Attempt of heterologous expression of mdh in Zymomonas mobilis for the use of mannitol as a carbon and energy source

Abstract

The global energy crisis and increased environmental concerns have led to great advances in the development of renewable and sustainable biofuels. As one of the first-generation biofuels, bioethanol production has grown rapidly around the world. Zymomonas mobilis has emerged as a potential alternative to presently used yeast for ethanol production, for its advantages over yeast, such as higher specific rate of sugar uptake, higher ethanol production yield and productivity, lower biomass production as compared to yeast, non-requirement of controlled addition of oxygen to maintain the cell viability and amenability to genetic improvement being a prokaryote. However, the apparent drawback of Z.mobilis, its narrow range of available substrate (only glucose, fructose, and sucrose), restricts its applications. Meanwhile, the conflict between food and fuel favors the search of novel renewable and cheap carbohydrate sources. Seaweeds are considered as one of the promising candidates because they have high productivity, high CO2 capture capacity and lignin-free composition

moreover, they don’t require arable land. Among various seaweeds, brown algae have fast growth rate and high carbohydrate contents. Laminaria japonica, in particular, is the major seaweed species cultivated in South Korea, which is mostly composed of carbohydrates (up to 60%-70% of the total dry weight), including mannitol, alginate, laminaran, and cellulose. In this research, to achieve high yield of bioethanol production from L. japonica, recombinant Z. mobilis strains have been developed because of the disability of the wild type strains to ferment mannitol, the most abundant carbohydrate in L. japonica. Before mannitol enters the glycolysis pathway through the intermediate fructose-6-phosphate, it can be firstly oxidized to fructose by mannitol 2-dehydrogenase (M2DH), with the conversion of NAD+ to NADH, followed by the conversion of fructose into fructose-6-phosphate by fructokinase. In this study, the MDH-expressing vector has been constructed using pBBR1MCS-3, pdc promoter from Z.mobilis, and rrnBT1T2 as a backbone plasmid, promoter, and terminator, respectively. Two kinds of mdh gene have been chosen as target gene: one from Z.mobilis CP4.66, the other from L.reuteri JCM1112. After transformation, recombinant screening has been performed and three strains have been selected: strain No.1 and 41 from Z.mobilis/mut-ZM-mdh (named as M1 and M41) and strain No.17 from Z.mobilis/LR-mdh (named as L17). The relative MDH activity of M1, M41 and L17 was 1.4, 1.04 and 4.9 times higher than that of each negative control (wild type strain), respectively. The growth of these recombinant Z.mobilis strains was higher than that of the wild type strain, which demonstrated that the heterologous mdh gene had positive effect on the growth of Z.mobilis in mannitol medium.However, all these strains still showed low growth in mannitol medium The possible reason for this failure should be no or low activity of importer protein, functioning in mannitol uptake in Z.mobilis, and several further strategies for investigating suitable importer and/or employing alternative PTS-M1PDH pathway in Z.mobilis have been discussed. Although enhanced ethanol production has not been achieved, this research provides a valuable guideline for engineering the strains using mannitol metabolism.