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.

Central and peripheral corneal thickness in Malays and its variation with age

“…Tomidokoro et al 7 found the mean central CT obtained in a Japanese population was associated significantly with corneal curvature but not with age. Lee et al 8 also found no significant difference in CT in a sample of Korean with respect to age except for the 20-26 year-old age group. Earlier studies 2,[5][6][9][10][11] and recently Hoffman et al 12 19 reported that African Americans had significantly thinner central CT than Caucasians, Asians, or Hispanics.…”

Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology

“…Methylomonas sp. DH-1 can convert methane into various value-added compounds and thus has become a promising platform strain for metabolic engineering 47,49,58 . In genome integration, homologous recombination is commonly used in Methylomonas sp.…”

“…Methylomonas sp. DH-1 has recently been shown to utilize methane as a sole carbon source and is gaining great interest as a platform for metabolic engineering [47][48][49] . However, the metabolic engineering of this methanotroph is challenging due to the lack of genetic manipulation tools, including the absence of artificial plasmids for genetic engineering.…”

Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

“…Adaptive laboratory evolution (ALE) is an important strategy to improve the fitness of microorganisms through selected environmental conditions and was widely used in metabolic engineering ( Papapetridis et al., 2018 ; Lee et al., 2016 ; Choe et al., 2019 ; Phaneuf et al., 2019 ; Gibson et al., 2020 ). Five major application areas of ALE are growth rate optimization ( Sandberg et al., 2014 ; Pfeifer et al., 2017 ), increasing tolerance of the strain ( Qi et al., 2019 ; Wang et al., 2018 ; Pereira et al., 2019 ), increase of substrate utilization ( Kawai et al., 2019 ; Gonzalez-Villanueva et al., 2019 ), increase of product yield/titer ( Gibson et al., 2020 ; Lee et al., 2019 ; Vasconcellos et al., 2019 ) and mechanism discovery ( Tenaillon et al., 2012 ; Kang et al., 2019 ). Fast growth phenotypes of Escherichia coli have been obtained through ALE, and the underlying mechanism has been studied ( LaCroix et al., 2015 ; Long et al., 2017 ; McCloskey et al., 2018 ; Wannier et al., 2018 ; Sandberg et al., 2016 ).…”