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Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Paper ID Volume ID Publish Year Pages File Format Full-Text
17272 42656 2012 6 PDF Available
Title
Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH
Abstract

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.

► Obtained increased aldehyde reductase activities of GRE2. ► Mutant Y62-G6 is able to use additional cofactor NADPH to reduce furfural and HMF. ► Amino acid substitution of Val285 to Asp285 is related to additional use of NADPH.

Keywords
Aldehyde reductase activity; Error-prone PCR; Evolutionary engineering; Stress tolerance
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Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 50, Issue 2, 10 February 2012, Pages 115–120
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us