Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Comparative Genomic and Genetic Functional Analysis of Industrial L-Leucine- and L-Valine-Producing Corynebacterium glutamicum Strains

  • Ma, Yuechao (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Chen, Qixin (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Cui, Yi (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Du, Lihong (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Shi, Tuo (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Xu, Qingyang (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Ma, Qian (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Xie, Xixian (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology) ;
  • Chen, Ning (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology)
  • Received : 2018.05.13
  • Accepted : 2018.09.12
  • Published : 2018.11.28
Download PDF
⟨ Previous Next ⟩

Abstract

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

Keywords

References

  1. Lee JY, Na YA, Kim E, Lee HS, Kim P. 2016. The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse. J. Microbiol. Biotechnol. 26: 807-822. https://doi.org/10.4014/jmb.1601.01053
  2. Kase H, Nakayama K. 1997. L-Isoleucine production by analog-resistant mutants derived from threonine-producing strain of Corynebacterium glutamicum. Agric. Biol. Chem. 41: 109-116.
  3. Vallino JJ, Stephanopoulos G. 1993. Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol. Bioeng. 41: 633-646. https://doi.org/10.1002/bit.260410606
  4. Park JH, Lee SY, Kim TY, Kim HU. 2008. Application of systems biology for bioprocess development. Trends Biotechnol. 26: 404-412. https://doi.org/10.1016/j.tibtech.2008.05.001
  5. Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, Sasamoto S, et al. 2002. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res. 9: 189-197. https://doi.org/10.1093/dnares/9.6.189
  6. Ikeda M, Ohnishi J, Hayashi M, Mitsuhashi S. 2006. A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient L-lysine production. J. Ind. Microbiol. Biotechnol. 33: 610-615. https://doi.org/10.1007/s10295-006-0104-5
  7. Becker J, Zelder O, Hafner S, Schroder H, Wittmann C. 2011. From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng. 13: 159-168. https://doi.org/10.1016/j.ymben.2011.01.003
  8. Ikeda M, Nakagawa S. 2003. The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl. Microbiol. Biotechnol. 62: 99-109. https://doi.org/10.1007/s00253-003-1328-1
  9. Wu Y, Li P, Zheng P, Zhou W, Chen N, Sun J. 2015. Complete genome sequence of Corynebacterium glutamicum B253, a Chinese lysine-producing strain. J. Biotechnol. 207: 10-11. https://doi.org/10.1016/j.jbiotec.2015.04.018
  10. Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, et al. 2003. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol. 104: 5-25. https://doi.org/10.1016/S0168-1656(03)00154-8
  11. Denisov G, Walenz B, Halpern AL, Miller J, Axelrod N, Levy S, et al. 2008. Consensus generation and variant detection by Celera Assembler. Bioinformatics 24: 1035-1040. https://doi.org/10.1093/bioinformatics/btn074
  12. Patel RK, Jain M. 2012. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7: e30619. https://doi.org/10.1371/journal.pone.0030619
  13. Seemann, T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30: 2068-2069. https://doi.org/10.1093/bioinformatics/btu153
  14. Salamov VSA, Solovyev A. 2011. Automatic annotation of microbial genomes and metagenomic sequences, pp. 61-78. In Li RW (ed.), Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies. Nova Science Publishers, Hauppauge, N.Y.
  15. Husemann P, Stoye J. 2010. r2cat: synteny plots and comparative assembly. Bioinformatics 26: 570-571. https://doi.org/10.1093/bioinformatics/btp690
  16. Darling AC, Mau B, Blattner FR, Perna NT. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14: 1394-1403. https://doi.org/10.1101/gr.2289704
  17. Zhao Y, Wu J, Yang J, Sun S, Xiao J, Yu J. 2012. PGAP: pangenomes analysis pipeline. Bioinformatics 28: 416-418. https://doi.org/10.1093/bioinformatics/btr655
  18. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754-1760. https://doi.org/10.1093/bioinformatics/btp324
  19. Milne I, Bayer M, Cardle L, Shaw P, Stephen G, Wright F, et al. 2010. Tablet--next generation sequence assembly visualization. Bioinformatics 26: 401-402. https://doi.org/10.1093/bioinformatics/btp666
  20. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25: 2078-2079. https://doi.org/10.1093/bioinformatics/btp352
  21. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, et al. 2004. Versatile and open software for comparing large genomes. Genome Biol. 5: R12. https://doi.org/10.1186/gb-2004-5-2-r12
  22. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, et al. 2012. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6: 80-92. https://doi.org/10.4161/fly.19695
  23. Kirchner O, Tauch A. 2003. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J. Biotechnol. 104: 287-299. https://doi.org/10.1016/S0168-1656(03)00148-2
  24. van der Rest ME, Lange C, Molenaar D. 1999. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 52: 541-545. https://doi.org/10.1007/s002530051557
  25. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145: 69-73. https://doi.org/10.1016/0378-1119(94)90324-7
  26. Cremer J, Eggeling L, Sahm H. 1991. Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Environ. Microbiol. 57: 1746-1752.
  27. Wendisch, Volker F. 2006. Genetic regulation of Corynebacterium glutamicum metabolism. J. Microbiol. Biotechnol. 16: 1010-1016.
  28. Gui Y, Ma Y, Xu Q, Zhang C, Xie X, Chen N. 2016. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain. J. Biotechnol. 220: 64-65. https://doi.org/10.1016/j.jbiotec.2016.01.010
  29. Baumgart M, Unthan S, Ruckert C, Sivalingam J, Grunberger A, Kalinowski J, et al. 2013. Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology. Appl. Environ. Microbiol. 79: 6006-6015. https://doi.org/10.1128/AEM.01634-13
  30. Vogt M, Haas S, Klaffl S, Polen T, Eggeling L, Ooyen JV, et al. 2014. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metab. Eng. 22: 40-52. https://doi.org/10.1016/j.ymben.2013.12.001
  31. Xie X, Xu L, Shi J, Xu Q, Chen N. 2012. Effect of transport proteins on L-isoleucine production with the L-isoleucineproducing strain Corynebacterium glutamicum YILW. J. Ind. Microbiol. Biotechnol. 39: 1549-1556. https://doi.org/10.1007/s10295-012-1155-4
  32. Yin L, Shi F, Hu X, Chen C, Wang X. 2013. Increasing L-isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two-component export system BrnFE. J. Appl. Microbiol. 114: 1369-1377. https://doi.org/10.1111/jam.12141
  33. Patek M. 2005. Regulation of Gene Expression, pp. 81-98. In Eggeling L, Bott M (eds.), Handbook of Corynebacterium glutamicum. CRC Press, Florida.
  34. Blombach B, Schreiner ME, Holatko J, Bartek T, Oldiges M, Eikmanns BJ. 2007. L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl. Environ. Microbiol. 73: 2079-2084. https://doi.org/10.1128/AEM.02826-06
  35. Chen C, Li Y, Hu J, Dong X, Wang X. 2015. Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production. Metab. Eng. 29: 66-75. https://doi.org/10.1016/j.ymben.2015.03.004
  36. Hua Q, Yang C, Baba T, Mori H, Shimizu K. 2003. Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts. J. Bacteriol. 185: 7053-7067. https://doi.org/10.1128/JB.185.24.7053-7067.2003
  37. Inui M, Murakami S, Okino S, Kawaguchi H, Vertes AA, Yukawa H. 2004. Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7: 182-196. https://doi.org/10.1159/000079827
  38. Peters-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L. 2005. Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl. Environ. Microbiol. 71: 7139-7144. https://doi.org/10.1128/AEM.71.11.7139-7144.2005
  39. Wagner T, Bellinzoni M, Wehenkel A, O'Hare HM, Alzari PM. 2011. Functional plasticity and allosteric regulation of ${\alpha}$-ketoglutarate decarboxylase in central mycobacterial metabolism. Chem. Biol. 18: 1011-1020. https://doi.org/10.1016/j.chembiol.2011.06.004
  40. Bunik VI, Fernie AR. 2009. Metabolic control exerted by the 2-oxoglutarate dehydrogenase reaction: a cross-kingdom comparison of the crossroad between energy production and nitrogen assimilation. Biochem. J. 422: 405-421. https://doi.org/10.1042/BJ20090722
  41. Tauch A, Hermann T, Burkovski A, Kramer R, Puhler A, Kalinowski J. 1998. Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product. Arch. Microbiol. 169: 303-312. https://doi.org/10.1007/s002030050576
  42. Bertioli DJ, Moretzsohn MC, Madsen LH, Sandal N, Leal- Bertioli SC, Guimarães PM, et al. 2009. An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes. BMC Genomics 10: 45. https://doi.org/10.1186/1471-2164-10-45
  43. Leuchtenberger W. 1996. Amino acids-technical production and use, pp. 465-502. In Rehm HJ, Reed G (eds.), Biotechnology: Products of primary metabolism, 2th Ed. Verlag-Chemie, Weinheim, Germany.

Cited by

  1. Transcriptomic and metabolomics analyses reveal metabolic characteristics of L-leucine- and L-valine-producing Corynebacterium glutamicum mutants vol.69, pp.5, 2018, https://doi.org/10.1007/s13213-018-1431-2
  2. Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production vol.12, pp.1, 2018, https://doi.org/10.1080/21655979.2021.1874693
  3. Effect of low-level ultrasound treatment on the production of L-leucine by Corynebacterium glutamicum in fed-batch culture vol.12, pp.1, 2018, https://doi.org/10.1080/21655979.2021.1906028
  4. Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids vol.20, pp.1, 2021, https://doi.org/10.1186/s12934-021-01721-0