A synthetic pathway for the production of 2-hydroxyisovaleric acid in Escherichia coli

Abstract

Synthetic biology, encompassing the design and construction of novel artificial biological pathways and organisms and the redesign of existing natural biological systems, is rapidly expanding the number of applications for which biological systems can play an integral role. In the context of chemical production, the combination of synthetic biology and metabolic engineering approaches continues to unlock the ability to biologically produce novel and complex molecules from a variety of feedstocks. Here, we utilize a synthetic approach to design and build a pathway to produce 2-hydroxyisovaleric acid in Escherichia coli and demonstrate how pathway design can be supplemented with metabolic engineering approaches to improve pathway performance from various carbon sources. Drawing inspiration from the native pathway for the synthesis of the 5-carbon amino acid l-valine, we exploit the decarboxylative condensation of two molecules of pyruvate, with subsequent reduction and dehydration reactions enabling the synthesis of 2-hydroxyisovaleric acid. Key to our approach was the utilization of an acetolactate synthase which minimized kinetic and regulatory constraints to ensure sufficient flux entering the pathway. Critical host modifications enabling maximum product synthesis from either glycerol or glucose were then examined, with the varying degree of reduction of these carbons sources playing a major role in the required host background. Through these engineering efforts, the designed pathway produced 6.2 g/L 2-hydroxyisovaleric acid from glycerol at 58% of maximum theoretical yield and 7.8 g/L 2-hydroxyisovaleric acid from glucose at 73% of maximum theoretical yield. These results demonstrate how the combination of synthetic biology and metabolic engineering approaches can facilitate bio-based chemical production.

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References

  1. 1.

    Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89. https://doi.org/10.1038/nature06450

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Atsumi S, Li Z, Liao JC (2009) Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli. Appl Environ Microbiol 75:6306–6311. https://doi.org/10.1128/aem.01160-09

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. 3.

    Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. https://doi.org/10.1038/msb4100050

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Ze Barak, Chipman DM (2012) Allosteric regulation in acetohydroxyacid synthases (AHASs)—different structures and kinetic behavior in isozymes in the same organisms. Arch Biochem Biophys 519:167–174. https://doi.org/10.1016/j.abb.2011.11.025

    Article  CAS  Google Scholar 

  5. 5.

    Burk MJ, Van Dien S (2016) Biotechnology for chemical production: challenges and opportunities. Trends Biotechnol 34:187–190. https://doi.org/10.1016/j.tibtech.2015.10.007

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Chambellon E, Rijnen L, Lorquet F, Gitton C, van Hylckama Vlieg JET, Wouters JA, Yvon M (2009) The D-2-Hydroxyacid dehydrogenase incorrectly annotated PanE is the sole reduction system for branched-chain 2-keto acids in Lactococcus lactis. J Bacteriol 191:873–881. https://doi.org/10.1128/jb.01114-08

    Article  PubMed  CAS  Google Scholar 

  7. 7.

    Cheong S, Clomburg JM, Gonzalez R (2016) Energy- and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions. Nat Biotechnol 34:556–561. https://doi.org/10.1038/nbt.3505

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Choi SY, Park SJ, Kim WJ, Yang JE, Lee H, Shin J, Lee SY (2016) One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli. Nat Biotechnol 34:435–440. https://doi.org/10.1038/nbt.3485

    Article  PubMed  CAS  Google Scholar 

  9. 9.

    Clomburg JM, Crumbley AM, Gonzalez R (2017) Industrial biomanufacturing: the future of chemical production. Science. https://doi.org/10.1126/science.aag0804

    PubMed  Article  Google Scholar 

  10. 10.

    Clomburg JM, Gonzalez R (2013) Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol 31:20–28. https://doi.org/10.1016/j.tibtech.2012.10.006

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    Clomburg JM, Vick JE, Blankschien MD, Rodriguez-Moya M, Gonzalez R (2012) A synthetic biology approach to engineer a functional reversal of the β-oxidation cycle. ACS Synth Biol 1:541–554. https://doi.org/10.1021/sb3000782

    Article  PubMed  CAS  Google Scholar 

  12. 12.

    Cohen-Arazi N, Katzhendler J, Kolitz M, Domb AJ (2008) Preparation of new α-hydroxy acids derived from amino acids and their corresponding polyesters. Macromolecules 41:7259–7263. https://doi.org/10.1021/ma8012477

    Article  CAS  Google Scholar 

  13. 13.

    Cordova LT, Alper HS (2016) Central metabolic nodes for diverse biochemical production. Curr Opin Chem Biol 35:37–42. https://doi.org/10.1016/j.cbpa.2016.08.025

    Article  PubMed  CAS  Google Scholar 

  14. 14.

    Cornils B, Fischer RW, Kohlpaintner C (2000) Butanals. In: Ullmann’s encyclopedia of industrial chemistry. Wiley-VCH Verlag GmbH & Co. KGaA. https://doi.org/10.1002/14356007.a04_447

  15. 15.

    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645. https://doi.org/10.1073/pnas.120163297

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    Dharmadi Y, Murarka A, Gonzalez R (2006) Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng 94:821–829. https://doi.org/10.1002/bit.21025

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Durnin G, Clomburg J, Yeates Z, Alvarez PJJ, Zygourakis K, Campbell P, Gonzalez R (2009) Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 103:148–161. https://doi.org/10.1002/bit.22246

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Erickson B, Nelson Winters P (2012) Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol J 7:176–185. https://doi.org/10.1002/biot.201100069

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Felice MD, Guardiola J, Esposito B, Iaccarino M (1974) Structural genes for a newly recognized acetolactate synthase in Escherichia coli K-12. J Bacteriol 120:1068–1077

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Fessenden RJ, Fesenden JS, Logue MW (1998) Organic chemistry, 6th edn. Brooks/Cole Publishing Company, Pacific Grove

    Google Scholar 

  21. 21.

    Furukawa N, Miyanaga A, Togawa M, Nakajima M, Taguchi H (2014) Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria. AMB Express 4:76. https://doi.org/10.1186/s13568-014-0076-1

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. 22.

    Gollop N, Damri B, Ze Barak, Chipman DM (1989) Kinetics and mechanism of acetohydroxy acid synthase isozyme III from Escherichia coli. Biochemistry 28:6310–6317. https://doi.org/10.1021/bi00441a024

    Article  PubMed  CAS  Google Scholar 

  23. 23.

    Gollop N, Damri B, Chipman DM, Barak Z (1990) Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms. J Bacteriol 172:3444–3449

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Guardiola J, De Felice M, Lamberti A, Iaccarino M (1977) The acetolactate synthase isoenzymes of Escherichia coli K-12. Mol Gen Genet 156:17–25. https://doi.org/10.1007/bf00272247

    Article  PubMed  CAS  Google Scholar 

  25. 25.

    Holtzclaw WD, Chapman LF (1975) Degradative acetolactate synthase of Bacillus subtilis: purification and properties. J Bacteriol 121:917–922

    PubMed  PubMed Central  CAS  Google Scholar 

  26. 26.

    Iwakura Y, Iwata K, Matsuo S, Tohara A (1971) Synthesis of optically active poly(L-α-hydroxyisovalerate) and poly(L-α-hydroxyisocaproate). Die Makromolekulare Chemie 146:21–32. https://doi.org/10.1002/macp.1971.021460103

    Article  CAS  Google Scholar 

  27. 27.

    Jiang GR, Nikolova S, Clark DP (2001) Regulation of the ldhA gene, encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 147:2437–2446. https://doi.org/10.1099/00221287-147-9-2437

    Article  PubMed  CAS  Google Scholar 

  28. 28.

    Kang YS, Durfee T, Glasner JD, Qiu Y, Frisch D, Winterberg KM, Blattner F (2004) Systematic mutagenesis of the Escherichia coli genome. J Bacteriol 186:4921–4930. https://doi.org/10.1128/jb.186.15.1921-1930.2004

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Kim S, Clomburg J, Gonzalez R (2015) Synthesis of medium-chain length (C6–C10) fuels and chemicals via β-oxidation reversal in Escherichia coli. J Ind Microbiol Biotechnol 42:465–475. https://doi.org/10.1007/s10295-015-1589-6

    Article  PubMed  CAS  Google Scholar 

  30. 30.

    Klingler FD, Ebertz W (2000) Oxocarboxylic acids. In: Ullmann’s encyclopedia of industrial chemistry. Wiley-VCH Verlag GmbH & Co. KGaA. https://doi.org/10.1002/14356007.a18_313

  31. 31.

    Lee JW, Na D, Park JM, Lee J, Choi S, Lee SY (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol 8:536–546

    Article  PubMed  CAS  Google Scholar 

  32. 32.

    Marubayashi H, Nojima S (2016) Crystallization and solid-state structure of poly(l-2-hydroxy-3-methylbutanoic acid). Macromolecules 49:5538–5547. https://doi.org/10.1021/acs.macromol.5b02774

    Article  CAS  Google Scholar 

  33. 33.

    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  34. 34.

    Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 74:1124–1135. https://doi.org/10.1128/aem.02192-07

    Article  PubMed  CAS  Google Scholar 

  35. 35.

    Myers JW (1961) Dihydroxy acid dehydrase: an enzyme involved in the biosynthesis of isoleucine and valine. J Biol Chem 236:1414–1418

    PubMed  CAS  Google Scholar 

  36. 36.

    Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747

    PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

    Neidhardt FC, Curtiss R (1996) Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, D.C.

    Google Scholar 

  38. 38.

    Ng C, M-y Jung, Lee J, Oh M-K (2012) Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering. Microb Cell Fact 11:68. https://doi.org/10.1186/1475-2859-11-68

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. 39.

    Nielsen J, Keasling Jay D (2016) Engineering cellular metabolism. Cell 164:1185–1197. https://doi.org/10.1016/j.cell.2016.02.004

    Article  PubMed  CAS  Google Scholar 

  40. 40.

    Nielsen J, Villadsen J, Liden G (2003) Bioreaction engineering principles. Kluwer Academic/Plenum Publishers, New York

    Google Scholar 

  41. 41.

    Nwaukwa SO, Keehn PM (1982) Oxidative cleavage of α-diols, α-diones, α-hydroxy-ketones and α-hydroxy- and α-keto acids with calcium hypochlorite [Ca(OCl)2]. Tetrahedron Lett 23:3135–3138. https://doi.org/10.1016/S0040-4039(00)88578-0

    Article  CAS  Google Scholar 

  42. 42.

    Ramakrishnan T, Adelberg EA (1965) Regulatory mechanisms in the biosynthesis of isoleucine and valine II. Identification of two operator genes. J Bacteriol 89:654–660

    PubMed  PubMed Central  CAS  Google Scholar 

  43. 43.

    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  44. 44.

    Sawers R, Blokesch M, Böck A (2004) Anaerobic formate and hydrogen metabolism. EcoSal Plus. https://doi.org/10.1128/ecosalplus.3.5.4

    Article  PubMed  Google Scholar 

  45. 45.

    Sawers R, Clark D (2004) Fermentative pyruvate and acetyl-coenzyme A metabolism. EcoSal Plus. https://doi.org/10.1128/ecosalplus.3.5.3

    Article  PubMed  Google Scholar 

  46. 46.

    Shiue E, Prather KLJ (2012) Synthetic biology devices as tools for metabolic engineering. Biochem Eng J 65:82–89. https://doi.org/10.1016/j.bej.2012.04.006

    Article  CAS  Google Scholar 

  47. 47.

    Tsuji H, Hayakawa T (2016) Heterostereocomplex- and homocrystallization and thermal properties and degradation of substituted poly(lactic acid)s, poly(l-2-hydroxybutanoic acid) and poly(d-2-hydroxy-3-methylbutanoic acid). Macromol Chem Phys 217:2483–2493. https://doi.org/10.1002/macp.201600359

    Article  CAS  Google Scholar 

  48. 48.

    Umbarger HE, Brown B, Eyring EJ (1960) Isoleucine and valine metabolism in Escherichia coli: IX. Utilization of acetolactate and acetohydroxybutyrate. J Biol Chem 235:1425–1432

    PubMed  CAS  Google Scholar 

  49. 49.

    Vick JE, Clomburg JM, Blankschien MD, Chou A, Kim S, Gonzalez R (2015) Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of the β-oxidation cycle. Appl Environ Microbiol 81:1406–1416. https://doi.org/10.1128/aem.03521-14

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Yang JE, Kim JW, Oh YH, Choi SY, Lee H, Park AR, Shin J, Park SJ, Lee SY (2016) Biosynthesis of poly(2-hydroxyisovalerate-co-lactate) by metabolically engineered Escherichia coli. Biotechnol J 11:1572–1585. https://doi.org/10.1002/biot.201600420

    Article  PubMed  CAS  Google Scholar 

  51. 51.

    Yazdani SS, Gonzalez R (2008) Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products. Metab Eng 10:340–351. https://doi.org/10.1016/j.ymben.2008.08.005

    Article  CAS  Google Scholar 

  52. 52.

    Zheng R, Blanchard JS (2003) Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase. Biochemistry 42:11289–11296. https://doi.org/10.1021/bi030101k

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported by Grants from the U.S. National Science Foundation (EEC-0813570, CBET-1134541, and CBET-1067565).

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Correspondence to Ramon Gonzalez.

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Cheong, S., Clomburg, J.M. & Gonzalez, R. A synthetic pathway for the production of 2-hydroxyisovaleric acid in Escherichia coli. J Ind Microbiol Biotechnol 45, 579–588 (2018). https://doi.org/10.1007/s10295-018-2005-9

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Keywords

  • Synthetic biology
  • Metabolic engineering
  • Fuels and chemicals
  • 2-Hydroxyisovaleric acid