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

Department of Applied Microbiology, Lund University, Lund, Sweden.
Applied and Environmental Microbiology (Impact Factor: 3.67). 10/2010; 76(23):7796-802. DOI: 10.1128/AEM.01505-10
Source: PubMed


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 Vmax 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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    • "S. cerevisiae TMB 3043 (Karhumaa et al. 2005) was transformed with the EcoRV-linearized vector YIpOB8 (Runquist et al. 2010), generating the strain TMB 4440. The yeast cells were transformed using the highefficiency Li-Ac method (Gietz and Schiestl 2007), and plated on YNB-xylose (20 g L "
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    • "By monitoring FI instead, we wanted to investigate whether the constructed biosensor could monitor less drastic changes in the redox balance. To validate this hypothesis, we introduced XR enzymes with slightly different catalytic activity for NADH oxidation, namely two xylose reductases having NADH/ NADPH selectivity ratios of 0.04 and 0.80 respectively (Runquist et al. 2010). As expected, the biggest difference in growth was observed in the double deletion strains between the control and the strain carrying the mutXR, since more carbon was made available for growth when xylitol formation from xylose was used as redox sink instead of glycerol formation from glucose. "
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