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

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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.

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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.

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Correspondence to Ramon Gonzalez.

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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

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Keywords

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