Advertisement

Transcriptomic and metabolomics analyses reveal metabolic characteristics of L-leucine- and L-valine-producing Corynebacterium glutamicum mutants

  • Yuechao Ma
  • Qian Ma
  • Yi Cui
  • Lihong Du
  • Xixian XieEmail author
  • Ning ChenEmail author
Original Article
  • 26 Downloads

Abstract

Industrial amino acid production strains of Corynebacterium glutamicum are usually obtained by mutagenesis. However, the genetic and metabolic characteristics and the efficient synthesis mechanism of the selected mutants are unclear. The aims of this study were (1) to determine the gene transcriptional patterns and intracellular metabolite levels of an L-leucine-producing mutant C. glutamicum CP and an L-valine-producing mutant C. glutamicum XV referring to wild type, and (2) to understand the efficient synthesis mechanism of target product of these mutants. For this purpose, transcriptomic and metabolomics analyses were combined to investigate the association between intracellular patterns and product synthesis. The high intracellular level of glucose and the low intracellular level of metabolites in the central carbon metabolism meant the glucose metabolism rate of two mutants was lower than wild type. However, the increased intracellular pentose level and gene transcription in the pentose phosphate pathway (PPP) indicated that the PPP of mutants was more active. Furthermore, the mutants showed higher intracellular level of NADPH, which was mainly generated in PPP. In the specific pathway for the synthesis of L-leucine and L-valine, the transcriptional level of most genes was upregulated in the mutants. However, the transcription of transaminase C coding gene Cgl2844 was downregulated in CP but upregulated in XV. The upregulation of Cgl2844 might benefit to the synthesis of L-valine and cause the significant decrease of intracellular level of L-alanine and L-glutamate of XV. These characteristics of the mutants provided insight into changes that could be made to systematically optimize the metabolic pathways for the production of L-leucine and L-valine.

Keywords

Corynebacterium glutamicum Transcriptomic Metabolomics L-valine L-leucine 

Notes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (31470211 and 31770053), Natural Science Foundation of Tianjin (17JCQNJC09500), and Tianjin Municipal Science and Technology Commission (17YFZCSY01050).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

13213_2018_1431_MOESM1_ESM.pdf (2.7 mb)
ESM 1 (PDF 2790 kb)

References

  1. Anders S (2010) Analysing RNA-Seq data with the DESeq package. Mol Biol 43(4):1–17Google Scholar
  2. Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169CrossRefGoogle Scholar
  3. Bartek T, Makus P, Klein B, Lang S, Oldiges M (2008) Influence of L-isoleucine and pantothenate auxotrophy for L-valine formation in Corynebacterium glutamicum revisited by metabolome analyses. Bioprocess Biosyst Eng 31:217–225CrossRefGoogle Scholar
  4. Becker J, Zelder O, Häfner S, Schröder H, Wittmann C (2011) From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab Eng 13:159–168CrossRefGoogle Scholar
  5. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple hypothesis testing. J R Stat Soc B 57:289–300Google Scholar
  6. Blombach B, Schreiner ME, Bartek T, Oldiges M, Eikmanns BJ (2008) Corynebacterium glutamicum tailored for high-yield L-valine production. Appl Microbiol Biotechnol 79:471–479CrossRefGoogle Scholar
  7. Bommareddy RR, Chen Z, Rappert S, Zeng AP (2014) A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase. Metab Eng 25:30–37CrossRefGoogle Scholar
  8. Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15(9):417–425CrossRefGoogle Scholar
  9. Chen C, Pan J, Yang X, Guo C, Ding W, Si M, Zhang Y, Shen X, Wang Y (2016) Global transcriptomic analysis of the response of Corynebacterium glutamicum to vanillin. PLoS One 11:e0164955CrossRefGoogle Scholar
  10. Gaigalat L, Schlüter JP, Hartmann M, Mormann S, Tauch A, Pühler A, Kalinowski J (2007) The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum. BMC Mol Biol 8:104CrossRefGoogle Scholar
  11. Hasegawa S, Suda M, Uematsu K, Natsuma Y, Hiraga K, Jojima T, Inui M, Yukawa H (2013) Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions. Appl Environ Microbiol 79:1250–1257CrossRefGoogle Scholar
  12. Huang Z, Lee DY, Yoon S (2017) Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures. Biotechnol Bioeng 114:2717–2728CrossRefGoogle Scholar
  13. Inui M, Suda M, Okino S, Nonaka H, Puskás LG, Vertès AA, Yukawa H (2007) Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions. Microbiology 153:2491–2504CrossRefGoogle Scholar
  14. Krömer JO, Sorgenfrei O, Klopprogge K, Heinzle E, Wittmann C (2004) In-depth profiling of lysine-producing Corynebacterium glutamicum by combined analysis of the transcriptome, metabolome, and fluxome. J Bacteriol 186:1769–1784CrossRefGoogle Scholar
  15. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25CrossRefGoogle Scholar
  16. Leuchtenberger W (1996) Amino acids–technical production and use. Biotechnology: Prod Primary Metab 6:465–502 (Second Edition)CrossRefGoogle Scholar
  17. Ma Y, Chen Q, Cui Y, Du L, Shi T, Xu Q, Ma Q, Xie X, Chen N (2018) Comparative genomic and genetic functional analysis of industrial L-leucine-and L-valine-producing Corynebacterium glutamicum strains. J Microbiol Biotechnol 28(11):1916–1927Google Scholar
  18. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7):621–628CrossRefGoogle Scholar
  19. Muffler A, Bettermann S, Haushalter M, Hörlein A, Neveling U, Schramm M, Sorgenfrei O (2002) Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose. J Biotechnol 98:255–268CrossRefGoogle Scholar
  20. Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480CrossRefGoogle Scholar
  21. Reimonn TM, Park SY, Agarabi CD, Brorson KA, Yoon S (2016) Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis. Biotechnol Prog 32:1163–1173CrossRefGoogle Scholar
  22. Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79:544–552CrossRefGoogle Scholar
  23. Solieri L, Dakal TC, Giudici P (2013) Next-generation sequencing and its potential impact on food microbial genomics. Ann Microbiol 63:21–37CrossRefGoogle Scholar
  24. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9):1105–1111CrossRefGoogle Scholar
  25. Wieschalka S, Blombach B, Eikmanns BJ (2012) Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol 94(2):449–459CrossRefGoogle Scholar
  26. Vogt M, Haas S, Klaffl S, Polen T, Eggeling L, Ooyen JV, Bott M (2014) Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metab Eng 22:40–52CrossRefGoogle Scholar
  27. Yang J, Yang S (2017) Comparative analysis of Corynebacterium glutamicum genomes: a new perspective for the industrial production of amino acids. BMC Genomics 18(1):940CrossRefGoogle Scholar
  28. Yin L, Hu X, Xu D, Ning J, Chen J, Wang X (2012) Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum. Metab Eng 14:542–550CrossRefGoogle Scholar
  29. Zhang H, Li Y, Wang C, Wang X (2018) Understanding the high l-valine production in Corynebacterium glutamicum VWB-1 using transcriptomics and proteomics. Sci Rep 8(1):3632CrossRefGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  1. 1.National and Local United Engineering Lab of Metabolic Control Fermentation TechnologyTianjin University of Science & TechnologyTianjinPeople’s Republic of China
  2. 2.Key Laboratory of Microbial Engineering of China Light IndustryTianjin University of Science & TechnologyTianjinPeople’s Republic of China
  3. 3.College of BiotechnologyTianjin University of Science & TechnologyTianjinPeople’s Republic of China

Personalised recommendations