Abstract
BACKGROUND: Methane is an abundant and low-cost carbon source with great potential for conversion into value-added chemicals. Methanotrophs, microorganisms that utilize methane as their sole carbon and energy source, present a promising platform for biotechnological applications. This study aimed to engineer Methylomonas sp. DH-1 to enhance D-LA production through metabolic pathway optimization during large-scale cultivation. RESULTS: In this study, we regulated the expression of D-lactate dehydrogenase (D-LDH) using a Ptac promoter with IPTG induction to mitigate the toxic effects of lactate accumulation. To further optimize carbon flow away from glycogen, the glgA gene was deleted. However, this modification led to growth inhibition, especially during scale-up, likely due to the accumulation of ADP-glucose caused by the rewired carbon flux under carbon-excess conditions. Deleting the glgC gene, which encodes glucose 1-phosphate adenylyltransferase, alleviated this issue. The final optimized strain, JHM805, achieved a D-LA production of 6.17 g/L in a 5-L bioreactor, with a productivity of 0.057 g/L/h, marking a significant improvement in D-LA production from methane. CONCLUSIONS: The metabolic engineering strategies employed in this study, including the use of an inducible promoter and alleviation of ADP-glucose accumulation toxicity, successfully enhanced the ability of the strain to produce D-LA from methane. Furthermore, optimizing the bioreactor fermentation process through methane and nitrate supplementation resulted in a significant increase in both the titer and productivity, exceeding previously reported values.