Metabolic engineering of the type I methanotroph Methylomonas sp. DH-1 for production of succinate from methane

Abstract

Methane-utilizing methanotrophs are fascinating systems for methane bioconversion. Methylomonas sp. DH-1, a novel type I methanotroph isolated from brewery sludge, has been evaluated as a promising candidate for an industrial bio-catalyst. Succinate has been considered one of the top building block chemicals for the agricultural, food, and pharmaceutical industries. In this study, Methylomonas sp. DH-1 was engineered to accumulate succinate as a desired product. The TCA cycle and enzymes diverting carbon flux to acetate or formate were modified or deleted to improve succinate productivity. By deleting succinate dehydrogenase (sdh) in the TCA cycle, succinate production increased dramatically ∼10 times compared to that of the wild type. In addition, the maximum succinate titer of ∼134 mg/L (DS-GL) was achieved by integrating glyoxylate shunt enzymes from the E. coli MG1655 strain. Pyruvate formate lyase (pfl) and acetate kinase-phosphotransacetylase (ack-pta) genes were disrupted to further concentrate carbon flux to the TCA cycle. However, these additional disruptions of competitive pathways did not affect cell growth or succinate production positively. The mutant strain DS-GL, which showed the best succinate production, was grown in a fed-batch bioreactor, and higher cell growth and succinate production (∼195 mg/L succinate with 0.0789 g-succinate/g-methane yield) were achieved. In this study, we demonstrated a novel platform for microbial conversion of methane to succinate using methanotroph.

Keywords: Metabolic engineering; Methane; Methanotroph; Methylomonas sp. DH-1; Succinate production.