(380f) Molecular Engineering of Xylose Transport and Metabolism in Escherichia Coli and Saccharomyces Cerevisiae

Efficient utilization of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials[1]. Escherichia coli and Saccharomyces cerevisiae are two of the most important workhorses for bio-refinery of biomass. Xylose transport and metabolism process are important for their efficient use of xylose in the lignocellulosic hydrolysate[2-4]. In the presence of glucose, the xylose transport and metabolism in microorganisms is often inhibited, due to the carbon catabolite repression (CCR) mechanism. In this work, we introduced a heterologous xylose transporter, glf from Zymomonas mobilis, into E .coli, and carried out directed evolution of this transporter, using a high throughput assay of xylose transport[5]. After 2 rounds of evolution, we obtained a mutant RD5, the xylose transport activity of which increased more than 7 folds in the presence of glucose. Fermentation tests showed that RD5 significantly improved sugar utilization in media supplemented with xylose or a mixture of xylose and glucose, compared with the wild type strain. To make S.cerevisiae be able to metabolize xylose efficiently, XylA, xylose isomerase from E. coli was cloned into S.cerevisiae, and then evolved by error-prone PCR and selection on plates containing xylose as a sole carbon source. After two rounds of selection, a mutant 1-M13 was obtained. Fermentation tests showed that the xylose utilization for this mutant improved to about 1.7 folds of the wild type strain in media supplemented with mixtures of xylose and glucose. The xylose isomerase activity profile showed that the optimal pH value for 1-M13 has shifted to a more acidic range than the wild type.

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