A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1

Abstract

Convergence of market drivers such as abundant availability of inexpensive natural gas and increasing awareness of its global warming effects have created new opportunities for the development of small-scale gas-to-liquid (GTL) conversion technologies that can efficiently utilize methane, the primary component of natural gas. Leveraging the unique ability of methanotrophs that use methane as carbon and energy source, biological GTL platforms can be envisioned that are readily deployable at remote petroleum drilling sites where large chemical GTL infrastructure is uneconomical to set-up. Methylomicrobium buryatense, an obligate methanotroph, has gained traction as a potential industrial methanotrophic host because of availability of genetic tools and recent advances in its metabolic engineering. However, progress is impeded by low strain performance and lack of an industrial medium. In this study, we first established a small-scale cultivation platform using Hungate tubes for growth of M. buryatense at medium-to-high-throughput that also enabled 2X faster growth compared to that obtained in traditional glass serum bottles. Then, employing a synthetic biology approach we engineered M. buryatense with varying promoter (inducible and constitutive) and ribosome-binding site combinations, and obtained a strain capable of producing l-lactate from methane at a flux 14-fold higher than previously reported. Finally, we demonstrated l-lactate production in an industrial medium by replacing nitrate with less-expensive ammonium as the nitrogen source. Under these conditions, l-lactate was synthesized at a flux approximately 50-fold higher than that reported previously in a bioreactor system while achieving a titer of 0.6 g/L. These findings position M. buryatense closer to becoming an industrial host strain of choice, and pave new avenues for accelerating methane-to-chemical conversion using synthetic biology.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. 1.

    Altaf M, Naveena BJ, Reddy G (2007) Use of inexpensive nitrogen sources and starch for L(+) lactic acid production in anaerobic submerged fermentation. Bioresour Technol 98:498–503

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Anthony C (1982) The biochemistry of methylotrophs. Academic Press, New York

    Google Scholar 

  3. 3.

    Bothe H, Møller Jensen K, Mergel A, Larsen J, Jørgensen C, Jørgensen L (2002) Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process. Appl Microbiol Biotechnol 59:33–39

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Brandt AR, Heath GA, Kort EA, O’Sullivan F, Pétron G, Jordaan SM, Tans P, Wilcox J, Gopstein AM, Arent D, Wofsy S, Brown NJ, Bradley R, Stucky GD, Eardley D, Harriss R (2014) Energy and environment. Methane leaks from North American natural gas systems. Science 343:733–735

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Bédard C, Knowles R (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH4 + , and CO oxidation by methanotrophs and nitrifiers. Microbiol Rev 53:68–84

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Choi YJ, Morel L, Le François T, Bourque D, Bourget L, Groleau D, Massie B, Míguez CB (2010) Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Appl Environ Microbiol 76:5058–5066

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. 7.

    Clomburg JM, Blankschien MD, Vick JE, Chou A, Kim S, Gonzalez R (2015) Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids. Metab Eng 28:202–212

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Clomburg JM, Crumbley AM, Gonzalez R (2017) Industrial biomanufacturing: the future of chemical production. Science 355:aag0804

    Article  PubMed  CAS  Google Scholar 

  9. 9.

    Clomburg JM, Vick JE, Blankschien MD, Rodríguez-Moyá M, Gonzalez R (2012) A synthetic biology approach to engineer a functional reversal of the β-oxidation cycle. ACS Synth Biol 1:541–554

    Article  PubMed  CAS  Google Scholar 

  10. 10.

    Conrado RJ, Gonzalez R (2014) Chemistry. Envisioning the bioconversion of methane to liquid fuels. Science 343:621–623

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    de la Torre A, Metivier A, Chu F, Laurens LM, Beck DA, Pienkos PT, Lidstrom ME, Kalyuzhnaya MG (2015) Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1). Microb Cell Fact 14:188

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Demidenko A, Akberdin IR, Allemann M, Allen EE, Kalyuzhnaya MG (2016) Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1). Front Microbiol 7:2167

    PubMed  Google Scholar 

  13. 13.

    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

    Article  PubMed  CAS  Google Scholar 

  14. 14.

    Dunfield P, Knowles R (1995) Kinetics of inhibition of methane oxidation by nitrate, nitrite, and ammonium in a humisol. Appl Environ Microbiol 61:3129–3135

    PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

    Environmental Protection Agency (2017) Greenhouse gas emissions: understanding global warming potentials

  16. 16.

    Espah Borujeni A, Channarasappa AS, Salis HM (2014) Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res 42:2646–2659

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Fei Q, Guarnieri MT, Tao L, Laurens LM, Dowe N, Pienkos PT (2014) Bioconversion of natural gas to liquid fuel: opportunities and challenges. Biotechnol Adv 32:596–614

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Gilman A, Laurens LM, Puri AW, Chu F, Pienkos PT, Lidstrom ME (2015) Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microb Cell Fact 14:182

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Hanson RS, Hanson TE (1996) Methanotrophic bacteria. Microbiol Rev 60:439–471

    PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Haroon MF, Hu S, Shi Y, Imelfort M, Keller J, Hugenholtz P, Yuan Z, Tyson GW (2013) Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage. Nature 500:567–570

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Haynes CA, Gonzalez R (2014) Rethinking biological activation of methane and conversion to liquid fuels. Nat Chem Biol 10:331–339

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT (2016) Bioconversion of methane to lactate by an obligate methanotrophic bacterium. Sci Rep 6:21585

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. 23.

    Henard CA, Smith HK, Guarnieri MT (2017) Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst. Metab Eng 41:152–158

    Article  PubMed  CAS  Google Scholar 

  24. 24.

    Holmes AJ, Costello A, Lidstrom ME, Murrell JC (1995) Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett 132:203–208

    Article  PubMed  CAS  Google Scholar 

  25. 25.

    Hujanen M, Linko Y-Y (1996) Effect of Temperature and Various Nitrogen Sources on L(+)-Lactic Acid Production by Lactobacillus casei. Appl Microbiol Biotechnol 45:307–313

    Article  CAS  Google Scholar 

  26. 26.

    Hwang IY, Lee SH, Choi YS, Park SJ, Na JG, Chang IS, Kim C, Kim HC, Kim YH, Lee JW, Lee EY (2014) Biocatalytic conversion of methane to methanol as a key step for development of methane-based biorefineries. J Microbiol Biotechnol 24:1597–1605

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Kaluzhnaya M, Khmelenina V, Eshinimaev B, Suzina N, Nikitin D, Solonin A, Lin JL, McDonald I, Murrell C, Trotsenko Y (2001) Taxonomic characterization of new alkaliphilic and alkalitolerant methanotrophs from soda lakes of the Southeastern Transbaikal region and description of Methylomicrobium buryatense sp.nov. Syst Appl Microbiol 24:166–176

    Article  PubMed  CAS  Google Scholar 

  28. 28.

    Kalyuzhnaya MG, Puri AW, Lidstrom ME (2015) Metabolic engineering in methanotrophic bacteria. Metab Eng 29:142–152

    Article  PubMed  CAS  Google Scholar 

  29. 29.

    Kalyuzhnaya MG, Yang S, Rozova ON, Smalley NE, Clubb J, Lamb A, Gowda GA, Raftery D, Fu Y, Bringel F, Vuilleumier S, Beck DA, Trotsenko YA, Khmelenina VN, Lidstrom ME (2013) Highly efficient methane biocatalysis revealed in a methanotrophic bacterium. Nat Commun 4:2785

    Article  PubMed  CAS  Google Scholar 

  30. 30.

    Khmelenina VN, Beck DA, Munk C, Davenport K, Daligault H, Erkkila T, Goodwin L, Gu W, Lo CC, Scholz M, Teshima H, Xu Y, Chain P, Bringel F, Vuilleumier S, Dispirito A, Dunfield P, Jetten MS, Klotz MG, Knief C, Murrell JC, Op den Camp HJ, Sakai Y, Semrau J, Svenning M, Stein LY, Trotsenko YA, Kalyuzhnaya MG (2013) Draft genome sequence of methylomicrobium buryatense Strain 5G, a haloalkaline-tolerant methanotrophic bacterium. Genome Announc 1:e00053

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    King GM, Schnell S (1994) Ammonium and nitrite inhibition of methane oxidation by Methylobacter albus BG8 and Methylosinus trichosporium OB3b at low methane concentrations. Appl Environ Microbiol 60:3508–3513

    PubMed  PubMed Central  CAS  Google Scholar 

  32. 32.

    Leak DJ, Dalton H (1986) Growth yields of methanotrophs 1. Appl Microbiol Biotechnol 23:470–476

    Article  CAS  Google Scholar 

  33. 33.

    Mazumdar S, Blankschien MD, Clomburg JM, Gonzalez R (2013) Efficient synthesis of l-lactic acid from glycerol by metabolically engineered Escherichia coli. Microb Cell Fact 12:7

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. 34.

    Miller SM, Wofsy SC, Michalak AM, Kort EA, Andrews AE, Biraud SC, Dlugokencky EJ, Eluszkiewicz J, Fischer ML, Janssens-Maenhout G, Miller BR, Miller JB, Montzka SA, Nehrkorn T, Sweeney C (2013) Anthropogenic emissions of methane in the United States. Proc Natl Acad Sci U S A 110:20018–20022

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. 35.

    Murrell JC, Dalton H (1983) Ammonia Assimilation in Methylococcus capsulatus (Bath) and other Obligate Methanotrophs. J Gen Microbiol 129:1197–1206

    CAS  Google Scholar 

  36. 36.

    Nancib A, Nancib N, Meziane-Cherif D, Boubendir A, Fick M, Boudrant J (2005) Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp. rhamnosus. Bioresour Technol 96:63–67

    Article  PubMed  CAS  Google Scholar 

  37. 37.

    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 98:1567–1581

    Article  PubMed  CAS  Google Scholar 

  38. 38.

    O’Neill JG, Wilkinson JF (1977) Oxidation of ammonia by methane-oxidizing bacteria and the effects of ammonia on methane oxidation. J Gen Microbiol 100:407–412

    Article  Google Scholar 

  39. 39.

    Pieja AJ, Rostkowski KH, Criddle CS (2011) Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria. Microb Ecol 62:564–573

    Article  PubMed  CAS  Google Scholar 

  40. 40.

    Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, Lidstrom ME (2015) Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense. Appl Environ Microbiol 81:1775–1781

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. 41.

    Reeve B, Hargest T, Gilbert C, Ellis T (2014) Predicting translation initiation rates for designing synthetic biology. Front Bioeng Biotechnol 2:1

    Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. 43.

    Shindell D, Kuylenstierna JC, Vignati E, van Dingenen R, Amann M, Klimont Z, Anenberg SC, Muller N, Janssens-Maenhout G, Raes F, Schwartz J, Faluvegi G, Pozzoli L, Kupiainen K, Höglund-Isaksson L, Emberson L, Streets D, Ramanathan V, Hicks K, Oanh NT, Milly G, Williams M, Demkine V, Fowler D (2012) Simultaneously mitigating near-term climate change and improving human health and food security. Science 335:183–189

    Article  PubMed  CAS  Google Scholar 

  44. 44.

    Strong PJ, Kalyuzhnaya M, Silverman J, Clarke WP (2016) A methanotroph-based biorefinery: potential scenarios for generating multiple products from a single fermentation. Bioresour Technol 215:314–323

    Article  PubMed  CAS  Google Scholar 

  45. 45.

    Strong PJ, Xie S, Clarke WP (2015) Methane as a resource: can the methanotrophs add value? Environ Sci Technol 49:4001–4018

    Article  PubMed  CAS  Google Scholar 

  46. 46.

    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 β-oxidation cycle. Appl Environ Microbiol 81:1406–1416

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. 47.

    Yan X, Chu F, Puri AW, Fu Y, Lidstrom ME (2016) Electroporation-Based Genetic Manipulation in Type I Methanotrophs. Appl Environ Microbiol 82:2062–2069

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. 48.

    Ye RW, Kelly K (2012) Construction of carotenoid biosynthetic pathways through chromosomal integration in methane-utilizing bacterium Methylomonas sp. strain 16a. Methods Mol Biol 892:185–195

    Article  PubMed  CAS  Google Scholar 

  49. 49.

    Ye RW, Yao H, Stead K, Wang T, Tao L, Cheng Q, Sharpe PL, Suh W, Nagel E, Arcilla D, Dragotta D, Miller ES (2007) Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a. J Ind Microbiol Biotechnol 34:289–299

    Article  PubMed  CAS  Google Scholar 

  50. 50.

    Zhang T, Zhou J, Wang X, Zhang Y (2017) Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b. J Environ Sci (China) 52:49–57

    Article  Google Scholar 

Download references

Acknowledgements

This work was funded by an award from the U.S. Department of Energy Advanced Research Projects Agency-Energy (ARPA-E) (Award No. DE-AR0000762). We would like to thank Dr. G. Bennett and Dr. M. Lidstrom for kindly sharing strain Methylomicrobium buryatense 5GB1 and Dr. M. Guarnieri for sharing plasmid pCAH01.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ramon Gonzalez.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 94 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Garg, S., Clomburg, J.M. & Gonzalez, R. A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1. J Ind Microbiol Biotechnol 45, 379–391 (2018). https://doi.org/10.1007/s10295-018-2035-3

Download citation

Keywords

  • Methanotroph
  • Metabolic engineering
  • Lactate
  • Methylomicrobium buryatense
  • Industrial medium