Title: Plasmidic Expression of nemA and yafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

Abstract

ABSTRACTHydrolysate-resistantEscherichia coliSL100 was previously isolated from ethanologenic LY180 after sequential transfers in AM1 medium containing a dilute acid hydrolysate of sugarcane bagasse and was used as a source of resistance genes. Many genes that affect tolerance to furfural, the most abundant inhibitor, have been described previously. To identify genes associated with inhibitors other than furfural, plasmid clones were selected in an artificial hydrolysate that had been treated with a vacuum to remove furfural. Two new resistance genes were discovered from Sau3A1 libraries of SL100 genomic DNA:nemA(N-ethylmaleimide reductase) and a putative regulatory gene containing a mutation in the coding region,yafC*. The presence of these mutations in SL100 was confirmed by sequencing. A single mutation was found in the upstream regulatory region ofnemR(nemRAoperon) in SL100. This mutation increasednemAactivity 20-fold over that of the parent organism (LY180) in AM1 medium without hydrolysate and increasednemAmRNA levels >200-fold. Addition of hydrolysates inducednemAexpression (mRNA and activity), in agreement with transcriptional control. NemA activity was stable in cell extracts (9 h, 37°C), eliminating a role for proteinase in regulation. LY180 with a plasmid expressingnemAoryafC*was more resistant to a vacuum-treated sugarcane bagasse hydrolysate and to a vacuum-treated artificial hydrolysate than LY180 with an empty-vector control. Neither gene affectedmore » furfural tolerance. The vacuum-treated hydrolysates inhibited the reduction ofN-ethylmaleimide by NemA while also serving as substrates. Expression of thenemAoryafC*plasmid in LY180 doubled the rate of ethanol production from the vacuum-treated sugarcane bagasse hydrolysate.« less

Authors:

Shi, Aiqin ^[1]; Zheng, Huabao ^[1]; Yomano, Lorraine P. ^[1]; York, Sean W. ^[1]; Shanmugam, Keelnatham T. ^[1]; Ingram, Lonnie O. ^[1]

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+ Show Author Affiliations

1. Univ. of Florida, Gainesville, FL (United States). Dept of Microbiology and Cell Science

Publication Date:

Mon Mar 21 00:00:00 EDT 2016

Research Org.:

Univ. of Florida, Gainesville, FL (United States).; Univ. of Florida, Gainesville, FL (United States)

Sponsoring Org.:

USDOE Office of International Affairs (IA)

OSTI Identifier:

1470725

Alternate Identifier(s):

OSTI ID: 1770943

Grant/Contract Number:  

PI0000031

Resource Type:

Accepted Manuscript

Journal Name:

Applied and Environmental Microbiology

Additional Journal Information:

Journal Volume: 82; Journal Issue: 7; Journal ID: ISSN 0099-2240

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES