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.

Molecular Mechanism Associated With the Impact of Methane/Oxygen Gas Supply Ratios on Cell Growth of Methylomicrobium buryatense 5GB1 Through RNA-Seq

“…Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N 2 as the nitrogen source (Auman et al, 2001;Matsen et al, 2013). From the genome annotation and metabolic modeling, M. buryatense 5GB1 was also predicted to be able to assimilate N 2 into ammonia, which can be used as a nitrogen source (de la Torre et al, 2015;Garg et al, 2018). The intriguing discovery from our transcriptomic analysis was that thirty-four nitrogen assimilation related genes were significantly up-regulated in the #3 condition compared with other conditions, including nitrogen fixation structural genes molybdenum nitrogenase genes (nifHDK), nitrogenase MoFe maturation genes (NifPZ), cofactor biosynthesis genes (nifBEMNXQTUVY), ferredoxin genes (fdxBCD), as well as genes involved in electron supply (rnfCH, nifF, rsxABDEG), nitrogen fixation regulation (nifLA, glnGLK), and post-translational modification (draTG) (Figure 3 and Supplementary Table S2).…”

“…A series of genetic constructions and metabolic engineering works of M. buryatense 5GB1 have been carried out to enhance the carbon flux for the production of biomass, lipids, lactic acid, fatty acids, etc. (Dong et al, 2017;Fu et al, 2017;Hu et al, 2017;Garg et al, 2018). The highest lipid productivity of 45 mg/L/h along with the growth rate of 0.22 h −1 and DCW of 21 g/L has been achieved by knocking out glycogen biosynthesis genes of M. buryatense 5GB1 in batch cultures .…”

“…The flux balance model of M. buryatense 5GB1 also suggests that the maximum carbon conversion efficiency (CCE) can be as high as 60∼70% on molar basis. Garg et al (2018) have successfully improved the CCE 50 times by using suitable promoters and RBS combinations for lactic acid production by M. buryatense 5GB1.…”

Accumulation of ammonium owing to the metabolic imbalance of carbon and nitrogen might inhibit the central metabolism inMethylomonassp. ZR1

“…At the beginning methanotrophs were used to produce single cell protein from methane or methanol. Nowadays, with the development of biotechnology, methanotrophs were genetic modified to produce lactic acid (Henard et al, 2016; Garg et al, 2018), astaxanthin (Ye and Kelly, 2012) and α-hemelune (Sonntag et al, 2015) from methane. However, the slow growth nature of methanotrophs has constrained its application in industrial biotechnology.…”

Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts

“…Indeed, recent efforts have demonstrated the potential utility of methanotrophic biocatalysts for production of platform chemicals from diverse waste streams. [19][20][21][22][23][24][25] Here, we present the first heterologous production of MA from CH 4 using methanotrophic bacteria as a production chassis. Further, we leveraged the M. alcaliphilum 20Z genome-scale model 26 to guide rational metabolic engineering to increase carbon flux to the MA pathway, which revealed a yet unidentified shikimate biosynthetic enzyme or pathway in this organism.…”