The L-arabinose utilization pathway was established in Saccharomyces cerevisiae, by expressing the codon-optimized araA, araB, and araD genes of Lactobacillus plantarum. After overexpressing the TAL1, TKL1, RPE1, RKI1, and GAL2 genes and adaptive evolution, the L-arabinose utilization of the recombinant strain became efficient. The resulting strain displayed a maximum specific growth rate of 0.075 h−1, a maximum specific L-arabinose consumption rate of 0.61 g h−1 g−1 dry cell weight, and a promising ethanol yield of 0.43 g g−1 from L-arabinose fermentation.

BackgroundEfficiently utilizing all available carbon from lignocellulosic feedstock presents a major barrier to the production of economically feasible biofuel. Previously, to enable xylose utilization, we introduced a cofactor-dependent xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway, or a cofactor-independent xylose isomerase (XI) pathway, into Saccharomyces cerevisiae. The resulting strains metabolized xylose with high efficiency. However, in both pathway recombinant strains, the cofactor imbalance caused accumulation of the byproducts glycerol and/or xylitol and reduced the ethanol production efficiency.ResultsIn this study, we introduced NADH oxidase from Lactococcus lactis into both XI and XR-XDH pathway recombinant strains. To reduce byproduct accumulation while maintaining xylose metabolism, we optimized the expression level of NADH oxidase by comparing its expression under the control of different promoters and plasmids. In recombinant XI strains, NADH oxidase was expressed at different levels, regulated by the GPD2 promoter or TEF1 promoter in the 2 μ plasmid. The expression under the control of GPD2 promoter decreased glycerol production by 84% and increased the ethanol yield and specific growth rate by 8% and 12%, respectively. In contrast, in the recombinant XR-XDH strains, such expression level was not efficient enough to decrease the byproduct accumulation. Therefore, higher NADH oxidase expression levels were tested. In the strain expressing NADH oxidase under the control of the TEF1 promoter in the centromeric plasmids, xylitol and glycerol production were reduced by 60% and 83%, respectively, without significantly affecting xylose consumption.ConclusionsBy fine-tuning NADH oxidase expression, we decreased the glycerol or/and xylitol production in both recombinant XI and XR-XDH xylose-metabolizing yeast strains. The optimal NADH oxidase expression levels depend on metabolic pathways. Similar cofactor engineering strategies could maximize the production of other redox dependent metabolites.

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Microbial production of commodity chemicals has gained increasing attention and most of the focus has been on reducing the production cost. Selecting a suitable microorganism, which can grow rapidly on cheap feedstocks, is of key importance when developing an economically feasible bioprocess. We chose Lactococcus lactis, a well-characterized lactic acid bacterium, as our microbial host to produce pyruvate, which is a commodity chemical with various important applications. Here we report the engineering of Lactococcus lactis into becoming an efficient microbial platform for producing pyruvate. The strain obtained, FS1076 (MG1363 Δ3ldh Δpta ΔadhE Δals), was able to produce pyruvate as the sole product. Since all the competitive pathways had been knocked out, we achieved growth-coupled production of pyruvate with high yield. More than 80 percent of the carbon flux was directed toward pyruvate, and a final titer of 54.6 g/L was obtained using a fed-batch fermentation setup. By introducing lactose catabolism into FS1076, we obtained the strain FS1080, which was able to generate pyruvate from lactose. We then demonstrated the potential of FS1080 for valorizing lactose contained in dairy side-streams, by achieving a high titer (40.1 g/L) and high yield (78.6%) of pyruvate using residual whey permeate (RWP) as substrate. The results obtained, show that the L. lactis platform is well-suited for transforming lactose in dairy waste into food-grade pyruvate, and the yields obtained are the highest reported in the literature. These results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) to valuable products.

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