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2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae

  • Biotechnological products and process engineering
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Abstract

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process.

Key Points

• A biological route of 2,3-dihydroxyisovalerate production was setup.

• Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae.

• Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell.

• 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation.

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References

  • Arfin SM, Ratzkin B, Umbarger HE (1969) The metabolism of valine and isoleucine in Escherichia coli. XVII. The role of induction in the derepression of acetohydroxy acid isomeroreductase. Biochem Biophys Res Commun 37:902–908

    Article  CAS  PubMed  Google Scholar 

  • Biebl H, Zeng AP, Menzel K, Deckwer WD (1998) Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae. Appl Microbiol Biotechnol 50:24–29

    Article  CAS  PubMed  Google Scholar 

  • Blombach B, Schreiner ME, Holátko J, Bartek T, Oldiges M, Eikmanns BJ (2007) L-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol 73(7):2079–2084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blomqvist K, Nikkola M, Lehtovaara P, Suihko M, Airaksinen U, Stråby K, Knowles J, Penttilä M (1993) Characterization of the genes of the 2, 3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes. J Bacteriol 175(5):1392–1404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bryant H, Dardonville C, Hodgkinson T, Hursthouse M, Malik KA (1998) Asymmetric synthesis of the left hand portion of the azinomycins. J Chem Soc Perk T 1(7):1249–1256

    Article  Google Scholar 

  • Chandresh T, Bhosale S, Ranade D (2006) Formation of succinic acid by Klebsiella pneumoniae MCM B-325 under aerobic and anaerobic conditions. J Microbiol Biotechnol 16(6):870–879

    Google Scholar 

  • Chen C, Wei D, Shi J, Wang M, Hao J (2014) Mechanism of 2, 3-butanediol stereoisomer formation in Klebsiella pneumoniae. Appl Microbiol Biotechnol 98(10):4603–4613

    Article  CAS  PubMed  Google Scholar 

  • Chunduru SK, Mrachko GT, Calvo KC (1989) Mechanism of ketol acid reductoisomerase - steady-state analysis and metal ion requirement. Biochem 28(2):486–493

    Article  CAS  Google Scholar 

  • Cioffi EA, Shaw KJ, Bailey WF, Berg CM (1980) Improved synthesis of the sodium salt of DL-α, β-dihydroxyisovaleric acid. Anal Biochem 104(2):485–488

    Article  CAS  PubMed  Google Scholar 

  • Dumas R, Biou V, Halgand F, Douce R, Duggleby RG (2001) Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase. Acc Chem Res 34(5):399–408

    Article  CAS  PubMed  Google Scholar 

  • Esener AA, Roels JA, Kossen NWF (1982) Dependence of elemental composition of K. pneumoniae on the steady-state specific growth rate. Biotechnol Bioeng 24:1445–1449

    Article  CAS  PubMed  Google Scholar 

  • Generoso WC, Brinek M, Dietz H, Oreb M, Boles E (2017) Secretion of 2, 3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae. FEMS Yeast Res 17(3)

  • Gu J, Zhou J, Zhang Z, Kim CH, Jiang B, Shi J, Hao J (2017) Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway. Metab Eng 43:71–84

    Article  CAS  PubMed  Google Scholar 

  • Guo X, Cao C, Wang Y, Li C, Wu M, Chen Y, Zhang C, Pei H, Xiao D (2014) Effect of the inactivation of lactate dehydrogenase, ethanol dehydrogenase, and phosphotransacetylase on 2, 3-butanediol production in Klebsiella pneumoniae strain. Biotechnol Biofuels 7(1):44

    Article  PubMed  PubMed Central  Google Scholar 

  • Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. P Natl Acad Sci USA 100(4):1541–1546

    Article  CAS  Google Scholar 

  • Hao J, Lin R, Zheng Z, Liu H, Liu D (2008) Isolation and characterization of microorganisms able to produce 1, 3-propanediol under aerobic conditions. World J Microbiol Biotechnol 24(9):1731–1740

    Article  CAS  Google Scholar 

  • Hill RK, Yan S-J (1971) Stereochemistry of valine and isoleucine biosynthesis II. Absolute configuration of (-)α,β-dihydroxyisovaleric acid and (-)α,β-dihydroxy-β-methylvaleric acid. Bioorg Chem 1(4):446–456

    Article  CAS  Google Scholar 

  • Holmes E, Foxall PJ, Spraul M, Farrant RD, Nicholson JK, Lindon JC (1997) 750 MHz 1H NMR spectroscopy characterisation of the complex metabolic pattern of urine from patients with inborn errors of metabolism: 2-hydroxyglutaric aciduria and maple syrup urine disease. J Pharm Biomed Anal 15(11):1647–1659

    Article  CAS  PubMed  Google Scholar 

  • Huang Q-G, Zeng B-D, Liang L, Wu S-G, Huang J-Z (2018) Genome shuffling and high-throughput screening of Brevibacterium flavum MDV1 for enhanced L-valine production. World J Microbiol Biotechnol 34(8):121

    Article  PubMed  Google Scholar 

  • Kosaric N, Magee R, Blaszczyk R (1992) Redox potential measurement for monitoring glucose and xylose conversion by K. pneumoniae. Chem Biochem Eng Q 6:145–151

    CAS  Google Scholar 

  • Lawther RP, Calhoun DH, Adams CW, Hauser CA, Gray J, Hatfield GW (1981) Molecular basis of valine resistance in Escherichia coli K-12. P Natl Acad Sci USA 78(2):922–925

    Article  CAS  Google Scholar 

  • Lawther RP, Wek RC, Lopes JM, Perira R, Taillon BE, Wesley G (1987) The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12. Nucleic Acids Res 15(5):2137–2155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ma C, Wang A, Qin J, Li L, Ai X, Jiang T, Tang H, Xu P (2009) Enhanced 2,3-butanediol production by Klebsiella pneumoniae SDM. Appl Microbiol Biotechnol 82:49–57

    Article  CAS  PubMed  Google Scholar 

  • Oh BR, Hong WK, Heo SY, Joe M, Seo JW, Kim CH (2013) The role of aldehyde/alcohol dehydrogenase (AdhE) in ethanol production from glycerol by Klebsiella pneumoniae. J Ind Microbiol Biotechnol 40:227–233

    Article  CAS  PubMed  Google Scholar 

  • Platko JV, Willins DA, Calvo JM (1990) The ilvIH operon of Escherichia coli is positively regulated. J Bacteriol 172(8):4563–4570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Priya A, Dureja P, Talukdar P, Rathi R, Lal B, Sarma PM (2016) Microbial production of 2,3-butanediol through a two-stage pH and agitation strategy in 150 l bioreactor. Biochem Eng J 105:159–167

    Article  CAS  Google Scholar 

  • Rathnasingh C, Park JM, Kim D, Song H, Chang YK (2016) Metabolic engineering of Klebsiella pneumoniae and in silico investigation for enhanced 2,3-butanediol production. Biotechnol Lett 38:975–982

    Article  CAS  PubMed  Google Scholar 

  • Silveira MM, Schmidell W, Berbert MA (1993) Effect of the air supply on the production of 2, 3-butanediol by Klebsiella pneumoniae NRRL B199. J Biotechnol 31(1):93–102

    Article  CAS  Google Scholar 

  • Sun Y, Wei D, Shi J, Mojović L, Han Z, Hao J (2014) Two-stage fermentation for 2-ketogluconic acid production by Klebsiella pneumoniae. J Microbiol Biotechnol 24(6):781–787

    Article  CAS  PubMed  Google Scholar 

  • Van Houdt R, Aertsen A, Michiels CW (2007) Quorum-sensing-dependent switch to butanediol fermentation prevents lethal medium acidification in Aeromonas hydrophila AH-1 N. Res Microbiol 158(4):379–385

    Article  PubMed  Google Scholar 

  • Wang D, Zhou J, Chen C, Wei D, Shi J, Jiang B, Liu P, Hao J (2015) R-acetoin accumulation and dissimilation in Klebsiella pneumoniae. J Ind Microbiol Biotechnol 42(8):1105–1115

    Article  CAS  PubMed  Google Scholar 

  • Wang D, Wang C, Wei D, Shi J, Kim CH, Jiang B, Han Z, Hao J (2016) Gluconic acid production by gad mutant of Klebsiella pneumoniae. World J Microbiol Biotechnol 32(8):132

    Article  PubMed  Google Scholar 

  • Wei D, Wang M, Shi J, Hao J (2012) Red recombinase assisted gene replacement in Klebsiella pneumoniae. J Ind Microbiol Biotechnol 39(8):1219–1226

    Article  CAS  PubMed  Google Scholar 

  • Wei D, Xu J, Sun J, Shi J, Hao J (2013) 2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366. J Ind Microbiol Biotechnol 40(6):561–570

    Article  CAS  PubMed  Google Scholar 

  • Xiao Z, Xu P (2007) Acetoin metabolism in bacteria. Crit Rev Microbiol 33:127–140

    Article  CAS  PubMed  Google Scholar 

  • Xu YZ, Guo NN, Zheng ZM, Ou XJ, Liu HJ, Liu DH (2009) Metabolism in 1, 3-propanediol fed-batch fermentation by ad-lactate deficient mutant of Klebsiella pneumoniae. Biotechnol Bioeng 104(5):965–972

    Article  CAS  PubMed  Google Scholar 

  • Zeng A-P, Biebl H, Deckwer W-D (1990) Effect of pH and acetic acid on growth and 2, 3-butanediol production of Enterobacter aerogenes in continuous culture. Appl Microbiol Biotechnol 33(5):485–489

    Article  CAS  Google Scholar 

  • Zhou J, Wang D, Wang C, Gu J, Kim CH, Shi J, Jiang B, Wang M, Hao J (2017) The role of the pyruvate acetyl-CoA switch in the production of 1, 3-propanediol by Klebsiella pneumoniae. Appl Biochem Biotechnol 181(3):1199–1210

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This study was funded by the National Key R&D Program of China (Grant No. 2017YFE0112700), Royal Society-Newton Advanced Fellowship (Grant No. NAF\R2\180721), Natural Science Foundation of Shanghai (Grant No. 19ZR1463600), and National Natural Science Foundation of China (Grant No. 21576279). FB would like to thank the Chinese Academy of Sciences for the award of a President’s International Fellowship Initiative (Grant No. 2019VCB0007).

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Author notes

  1. Yike Wang and Jinjie Gu contributed equally to this work.

Authors and Affiliations

  1. Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, 201210, Shanghai, People’s Republic of China

    Yike Wang, Jinjie Gu, Xiyang Lu, Zhongxi Zhang, Yang Yang, Shaoqi Sun, Jiping Shi & Jian Hao

  2. Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, UK

    Emily T. Kostas, Frank Baganz, Gary J. Lye & Jian Hao

  3. School of Life Science, Shanghai University, Shanghai, 200444, People’s Republic of China

    Yike Wang, Zhongxi Zhang, Yang Yang, Shaoqi Sun & Mintian Gao

  4. University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Yike Wang, Yang Yang & Shaoqi Sun

  5. School of life science and technology, ShanghaiTech University, Pudong, Shanghai, People’s Republic of China

    Jinjie Gu & Jiping Shi

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  1. Yike Wang
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Contributions

JH, GL, and FB designed this study. YW, JG, XL, ZZ, YY, and SS conducted the research. YW, JG, JP, MG, FB, GL, and JH analyzed the data. YW, EK, FB, and JH wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Frank Baganz, Gary J. Lye or Jian Hao.

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Wang, Y., Gu, J., Lu, X. et al. 2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae. Appl Microbiol Biotechnol 104, 6601–6613 (2020). https://doi.org/10.1007/s00253-020-10711-y

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