Shufang WangEmail author
Applied genetics and molecular biotechnology

Abstract

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.

Keywords

Poly-γ-glutamic acid γ-PGA-degrading enzymes Glutamate-independent synthesis Gene markerless deletion 

Notes

Acknowledgments

This work was supported by the National key Basic Research Program of China (“973”-Program) 2012CB725204, National High Technology Research and Development Program of China (“863”-Program) 2012AA021505, Natural Science Foundation of China Grant Nos. 31070039, 31170030, 31300032, and 51073081, Project of Tianjin, China (13JCZDJC27800, 13JCYBJC24900). The Project of Tianjin, China (13JCQNJC09700).

Supplementary material

253_2014_5729_MOESM1_ESM.pdf (212 kb)
ESM 1 (PDF 211 kb)

References

  1. Abe K, Ito Y, Ohmachi T, Asada Y (1997) Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 61:1621–1625PubMedCrossRefGoogle Scholar
  2. Abe S, Yasumura A, Tanaka T (2009) Regulation of Bacillus subtilis aprE expression by glnA through inhibition of scoC and σD-dependent degR expression. J Bacteriol 191:3050–3058PubMedCentralPubMedCrossRefGoogle Scholar
  3. Ashiuchi M, Misono H (2002) Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl Microbiol Biotechnol 59:9–14PubMedCrossRefGoogle Scholar
  4. Ashiuchi M, Nakamura H, Yamamoto T, Kamei T, Soda K, Park C, Sung MH, Yagi T, Misono H (2003) Poly-γ-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity. J Mol Catal B Enzym 23:249–255CrossRefGoogle Scholar
  5. Bisicchia P, Noone D, Lioliou E, Howell A, Quigley S, Jensen T, Jarmer H, Devine KM (2007) The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis. Mol Microbiol 65:180–200PubMedCrossRefGoogle Scholar
  6. Cao MF, Song CJ, Jin YH, Liu L, Liu J, Xie H, Guo WB, Wang SF (2010) Synthesis of poly(γ-glutamic acid) and heterologous expression of pgsBCA genes. J Mol Catal B Enzym 67:111–116CrossRefGoogle Scholar
  7. Cao MF, Geng WT, Liu L, Song CJ, Xie H, Guo WB, Jin YH, Wang SF (2011) Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. Bioresour Technol 102:4251–4257PubMedCrossRefGoogle Scholar
  8. Domínguez-Cuevas P, Porcelli I, Daniel RA, Errington J (2013) Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis. Mol Microbiol 89:1084–1098PubMedCentralPubMedCrossRefGoogle Scholar
  9. Feng J, Gu YY, Wang JQ, Song CJ, Yang C, Xie H, Zhang W, Wang SF (2013) Curing the plasmid pMC1 from the poly (γ-glutamic acid) producing Bacillus amyloliquefaciens LL3 strain using plasmid incompatibility. Appl Biochem Biotechnol 171:532–542PubMedCrossRefGoogle Scholar
  10. Geng WT, Cao MF, Song CJ, Xie H, Liu L, Yang C, Feng J, Zhang W, Jin YH, Du Y, Wang SF (2011) Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid. J Bacteriol 193:3393–3394PubMedCentralPubMedCrossRefGoogle Scholar
  11. Goto A, Kunioka M (1992) Biosynthesis and hydrolysis of poly(γ-glutamic acid) from Bacillus subtilis IFO3335. Biosci Biotechnol Biochem 56:1031–1035CrossRefGoogle Scholar
  12. Hashimoto M, Ooiwa S, Sekiguchi J (2012) Synthetic lethality of the lytE cwlO genotype in Bacillus subtilis is caused by lack of D, L-endopeptidase activity at the lateral cell wall. J Bacteriol 194:796–803PubMedCentralPubMedCrossRefGoogle Scholar
  13. Keller KL, Bender KS, Wall JD (2009) Development of a markerless genetic exchange system for Desulfovibrio vulgaris hildenborough and its use in generating a strain with increased transformation efficiency. Appl Environ Microbiol 75:7682–7691PubMedCentralPubMedCrossRefGoogle Scholar
  14. Kimura K, Tran LS, Uchida I, Itoh Y (2004) Characterization of Bacillus subtilis gamma-glutamyltransferase and its involvement in the degradation of capsule poly-gamma-glutamate. Microbiology 150:4115–4123PubMedCrossRefGoogle Scholar
  15. Kimura K, Tran LS, Do TH, Itoh Y (2009) Expression of the pgsB encoding the poly-gamma-DL-glutamate syhthetase of Bacillus subtilis (natto). Biosci Biotechnol Biochem 73:1149–1155PubMedCrossRefGoogle Scholar
  16. Liu J, Ma X, Wang Y, Liu F, Qia JQ, Li XZ, Gao XW, Zhou T (2011) Depressed biofilm production in Bacillus amyloliquefaciens C06 causes γ-polyglutamic acid (γ-PGA) overproduction. Curr Microbiol 62:235–241PubMedCrossRefGoogle Scholar
  17. Mitsui N, Murasawa H, Sekiguchi J (2011) Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto). J Gen Appl Microbiol 57:35–43PubMedCrossRefGoogle Scholar
  18. Ohsawa T, Tsukahara K, Ogura M (2009) Bacillus subtilis response regulator DegU is a direct activator of pgsB transcription involved in γ-poly-glutamic acid synthesis. Biosci Biotechnol Biochem 73:2096–2102PubMedCrossRefGoogle Scholar
  19. Osera C, Amati G, Calvio C, Galizzi A (2009) SwrAA activates poly-γ-glutamate synthesis in addition to swarming in Bacillus subtilis. Microbiology 155:2282–2287PubMedCrossRefGoogle Scholar
  20. Reiter L, Kolstø AB, Piehler AP (2011) Reference genes for quantitative, reverse-transcription PCR in Bacillus cereus group strains throughout the bacterial life cycle. J Microbiol Methods 86:210–217PubMedCrossRefGoogle Scholar
  21. Richard A, Margaritis A (2003) Rheology, oxygen transfer, and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis. Biotechnol Bioeng 82:299–305PubMedCrossRefGoogle Scholar
  22. Scoffone V, Dondi D, Biino G, Borghese G, Pasini D, Galizzi A, Calvio C (2013) Knockout of pgdS and ggt genes improves γ-PGA yield in B. subtilis. Biotechnol Bioeng 110:2006–2012PubMedCrossRefGoogle Scholar
  23. Shih IL, Van YT (2001) The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour Technol 79:207–225PubMedCrossRefGoogle Scholar
  24. Smith K, Youngman P (1992) Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74:705–711PubMedCrossRefGoogle Scholar
  25. Smith TJ, Blackman SA, Foster SJ (2000) Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146:249–262PubMedGoogle Scholar
  26. Soliman NA, Berekaa MM, Abdel-Fattah YR (2005) Polyglutamic acid (PGA) production by Bacillus sp. SAB-26: application of Plackett–Burman experimental design to evaluate culture requirements. Appl Microbiol Biotechnol 69:259–267PubMedCrossRefGoogle Scholar
  27. Stanley NR, Lazazzera BA (2005) Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-γ-DL-glutamic acid production and biofilm formation. Mol Microbiol 57:1143–1158PubMedCrossRefGoogle Scholar
  28. Su YS, Li X, Liu QZ, Hou ZW, Zhu XQ, Guo XP, Ling PX (2010) Improved poly-γ-glutamic acid production by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. Bioresour Technol 101:4733–4736PubMedCrossRefGoogle Scholar
  29. Sung MH, Park C, Kim CJ, Poo H, Soda K, Ashiuchi M (2005) Natural and edible biopolymer poly-γ-glutamic acid: synthesis, production, and applications. Chem Rec 5:352–366PubMedCrossRefGoogle Scholar
  30. Suzuki T, Tahara Y (2003) Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation. J Bacteriol 185:2379–2382PubMedCentralPubMedCrossRefGoogle Scholar
  31. Tran LS, Nagai T, Itoh Y (2000) Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis. Mol Microbiol 37:1159–1171PubMedCrossRefGoogle Scholar
  32. Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32:259–286PubMedCrossRefGoogle Scholar
  33. Wu Q, Xu H, Shi N, Yao J, Li S, Ouyang P (2008) Improvement of poly(γ-glutamic acid) biosynthesis and redistribution of metabolic flux with the presence of different additives in Bacillus subtilis CGMCC 0833. Appl Microbiol Biotechnol 79:527–535PubMedCrossRefGoogle Scholar
  34. Yamaguchi H, Furuhata K, Fukushima T, Yamamoto H, Sekiguchi J (2004) Characterization of a new Bacillus subtilis peptidoglycan hydrolase gene, yvcE (named cwlO), and the enzymatic properties of its encoded protein. J Biosci Bioeng 98:174–181PubMedCrossRefGoogle Scholar
  35. Yao J, Jing J, Xu H, Liang JF, Wu Q, Feng XH, Ouyang PK (2009) Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2. J Mol Catal B Enzym 56:158–164CrossRefGoogle Scholar
  36. Yasumura A, Abe S, Tanaka T (2008) Involvement of nitrogen regulation in Bacillus subtilis degU expression. J Bacteriol 190:5162–5171PubMedCentralPubMedCrossRefGoogle Scholar
  37. Yeh CM, Wang JP, Lo SC, Chan WC, Lin MY (2010) Chromosomal integration of a synthetic expression control sequence achieves poly-γ-glutamate production in a Bacillus subtilis strain. Biotechnol Prog 24:1001–1007Google Scholar
  38. Zhang L, Li Y, Wang Z, Xia Y, Chen W, Tang K (2007) Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnol Adv 25:123–136PubMedCrossRefGoogle Scholar
  39. Zhang W, Xie H, He Y, Feng J, Gao WX, Gu YY, Wang SF, Song CJ (2013) Chromosome integration of the Vitreoscilla hemoglobin gene (vgb) mediated by temperature-sensitive plasmid enhances γ-PGA production in Bacillus amyloliquefaciens. FEMS Microbiol Lett 343:127–134PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jun Feng
    • 1
    • 2
  • Weixia Gao
    • 1
  • Yanyan Gu
    • 1
  • Wei Zhang
    • 1
  • Mingfeng Cao
    • 3
  • Cunjiang Song
    • 1
    Email author
  • Peng Zhang
    • 1
  • Min Sun
    • 1
  • Chao Yang
    • 1
  • Shufang Wang
    • 2
    Email author
  1. 1.Key Laboratory of Molecular Microbiology and Technology of Ministry of EducationNankai UniversityTianjinChina
  2. 2.State Key Laboratory of Medicinal Chemical BiologyNankai UniversityTianjinChina
  3. 3.Department of Chemical and Biological EngineeringIowa State UniversityAmesUSA

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