Abstract
BackgroundMethane, 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.ResultsIn 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.ConclusionsJHM80, 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.
Highlights
Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source
Methane is oxidized to methanol by methane monooxygenase (MMO) and methanol is further oxidized to formaldehyde by methanol dehydrogenase (MDH) (Fig. 1a)
Wild-type strain integrated with Lm.lactate dehydrogenase (LDH) (JHM14) showed very low growth rate and produced only 100 mg/L d-lactic acid (LA) (Fig. 6b and c). These results clearly demonstrate that the increase in LA tolerance plays a key role in improving LA production in Methylomonas sp
Summary
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 promis‐ ing 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 contributes to understanding novel LA tolerance mechanisms. Only a few chemicals such as astaxanthin, butyrate, 2,3-butanediol, succinic acid, and LA were produced using genetically engineered methanotrophs, but with very low titers of less than 1 g/L [17,18,19,20]
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