Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli.

Abstract:

:Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K(m) values for NADPH (8 microM and 23 microM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K(m) for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

journal_name

Appl Environ Microbiol

authors

Miller EN,Jarboe LR,Yomano LP,York SW,Shanmugam KT,Ingram LO

doi

10.1128/AEM.00567-09

subject

Has Abstract

pub_date

2009-07-01 00:00:00

pages

4315-23

issue

13

eissn

0099-2240

issn

1098-5336

pii

AEM.00567-09

journal_volume

75

pub_type

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