Advertisement

Applied Microbiology and Biotechnology

, Volume 102, Issue 9, pp 4117–4130 | Cite as

Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum

  • Liang Wei
  • Ning Xu
  • Yiran Wang
  • Wei Zhou
  • Guoqiang Han
  • Yanhe Ma
  • Jun Liu
Applied genetics and molecular biotechnology

Abstract

Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative − 10 (NNTANANT) and − 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.

Keywords

Promoter library Multi-module combination technology Optimization of multi-gene pathway Threonine biosynthesis C. glutamicum 

Notes

Acknowledgements

We are grateful to Prof. Masayuki Inui (Research Institute of Innovative Technology for the Earth, Japan) for generously providing strains and plasmids. This study was supported by the National Natural Science Foundation of China (Nos. 31500044 and 31601460), the Natural Science Foundation of Tianjin (Nos. 17JCQNJC09600 and 17JCYBJC24000), and “Hundred Talents Program” of the Chinese Academy of Sciences.

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.

Supplementary material

253_2018_8911_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1220 kb)

References

  1. Ajikumar PK, Xiao WH, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330(6000):70–74.  https://doi.org/10.1126/science.1191652 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 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(2):159–168.  https://doi.org/10.1016/j.ymben.2011.01.003. CrossRefPubMedGoogle Scholar
  3. Biggs BW, De Paepe B, Santos CN, De Mey M, Kumaran Ajikumar P (2014) Multivariate modular metabolic engineering for pathway and strain optimization. Curr Opin Biotechnol 29:156–162.  https://doi.org/10.1016/j.copbio.2014.05.005 CrossRefPubMedGoogle Scholar
  4. Reinscheid DJ, Kronemeyer W, Eggeling L, Eikmanns BJ, Sahm H (1994) Stable expression of hom-1-thrB in Corynebacterium glutamicum and its effect on the carbon flux to threonine and related amino acids. Appl Environ Microbiol 60:126–132PubMedPubMedCentralGoogle Scholar
  5. Dong X, Zhao Y, Zhao J, Wang X (2016) Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis. J Ind Microbiol Biotechnol 43(6):873–885.  https://doi.org/10.1007/s10295-016-1763-5 CrossRefPubMedGoogle Scholar
  6. Dong XY, Quinn PJ, Wang XY (2011) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of L-threonine. Biotechnol Adv 29(1):11–23.  https://doi.org/10.1016/j.biotechadv.2010.07.009 CrossRefPubMedGoogle Scholar
  7. Du J, Yuan YB, Si T, Lian JZ, Zhao HM (2012) Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Res 40(18):e142.  https://doi.org/10.1093/nar/gks549 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Eikmanns BJ, Metzger M, Reinscheid D, Kircher M, Sahm H (1991) Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol 34(5):617–622.  https://doi.org/10.1007/BF00167910 CrossRefPubMedGoogle Scholar
  9. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol 3(11):839–843.  https://doi.org/10.1021/sb4001504 CrossRefPubMedGoogle Scholar
  10. Feng L, Zhang Y, Fu J, Mao Y, Chen T, Zhao X, Wang Z (2016) Metabolic engineering of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid. Biotechnol Bioeng 113(6):1284–1293.  https://doi.org/10.1002/bit.25886 CrossRefPubMedGoogle Scholar
  11. Islam ZU, Klein M, Aßkamp MR, ASR Ø, Nevoigt E (2017) A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae. Metab Eng doi:  https://doi.org/10.1016/j.ymben.2017.10.002
  12. Israelsen H, Madsen SM, Vrang A, Hansen EB, Johansen E (1995) Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol 61(7):2540–2547PubMedPubMedCentralGoogle Scholar
  13. Jeschek M, Gerngross D, Panke S (2017) Combinatorial pathway optimization for streamlined metabolic engineering. Curr Opin Biotechnol 47:142–151.  https://doi.org/10.1016/j.copbio.2017.06.014 CrossRefPubMedGoogle Scholar
  14. Juminaga D, Baidoo EE, Reddingjohanson AM, Batth TS, Burd H, Mukhopadhyay A, Petzold CJ, Keasling JD (2012) Modular engineering of L-tyrosine production in Escherichia coli. Appl Environ Microbiol 78(1):89–98.  https://doi.org/10.1128/AEM.06017-11 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kanagawa T (2003) Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng 96(4):317–323.  https://doi.org/10.1016/S1389-1723(03)90130-7 CrossRefPubMedGoogle Scholar
  16. Keasling JD (2012) Synthetic biology and the development of tools for metabolic engineering. Metab Eng 14(3):189–195.  https://doi.org/10.1016/j.ymben.2012.01.004 CrossRefPubMedGoogle Scholar
  17. Kim HI, Kim JH, Park YJ (2016) Transcriptome and gene ontology (GO) enrichment analysis reveals genes involved in biotin metabolism that affect l-lysine production in Corynebacterium glutamicum. Int J Mol Sci 17(3):353.  https://doi.org/10.3390/ijms17030353 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kirchner O, Tauch A (2003) Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol 104(1–3):287–299.  https://doi.org/10.1016/s0168-1656(03)00148-2 CrossRefPubMedGoogle Scholar
  19. Lee JH, Jung SC, Bui LM, Kang KH, Song JJ, Kim SC (2013) Improved production of L-threonine in Escherichia coli by use of a DNA scaffold system. Appl Environ Microbiol 79(3):774–782.  https://doi.org/10.1128/aem.02578-12 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li M, Borodina I (2015) Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae. FEMS Yeast Res 15(1):1–12.  https://doi.org/10.1111/1567-1364.12213 CrossRefPubMedGoogle Scholar
  21. Liu Y, Li Q, Zheng P, Zhang Z, Liu Y, Sun C, Cao G, Zhou W, Wang X, Zhang D, Zhang T, Sun J, Ma Y (2015) Developing a high-throughput screening method for threonine overproduction based on an artificial promoter. Microb Cell Factories 14:121.  https://doi.org/10.1186/s12934-015-0311-8 CrossRefGoogle Scholar
  22. Lo TM, Teo WS, Ling H, Chen B, Kang A, Chang MW (2013) Microbial engineering strategies to improve cell viability for biochemical production. Biotechnol Adv 31(6):903–914.  https://doi.org/10.1016/j.biotechadv.2013.02.001 CrossRefPubMedGoogle Scholar
  23. Mahr R, Gatgens C, Gatgens J, Polen T, Kalinowski J, Frunzke J (2015) Biosensor-driven adaptive laboratory evolution of L-valine production in Corynebacterium glutamicum. Metab Eng 32:184–194.  https://doi.org/10.1016/j.ymben.2015.09.017 CrossRefPubMedGoogle Scholar
  24. Mao Y, Fu J, Tao R, Huang C, Wang Z, Tang YJ, Chen T, Zhao X (2017) Systematic metabolic engineering of Corynebacterium glutamicum for industrial-level production of optically pure D-(−)-acetoin. Green Chem 19:5691–5702.  https://doi.org/10.1039/C7GC02753B CrossRefGoogle Scholar
  25. Nowroozi FF, Baidoo EE, Ermakov S, Redding-Johanson AM, Batth TS, Petzold CJ, Keasling JD (2014) Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly. Appl Microbiol Biotechnol 98(4):1567–1581.  https://doi.org/10.1007/s00253-013-5361-4 CrossRefPubMedGoogle Scholar
  26. Okibe N, Suzuki N, Inui M, Yukawa H (2010) Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum. Microbiology 156(Pt 12):3609–3623.  https://doi.org/10.1099/mic.0.043745-0 CrossRefPubMedGoogle Scholar
  27. Okibe N, Suzuki N, Inui M, Yukawa H (2011) Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid. J Microbiol Methods 85(2):155–163.  https://doi.org/10.1016/j.mimet.2011.02.012 CrossRefPubMedGoogle Scholar
  28. Pátek M, Nešvera J, Guyonvarch A, Reyes O, Leblon G (2003) Promoters of Corynebacterium glutamicum. J Biotechnol 104(1–3):311–323.  https://doi.org/10.1016/s0168-1656(03)00155-x CrossRefPubMedGoogle Scholar
  29. Patek M, Holatko J, Busche T, Kalinowski J, Nesvera J (2013) Corynebacterium glutamicum promoters: a practical approach. Microb Biotechnol 6(2):103–117.  https://doi.org/10.1111/1751-7915.12019 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Patek M, Nesvera J (2011) Sigma factors and promoters in Corynebacterium glutamicum. J Biotechnol 154(2–3):101–113.  https://doi.org/10.1016/j.jbiotec.2011.01.017 CrossRefPubMedGoogle Scholar
  31. Peccoud J, Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6(2):e16765.  https://doi.org/10.1371/journal.pone.0016765 CrossRefGoogle Scholar
  32. Rytter JV, Helmark S, Chen J, Lezyk MJ, Solem C, Jensen PR (2014) Synthetic promoter libraries for Corynebacterium glutamicum. Appl Microbiol Biotechnol 98(6):2617–2623.  https://doi.org/10.1007/s00253-013-5481-x CrossRefPubMedGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  34. Sang-Woo L, Min-Kyu O (2015) A synthetic suicide riboswitch for the high-throughput screening of metabolite production in Saccharomyces cerevisiae. Metab Eng 28:143–150.  https://doi.org/10.1016/j.ymben.2015.01.004 CrossRefGoogle Scholar
  35. Tauch A, Kirchner O, Loffler B, Gotker S, Puhler A, Kalinowski J (2002) Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr Microbiol 45(5):362–367.  https://doi.org/10.1007/s00284-002-3728-3 CrossRefPubMedGoogle Scholar
  36. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S (2012) Fast track assembly of multigene constructs using golden gate cloning and the MoClo system. Bioeng Bugs 3(1):38–43.  https://doi.org/10.4161/bbug.3.1.18223 PubMedGoogle Scholar
  37. Xu P, Gu Q, Wang WY, Wong L, Bower AGW, Collins CH, Koffas MAG (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun 4:1409.  https://doi.org/10.1038/ncomms2425 CrossRefPubMedGoogle Scholar
  38. Xu P, Vansiri A, Bhan N, Koffas MA (2012) ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli. ACS Synth Biol 1(7):256–266.  https://doi.org/10.1021/sb300016b CrossRefPubMedGoogle Scholar
  39. Yang TT, Cheng L, Kain SR (1996) Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 24(22):4592–4593.  https://doi.org/10.1093/nar/24.22.4592 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Yim SS, An SJ, Kang M, Lee J, Jeong KJ (2013) Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol Bioeng 110(11):2959–2969.  https://doi.org/10.1002/bit.24954 CrossRefPubMedGoogle Scholar
  41. Zhang B, Zhou N, Liu YM, Liu C, Lou CB, Jiang CY, Liu SJ (2015a) Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum. Microb Cell Factories 14:14.  https://doi.org/10.1186/s12934-015-0254-0 CrossRefGoogle Scholar
  42. Zhang C, Zhang J, Kang Z, Du G, Chen J (2015b) Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 42(5):787–797.  https://doi.org/10.1007/s10295-015-1593-x CrossRefPubMedGoogle Scholar
  43. Zhang Y, Cai J, Shang X, Wang B, Liu S, Chai X, Tan T, Zhang Y, Wen T (2017) A new genome-scale metabolic model of Corynebacterium glutamicum and its application. Biotechnol Biofuels 10:169.  https://doi.org/10.1186/s13068-017-0856-3 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zhang Y, Meng Q, Ma H, Liu Y, Cao G, Zhang X, Zheng P, Sun J, Zhang D, Jiang W, Ma Y (2015c) Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis. Microb Cell Factories 14:86.  https://doi.org/10.1186/s12934-015-0275-8 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Liang Wei
    • 1
    • 2
    • 3
  • Ning Xu
    • 1
    • 2
  • Yiran Wang
    • 4
  • Wei Zhou
    • 1
    • 2
  • Guoqiang Han
    • 5
  • Yanhe Ma
    • 1
    • 2
  • Jun Liu
    • 1
    • 2
  1. 1.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  2. 2.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.Key Laboratory of Food Nutrition and Safety, Ministry of EducationTianjin University of Science and TechnologyTianjinChina
  5. 5.School of Life Science and BiotechnologyYangtze Normal UniversityChongqingChina

Personalised recommendations