Efficient production of D-lactate from methane using Methylomonas sp. DH-1 strain with improved lactate tolerance

Abstract

Lactic acid is a monomer of poly lactic acid, which is biodegradable, biocompatible, and renewable biopolymer with increasing demand. Accordingly, production of lactic acid using microorganisms is also in the spotlight. Recently, abundant and inexpensive methane gas has emerged as a promising carbon source in biorefinery since the development of shale gas sampling technology. In this study, we engineered Methylomonas sp. DH-1, a methanotrophic bacteria that can use methane as sole carbon and energy source, to produce D-lactic acid from methane. First, to increase lactic acid tolerance, adaptive laboratory evolution was carried out by gradual increase in lactic acid concentrations in the medium during serial subcultures. The evolved strain Ev 8.0 isolated from the culture with 8.0 g/L lactic acid showed higher tolerance to lactic acid than the wild-type strain. In addition, Ev 8.0 showed improved acid resistance to other weak acids such as formic acid, acetic acid, and propionic acid. In order to product D-lactic acid, D form-specific lactate dehydrogenase gene (Lm.ldh) from lactic acid bacteria, was integrated into the genome while replacing glgA gene encoding glycogen synthase. Ev 8.0 strain expressing Lm.ldh produced about 8-fold higher level of D-lactic acid than wild-type strain expressing the same gene, demonstrating the importance of lactic acid tolerance in lactic acid production in the bacteria.