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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS-TOLERANT MICROBES FOR LOWER COST PRODUCTION OF BIOFUELS AND BIOPRODUCTS

Location: Crop Bioprotection Research

Title: Genomic mechanisms of inhibitor-detoxification for low-cost lignocellulosic bioethanol conversion

Authors
item Liu, Zonglin
item Moon, Jaewoong
item Mingzhou, Song - NM STATE U, LAS CRUCES,NM

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 17, 2008
Publication Date: November 5, 2008
Citation: Liu, Z., Moon, J., Mingzhou, S.J. 2008. Genomic mechanisms of inhibitor-detoxification for low-cost lignocellulosic bioethanol conversion [abstract]. Journal of Biotechnology 136S:S218.

Technical Abstract: One major challenges of sustainable lignocellulosic biomass conversion to ethanol is to overcome inhibitors generated from biomass pretreatment. Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural, cinnamaldehyde, phenylacetylaldehyde, and 4-hydroxybenzaldehyde, are common and potent inhibitors present in biomass hydrolyzate that inhibiting fermentative microorganism growth and ethanol production. Recently, we developed a tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049 by directed evolution that can in situ detoxify aldehyde inhibitors while producing a normal yield of ethanol. To better understand genomic mechanisms of the detoxification, we investigated global gene expression profiling, identified regulatory elements and networks involving the tolerance and characterized functional genes and enzymes responsible for the detoxification. Gene chips of S. cerevisiae were fabricated using OmniGrid 300 GeneMachine microarray robust with 70-mer DNA oligos representing 6388 genes of the yeast genome. Universal RNA controls were applied to the microarray and used for data normalization and analysis. Replicated microarray experiments were conducted under furfural plus 5-hydroxymethylfurfural challenged conditions compared with a normal control for tolerant Y-50049 and a non tolerant strain NRRL Y-12632. Data were analyzed using GeneSpring and gene regulatory networks inferred using recently developed computational linear discrete dynamic system model. Candidate genes were cloned and yeast transformation made using pYes2/NT constructs. Gene products of recombinant proteins were induced for enzyme expression assays compared with specific enzyme activities of whole cell extract from strain Y-50049. We identified more than 300 genes that can be grouped into over 100 clusters were differentially expressed significantly in response to the inhibitor challenges. Among which, eight transcriptional factors were identified as significant for transcriptional regulations of gene temporal interactions. A group of candidate genes having aldehyde reduction activities were identified and their specific enzyme activities characterized. We found that each individual gene product or enzyme has specific NADH and/or NADPH co-factor preference for reduction of aldehyde inhibitors. In contract, whole yeast cell extract showed strong aldehyde reduction activities with either co-factors. Deletion of a single reductase gene did not significantly affect cell tolerance and detoxification. We conclude that multiple gene mediated NAD(P)H dependent aldehyde reduction is a mechanism of in situ detoxification of aldehyde inhibitors including furfural, 5-hydroxymethylfurfural, cinnamaldehyde, phenylacetylaldehyde, and 4-hydroxybenzaldehyde. In addition, members of pleiotropic drug resistance gene family appeared to play a significant role to cope with cell survival in response to the inhibitor stress conditions.

Last Modified: 8/27/2014
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