Long-term adaptive evolution of Leuconostoc mesenteroides for enhancement of lactic acid tolerance and production

Abstract

BackgroundLactic acid has been approved by the United States Food and Drug Administration as Generally Regarded As Safe (GRAS) and is commonly used in the cosmetics, pharmaceutical, and food industries. Applications of lactic acid have also emerged in the plastics industry. Lactic acid bacteria (LAB), such as Leuconostoc and Lactobacillus, are widely used as lactic acid producers for food-related and biotechnological applications. Nonetheless, industrial mass production of lactic acid in LAB is a challenge mainly because of growth inhibition caused by the end product, lactic acid. Thus, it is important to improve acid tolerance of LAB to achieve balanced cell growth and a high titer of lactic acid. Recently, adaptive evolution has been employed as one of the strategies to improve the fitness and to induce adaptive changes in bacteria under specific growth conditions, such as acid stress.ResultsWild-type Leuconostoc mesenteroides was challenged long term with exogenously supplied lactic acid, whose concentration was increased stepwise (for enhancement of lactic acid tolerance) during 1 year. In the course of the adaptive evolution at 70 g/L lactic acid, three mutants (LMS50, LMS60, and LMS70) showing high specific growth rates and lactic acid production were isolated and characterized. Mutant LMS70, isolated at 70 g/L lactic acid, increased d-lactic acid production up to 76.8 g/L, which was twice that in the wild type (37.8 g/L). Proteomic, genomic, and physiological analyses revealed that several possible factors affected acid tolerance, among which a mutation of ATPase ε subunit (involved in the regulation of intracellular pH) and upregulation of intracellular ammonia, as a buffering system, were confirmed to contribute to the observed enhancement of tolerance and production of d-lactic acid.ConclusionsDuring adaptive evolution under lethal stress conditions, the fitness of L. mesenteroides gradually increased to accumulate beneficial mutations according to the stress level. The enhancement of acid tolerance in the mutants contributed to increased production of d-lactic acid. The observed genetic and physiological changes may systemically help remove protons and retain viability at high lactic acid concentrations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0662-3) contains supplementary material, which is available to authorized users.

Microbial response to acid stress: mechanisms and applications

“…Sophisticated mechanisms at the physiological and molecular levels have been developed by microorganisms to survive and adapt to acid stress (Fernández-Niño et al 2015;Hosseini Nezhad et al 2015;Ju et al 2016;Liu et al 2015c;Matsui and Cvitkovitch 2010), and a variety of approaches has also been deployed to unveil acid tolerance mechanisms in different microbes at different levels (He et al 2016;Hu et al 2017;Lee et al 2015;Sandoval et al 2011;Zhai et al 2014). After understanding the patterns and mechanisms of microbial response to acid stress comprehensively, specific strategies may be tailored for improvement of microbial producers and biosynthesis of valuable chemicals.…”

Adaptive laboratory evolution of cadmium tolerance in Synechocystis sp. PCC 6803

“…ALE has been demonstrated as an effective approach to obtain desired biological properties of the evolved strain. The titer of d -lactic acid produced by the evolved strain has been increased 2.0-fold than the original strain in Leuconostoc mesenteroides [ 29 ]. In addition, adaptive evolution under thermal stress not only increased the survival temperature from 33 to 41.5 °C, but also conferred cross tolerance to isobutanol in Corynebacterium glutamicum [ 30 ].…”

Metabolic engineering strategies for consolidated production of lactic acid from lignocellulosic biomass

“…Improved LA tolerance phenotype corresponded also in this case to increased LA production (titer up to 76.8 g/L) that was twofold higher than in the wild‐type strain. Analysis of L. mesenteroides mutants revealed increased intracellular content of ammonia and a mutation in the gene encoding ε subunit of F 0 F 1 ATPase likely causing more efficient ATP‐dependent proton extrusion activity .…”

“…Furthermore, multiple gene disruption had cumulative effects [62]. Adaptive evolution approach was recently used to improve LA tolerance of Leuconostoc mesenteroides up to 70 g/L [63]. Improved LA tolerance phenotype corresponded also in this case to increased LA production (titer up to 76.8 g/L) that was twofold higher than in the wild-type strain.…”