Dataset Information

Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution.

ABSTRACT: Background:Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. Results:In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. Conclusions:JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

SUBMITTER: Lee JK

PROVIDER: S-EPMC6767647 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Efficient production of d-lactate from methane in a lactate-tolerant strain of <i>Methylomonas</i> sp. DH-1 generated by adaptive laboratory evolution.

Lee Jong Kwan JK Kim Sujin S Kim Wonsik W Kim Sungil S Cha Seungwoo S Moon Hankyeol H Hur Dong Hoon DH Kim Seon-Young SY Na Jeong-Geol JG Lee Jin Won JW Lee Eun Yeol EY Hahn Ji-Sook JS

Biotechnology for biofuels 20190930

<h4>Background</h4>Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an ...[more]

PMID: 31583020

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Project description:BackgroundMethylacidiphilum sp. IT6 has been validated its C3 substrate assimilation pathway via acetol as a key intermediate using the PmoCAB3, a homolog of the particulate methane monooxygenase (pMMO). From the transcriptomic data, the contribution of PmoD of strain IT6 in acetone oxidation was questioned. Methylomonas sp. DH-1, a type I methanotroph containing pmo operon without the existence of its pmoD, has been deployed as a biocatalyst for the gas-to-liquid bioconversion of methane and propane to methanol and acetone. Thus, Methylomonas sp. DH-1 is a suitable host for investigation. The PmoD-expressed Methylomonas sp. DH-1 can also be deployed for acetol production, a well-known intermediate for various industrial applications. Microbial production of acetol is a sustainable approach attracted attention so far.ResultsIn this study, bioinformatics analyses elucidated that novel protein PmoD is a C-terminal transmembrane-helix membrane with the proposed function as a transport protein. Furthermore, the whole-cell biocatalyst was constructed in Methylomonas sp. DH-1 by co-expression the PmoD of Methylacidiphilum sp. IT6 with the endogenous pMMO to enable acetone oxidation. Under optimal conditions, the maximum accumulation, and specific productivity of acetol were 18.291 mM (1.35 g/L) and 0.317 mmol/g cell/h, respectively. The results showed the first coupling activity of pMMO with a heterologous protein PmoD, validated the involvement of PmoD in acetone oxidation, and demonstrated an unprecedented production of acetol from acetone in type I methanotrophic biocatalyst. From the data achieved in batch cultivation conditions, an assimilation pathway of acetone via acetol as the key intermediate was also proposed.ConclusionUsing bioinformatics tools, the protein PmoD has been elucidated as the membrane protein with the proposed function as a transport protein. Furthermore, results from the assays of PmoD-heteroexpressed Methylomonas sp. DH-1 as a whole-cell biocatalyst validated the coupling activity of PmoD with pMMO to convert acetone to acetol, which also unlocks the potential of this recombinant biocatalyst for acetol production. The proposed acetone-assimilated pathway in the recombinant Methylomonas sp. DH-1, once validated, can extend the metabolic flexibility of Methylomonas sp. DH-1.

Project description:BackgroundMethanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain.ResultsWe integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden-Meyerhof-Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH.ConclusionsThis study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries.

Dataset Information

Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution.

Publications

Efficient production of d-lactate from methane in a lactate-tolerant strain of <i>Methylomonas</i> sp. DH-1 generated by adaptive laboratory evolution.

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