Advertisement

Journal of Industrial Microbiology & Biotechnology

, Volume 46, Issue 11, pp 1557–1568 | Cite as

Increasing l-threonine production in Escherichia coli by overexpressing the gene cluster phaCAB

  • Jianli Wang
  • Wenjian Ma
  • Yu Fang
  • Jun Yang
  • Jie Zhan
  • Shangwei Chen
  • Xiaoyuan WangEmail author
Bioenergy/Biofuels/Biochemicals - Original Paper
  • 188 Downloads

Abstract

l-Threonine is an important branched-chain amino acid and could be applied in feed, drugs, and food. In this study, l-threonine production in an l-threonine-producing Escherichia coli strain TWF001 was significantly increased by overexpressing the gene cluster phaCAB from Ralstonia eutropha. TWF001/pFW01-phaCAB could produce 96.4-g/L l-threonine in 3-L fermenter and 133.5-g/L l-threonine in 10-L fermenter, respectively. In addition, TWF001/pFW01-phaCAB produced 216% more acetyl-CoA, 43% more malate, and much less acetate than the vector control TWF001/pFW01, and meanwhile, TWF001/pFW01-phaCAB produced poly-3-hydroxybutyrate, while TWF001/pFW01 did not. Transcription analysis showed that the key genes in the l-threonine biosynthetic pathway were up-regulated, the genes relevant to the acetate formation were down-regulated, and the gene acs encoding the enzyme which converts acetate to acetyl-CoA was up-regulated. The results suggested that overexpression of the gene cluster phaCAB in E. coli benefits the enhancement of l-threonine production.

Keywords

l-Threonine production Escherichia coli Poly-3-hydroxybutyrate phaCAB Acetate 

Notes

Acknowledgements

This study was supported by the National Key R&D Program of China (2018YFA0900302), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-10), the post-graduate research and practice innovation program of Jiangsu Province (KYLX15_1141), and the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.

References

  1. 1.
    Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472PubMedPubMedCentralGoogle Scholar
  2. 2.
    Borchert AJ, Downs DM (2018) Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica. J Biol Chem 293:19240–19249.  https://doi.org/10.1074/jbc.RA118.005626 CrossRefPubMedGoogle Scholar
  3. 3.
    Chen D, Xu D, Li M, He J, Gong Y, Wu D, Sun M, Yu Z (2012) Proteomic analysis of Bacillus thuringiensis DeltaphaC mutant BMB171/PHB(-1) reveals that the PHB synthetic pathway warrants normal carbon metabolism. J Proteom 75:5176–5188.  https://doi.org/10.1016/j.jprot.2012.06.002 CrossRefGoogle Scholar
  4. 4.
    de Almeida A, Catone MV, Rhodius VA, Gross CA, Pettinari MJ (2011) Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli. Appl Environ Microbiol 77:6622–6629.  https://doi.org/10.1128/AEM.05469-11 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ding Z, Fang Y, Zhu L, Wang J, Wang X (2019) Deletion of arcA, iclR and tdcC in Escherichia coli to improve l-threonine production. Biotechnol Appl Biochem.  https://doi.org/10.1002/bab.1789 CrossRefPubMedGoogle Scholar
  6. 6.
    Dong X, Quinn PJ, Wang X (2011) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine. Biotechnol Adv 29:11–23.  https://doi.org/10.1016/j.biotechadv.2010.07.009 CrossRefPubMedGoogle Scholar
  7. 7.
    Dong X, Quinn PJ, Wang X (2012) Microbial metabolic engineering for l-threonine production. Subcell Biochem 64:283–302.  https://doi.org/10.1007/978-94-007-5055-5_14 CrossRefPubMedGoogle Scholar
  8. 8.
    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 43:873–885.  https://doi.org/10.1007/s10295-016-1763-5 CrossRefPubMedGoogle Scholar
  9. 9.
    Eggers J, Steinbuchel A (2014) Impact of Ralstonia eutropha’s poly(3-hydroxybutyrate) (PHB) depolymerases and phasins on PHB storage in recombinant Escherichia coli. Appl Environ Microbiol 80:7702–7709.  https://doi.org/10.1128/AEM.02666-14 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gu P, Kang J, Yang F, Wang Q, Liang Q, Qi Q (2013) The improved l-tryptophan production in recombinant Escherichia coli by expressing the polyhydroxybutyrate synthesis pathway. Appl Microbiol Biotechnol 97:4121–4127.  https://doi.org/10.1007/s00253-012-4665-0 CrossRefPubMedGoogle Scholar
  11. 11.
    Han MJ, Yoon SS, Lee SY (2001) Proteome analysis of metabolically engineered Escherichia coli producing poly(3-hydroxybutyrate). J Bacteriol 183:301–308.  https://doi.org/10.1128/JB.183.1.301-308.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kang Z, Du L, Kang J, Wang Y, Wang Q, Liang Q, Qi Q (2011) Production of succinate and polyhydroxyalkanoate from substrate mixture by metabolically engineered Escherichia coli. Bioresour Technol 102:6600–6604.  https://doi.org/10.1016/j.biortech.2011.03.070 CrossRefPubMedGoogle Scholar
  13. 13.
    Kang Z, Gao C, Wang Q, Liu H, Qi Q (2010) A novel strategy for succinate and polyhydroxybutyrate co-production in Escherichia coli. Bioresour Technol 101:7675–7678.  https://doi.org/10.1016/j.biortech.2010.04.084 CrossRefPubMedGoogle Scholar
  14. 14.
    Kőrös Á, Varga Z, Molnár-Perl I (2008) Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A 1203:146–152.  https://doi.org/10.1016/j.chroma.2008.07.035 CrossRefPubMedGoogle Scholar
  15. 15.
    Kruse D, Kramer R, Eggeling L, Rieping M, Pfefferle W, Tchieu JH, Chung YJ, Jr Saier MH, Burkovski A (2002) Influence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol 59:205–210.  https://doi.org/10.1007/s00253-002-0987-7 CrossRefPubMedGoogle Scholar
  16. 16.
    Lee JH, Lee DE, Lee BU, Kim HS (2003) Global analyses of transcriptomes and proteomes of a parent strain and an l-threonine-overproducing mutant strain. J Bacteriol 185:5442–5451.  https://doi.org/10.1128/JB.185.18.5442-5451.2003 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lee JH, Sung BH, Kim MS, Blattner FR, Yoon BH, Kim JH, Kim SC (2009) Metabolic engineering of a reduced-genome strain of Escherichia coli for l-threonine production. Microb Cell Fact 8:2.  https://doi.org/10.1186/1475-2859-8-2 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lee KH, Park JH, Kim TY, Kim HU, Lee SY (2007) Systems metabolic engineering of Escherichia coli for l-threonine production. Mol Syst Biol 3:149.  https://doi.org/10.1038/msb4100196 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lee MH, Lee HW, Park JH, Ahn JO, Jung JK, Hwang YI (2006) Improved l-threonine production of Escherichia coli mutant by optimization of culture conditions. J Biosci Bioeng 101:127–130.  https://doi.org/10.1263/jbb.101.127 CrossRefPubMedGoogle Scholar
  20. 20.
    Leong YK, Show PL, Ooi CW, Ling TC, Lan JC (2014) Current trends in polyhydroxyalkanoates (PHAs) biosynthesis: insights from the recombinant Escherichia coli. J Biotechnol 180:52–65.  https://doi.org/10.1016/j.jbiotec.2014.03.020 CrossRefPubMedGoogle Scholar
  21. 21.
    Lin JH, Lee MC, Sue YS, Liu YC, Li SY (2017) Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly(3-hydroxybutyrate) (PHB) production. Appl Microbiol Biotechnol 101:6419–6430.  https://doi.org/10.1007/s00253-017-8386-2 CrossRefPubMedGoogle Scholar
  22. 22.
    Lin Z, Zhang Y, Yuan Q, Liu Q, Li Y, Wang Z, Ma H, Chen T, Zhao X (2015) Metabolic engineering of Escherichia coli for poly(3-hydroxybutyrate) production via threonine bypass. Microb Cell Fact 14:185.  https://doi.org/10.1186/s12934-015-0369-3 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Liu J, Li H, Xiong H, Xie X, Chen N, Zhao G, Caiyin Q, Zhu H, Qiao J (2019) Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli. Biotechnol Bioeng 116:110–120.  https://doi.org/10.1002/bit.26844 CrossRefPubMedGoogle Scholar
  24. 24.
    Liu Q, Ouyang SP, Kim J, Chen GQ (2007) The impact of PHB accumulation on l-glutamate production by recombinant Corynebacterium glutamicum. J Biotechnol 132:273–279.  https://doi.org/10.1016/j.jbiotec.2007.03.014 CrossRefPubMedGoogle Scholar
  25. 25.
    Liu S, Liang Y, Liu Q, Tao T, Lai S, Chen N, Wen T (2013) Development of a two-stage feeding strategy based on the kind and level of feeding nutrients for improving fed-batch production of l-threonine by Escherichia coli. Appl Microbiol Biotechnol 97:573–583.  https://doi.org/10.1007/s00253-012-4317-4 CrossRefPubMedGoogle Scholar
  26. 26.
    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 Fact 14:121.  https://doi.org/10.1186/s12934-015-0311-8 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408.  https://doi.org/10.1006/meth.2001.1262 CrossRefGoogle Scholar
  28. 28.
    Livshits VA, Zakataeva NP, Aleshin VV, Vitushkina MV (2003) Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. Res Microbiol 154:123–135.  https://doi.org/10.1016/S0923-2508(03)00036-6 CrossRefPubMedGoogle Scholar
  29. 29.
    Ma W, Wang J, Li Y, Yin L, Wang X (2018) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-produced with l-isoleucine in Corynebacterium glutamicum WM001. Microb Cell Fact 17:93.  https://doi.org/10.1186/s12934-018-0942-7 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Mahishi LH, Tripathi G, Rawal SK (2003) Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources. Microbiol Res 158:19–27.  https://doi.org/10.1078/0944-5013-00161 CrossRefPubMedGoogle Scholar
  31. 31.
    Nolden L, Farwick M, Krämer R, Burkovski A (2001) Glutamine synthetases of Corynebacterium glutamicum: transcriptional control and regulation of activity. FEMS Microbiol Lett 201:91–98.  https://doi.org/10.1111/j.1574-6968.2001.tb10738.x CrossRefPubMedGoogle Scholar
  32. 32.
    Posfai G, Plunkett G 3rd, Feher T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, Blattner FR (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044–1046.  https://doi.org/10.1126/science.1126439 CrossRefPubMedGoogle Scholar
  33. 33.
    Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80CrossRefGoogle Scholar
  34. 34.
    Su Y, Guo QQ, Wang S, Zhang X, Wang J (2018) Effects of betaine supplementation on l-threonine fed-batch fermentation by Escherichia coli. Bioprocess Biosyst Eng 41:1509–1518.  https://doi.org/10.1007/s00449-018-1978-0 CrossRefPubMedGoogle Scholar
  35. 35.
    Tyo KE, Fischer CR, Simeon F, Stephanopoulos G (2010) Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations. Metab Eng 12:187–195.  https://doi.org/10.1016/j.ymben.2009.10.005 CrossRefPubMedGoogle Scholar
  36. 36.
    Wang J, Cheng LK, Chen N (2014) High-level production of l-threonine by recombinant Escherichia coli with combined feeding strategies. Biotechnol Biotechnol Equip 28:495–501.  https://doi.org/10.1080/13102818.2014.927682 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wang Q, Zhuang Q, Liang Q, Qi Q (2013) Polyhydroxyalkanoic acids from structurally-unrelated carbon sources in Escherichia coli. Appl Microbiol Biotechnol 97:3301–3307.  https://doi.org/10.1007/s00253-013-4809-x CrossRefPubMedGoogle Scholar
  38. 38.
    Wang RY, Shi ZY, Chen JC, Wu Q, Chen GQ (2012) Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase. Metab Eng 14:496–503.  https://doi.org/10.1016/j.ymben.2012.07.003 CrossRefPubMedGoogle Scholar
  39. 39.
    Xie X, Liang Y, Liu H, Liu Y, Xu Q, Zhang C, Chen N (2014) Modification of glycolysis and its effect on the production of l-threonine in Escherichia coli. J Ind Microbiol Biotechnol 41:1007–1015.  https://doi.org/10.1007/s10295-014-1436-1 CrossRefPubMedGoogle Scholar
  40. 40.
    Xu M, Qin J, Rao Z, You H, Zhang X, Yang T, Wang X, Xu Z (2016) Effect of polyhydroxybutyrate (PHB) storage on l-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation. Microb Cell Fact 15:15.  https://doi.org/10.1186/s12934-016-0414-x CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Yang J, Fang Y, Wang J, Wang C, Zhao L, Wang X (2019) Deletion of regulator-encoding genes fadR, fabR and iclR to increase l-threonine production in Escherichia coli. Appl Microbiol Biotechnol 103:4549–4564.  https://doi.org/10.1007/s00253-019-09818-8 CrossRefPubMedGoogle Scholar
  42. 42.
    Yuzbashev TV, Vybornaya TV, Larina AS, Gvilava IT, Voyushina NE, Mokrova SS, Yuzbasheva EY, Manukhov IV, Sineoky SP, Debabov VG (2013) Directed modification of Escherichia coli metabolism for the design of threonine-producing strains. Appl Biochem Microbiol 49:723–742.  https://doi.org/10.1134/S0003683813090056 CrossRefGoogle Scholar
  43. 43.
    Zakataeva NP, Aleshin VV, Tokmakova IL, Troshin PV, Livshits VA (1999) The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. FEBS Lett 452:228–232CrossRefGoogle Scholar
  44. 44.
    Zhang X, Zhang J, Xu J, Zhao Q, Wang Q, Qi Q (2018) Engineering Escherichia coli for efficient coproduction of polyhydroxyalkanoates and 5-aminolevulinic acid. J Ind Microbiol Biotechnol 45:43–51.  https://doi.org/10.1007/s10295-017-1990-4 CrossRefPubMedGoogle Scholar
  45. 45.
    Zhang Y, Meng Q, Ma H, Liu Y, Cao G, Zhang X, Zheng P, Sun J, Zhang D, Jiang W, Ma Y (2015) Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis. Microb Cell Fact 14:86.  https://doi.org/10.1186/s12934-015-0275-8 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Zhao H, Fang Y, Wang X, Zhao L, Wang J, Li Y (2018) Increasing l-threonine production in Escherichia coli by engineering the glyoxylate shunt and the l-threonine biosynthesis pathway. Appl Microbiol Biotechnol 102:5505–5518.  https://doi.org/10.1007/s00253-018-9024-3 CrossRefPubMedGoogle Scholar
  47. 47.
    Zhuang Q, Wang Q, Liang Q, Qi Q (2014) Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid beta-oxidation cycle in Escherichia coli. Metab Eng 24:78–86.  https://doi.org/10.1016/j.ymben.2014.05.004 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  • Jianli Wang
    • 1
    • 2
  • Wenjian Ma
    • 1
    • 2
  • Yu Fang
    • 1
    • 2
  • Jun Yang
    • 1
    • 3
  • Jie Zhan
    • 1
    • 2
  • Shangwei Chen
    • 1
  • Xiaoyuan Wang
    • 1
    • 2
    • 3
    Email author
  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina
  3. 3.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

Personalised recommendations