Increased ethanol productivity in xylose-utilizing Saccharomyces cerevisiae via a randomly mutagenized xylose reductase. Appl Environ Microbiol

Department of Applied Microbiology, Lund University, Lund, Sweden.
Applied and Environmental Microbiology (Impact Factor: 3.95). 10/2010; 76(23):7796-802. DOI: 10.1128/AEM.01505-10
Source: PubMed

ABSTRACT Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V(max) of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ∼0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

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    • "the gene of interest, or alternatively promoter regions or even whole genomes, is subjected to PCR amplification with an elevated error rate (by the lack of exonuclease activity and/or nonideal reaction conditions), followed by transformation of the fragments into the microorganism of interest and a screening to recover mutants that display phenotypic improvement. For instance, Runquist et al. (2010) applied epPCR to the cofactor-binding region of the Sc. stipitis gene XYL1, one of the enzymes needed for S. cerevisiae to consume xylose, and transformed this library into S. cerevisiae. "
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