Codon-Optimized Bacterial Genes Improve l -Arabinose Fermentation in Recombinant Saccharomyces cerevisiae

Abstract

Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like L-arabinose or D-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial L-arabinose utilization pathway consisting of L-arabinose isomerase from Bacillus subtilis and L-ribulokinase and L-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. L-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on L-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the L-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved L-arabinose conversion rates. With this rational approach, the ethanol production rate from L-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h ؊1 (g dry weight) Decreasing fossil energy resources and the climate change caused by emissions of CO 2 from their burning have led to a growing interest in renewable-energy alternatives. Bioethanol produced by microbial fermentations of plant biomass is an excellent alternative to fossil fuels. However, for economical ethanol production, it is not enough to convert only the starch and sucrose fractions of plant biomass as is mainly done in conventional ethanol plants. The importance of the conversion of the lignocellulosic fraction as well becomes more and more evident. Lignocellulosic hydrolysates consist of easily fermentable hexose sugars but also significant amounts of pentose sugars. Depending on the choice of raw material, the amounts of pentoses found in lignocellulosic hydrolysates range from, for example, 16% xylan and 5% arabinan in grass and 15% arabinan and 19% xylan in wheat bran (14). These numbers clearly indicate that both hexoses and pentoses must be fermented in an efficient ethanol production process. Even small increases in substrate utilization should significantly improve the overall process costs (28).Although Saccharomyces cerevisiae is the organism most widely used for ethanol production and is able to convert hexoses rapidly and with high ethanol yields, wild-type S. cerevisiae strains are not able to ferment pentose sugars, such as D-xylose and L-arabinose, efficiently. Even though xylose can be slowly metabolized, at least by adapted strains (2), the second most abundant pentose, L-arabinose, cannot be converted at all. Several different genetic-engineering approaches have been used in attempts to enable D-xylose and L-arabinose...

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“…The plasmids and primers used in this study are listed in Tables 2 and 3. Molecular techniques were performed according to previously published procedures (51). Bacterial genomic DNA was prepared for PCR amplification as described in reference 2.…”

Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

“…Codon-optimized gene versions were obtained from Geneart AG (Regensburg, Germany) after changing the original codons of the respective genes to those used in the genes encoding glycolytic enzymes in S. cerevisiae as described in the work of Wiedemann and Boles (35) and cloned into the vector p426H7 by recombination cloning as described above. Using the coding region of C. phytofermentans xylA and Piromyces sp.…”

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…The translation efficiency of heterologous genes depends on codon usage in yeast. In fact, it is highly recommended to adapt the codons of bacterial genes to those of highly expressed S. cerevisiae genes, thus ensuring maximal expression (387).…”