Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

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 ϳ40 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 ϳ0 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.

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

“…Second, the overexpression of a heterologous transaldolase, encoded by tal1, from S. stipitis was chosen, since this protein has been previously reported to be beneficial for xylose utilization in S. cerevisiae (14). The overexpression of downstream pathway enzymes to ensure proper flux is advantageous for selection phenotypes (37). To achieve overexpression of tal1, the gene was cloned behind a TEF promoter in the p415 plasmid.…”

“…However, this pathway is inherently limited by a cofactor imbalance with the xylose reductase-utilizing NADPH and the xylitol dehydrogenase-utilizing NAD ϩ , which leads to diversion of metabolic flux toward undesired products as a compensation reaction and decreases the ethanol yield (49). Recent work has focused on modifying the cofactor preferences of these enzymes to make them more compatible and to establish an oxidation-reduction cycle (37,47). However, even with matching cofactor specificities, the oxidoreductase pathway requires cofactors that may limit overall pathway throughput.…”

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

“…However, S. cerevisiae lacks an endogenous xylose catabolic pathway and thus is unable to natively use the second most abundant sugar in lignocellulosic biomass. Decades of research have been focused on improving xylose catabolic pathways in recombinant S. cerevisiae (17)(18)(19)(20)(21)(22), but less work has been focused on the first committed step of the process-xylose transport, an outstanding limitation in the efficient conversion of lignocellulosic sugars (23,24).…”

Metabolomics applied in bioenergy

“…Although the identified functional enzymes were expressed in recombinant Saccharomyces cerevisiae industrial strains, their efficiency in fermenting xylose was shown to still be quite low [87,88], suggesting the requirement of additional modifications. Overexpression of genes encoding enzymes of non-oxidative pentose phosphate pathway (PPP) [89], replacement of a small amount of enzymes of xylose metabolism [90], as well as isolation of xylose transporters [91,92] were pointed out as crucial factors for the adequate function of this pathway. However, a more holistic approach could also provide the metabolic reconstruction of most biologically relevant and predictive models to improve the fermentative ability of S. cerevisiae [93].…”