Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment

Abstract

BackgroundPhenolic aldehydes generated from lignocellulose pretreatment exhibited severe toxic inhibitions on microbial growth and fermentation. Numerous tolerance studies against furfural, 5-hydroxymethyl-2-furaldehyde (HMF), acetate, and ethanol were reported, but studies on inhibition of phenolic aldehyde inhibitors are rare. For ethanologenic strains, Zymomonas mobilis ZM4 is high in ethanol productivity and genetic manipulation feasibility, but sensitive to phenolic aldehyde inhibitors. Molecular mechanisms of tolerance for Z. mobilis toward phenolic aldehydes are not known.ResultsWe took the first insight into genomic response of Z. mobilis ZM4 to the phenolic aldehyde inhibitors derived from lignocellulose pretreatment. The results suggest that the toxicity to cells is caused by the functional group of phenolic aldehyde, similar to furfural and HMF, rather than aromatic groups or phenolic hydroxyl groups. Transcriptome response against 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, representing phenolic groups H, S, and G, respectively, was investigated. The atlas of the important genes responsible for significantly enhanced and repressed genes at the genomic level was illustrated. 272 genes with twofold greater expressions than non-treated controls and 36 gene clusters in response to challenges of these phenolic aldehydes were identified. Several reductases encoded by ZMO1116, ZMO1696, and ZMO1885 were found to play the key roles in reducing phenolic aldehydes into the corresponding phenolic alcohols. Reduction of phenolic aldehydes by overexpression of ZMO1116, ZMO1696, and ZMO1885 in Z. mobilis ZM4 resulted in the increased inhibitor conversion and ethanol productivity, especially for 4-hydroxybenzaldehyde and vanillin. Several transporter genes such as ZMO0282, ZMO0283, ZMO0798, ZMO0799, and ZMO0800 was also displayed significantly increased expressions against the phenolic aldehydes.ConclusionsThe genes encoding reductases are with potentials on phenolic aldehydes-tolerant genes contributing to the reduction of phenolic aldehydes into the corresponding phenolic alcohols forms for Z. mobilis ZM4. Overexpression of the key genes improved the conversion ratio and ethanol productivity of 4-hydroxybenzaldehyde and vanillin with high toxicity. New knowledge obtained from this research aids understanding the mechanisms of bacterial tolerance and the development of the next-generation biocatalysts for advanced biofuels production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0333-9) contains supplementary material, which is available to authorized users.

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

“…Furthermore, combinations of HMF, furfural, and acetate resulted in additive rather than synergistic inhibition to Z. mobilis cell growth (Franden et al 2013). It was also observed that Z. mobilis is capable of converting aldehydes of furfural, HMF, vanillin, 4-hydroxybenzaldehyde, and syringaldehyde to their corresponding alcohol forms of furfuryl, 5-hydroxymethylfurfural, vanillyl, 4-hydroxybenzyl, and syringyl alcohol, respectively (Franden et al 2013;Yi et al 2015). These studies suggested that the enhanced conversion of toxic aldehydes into the relatively benign alcohol forms with engineered strains could reduce process cost by improving ethanol yields and/or by reducing fermentation time.…”

“…Using conventional and high-throughput growth assays, effects of inhibitory hydrolysate compounds on Z. mobilis growth and fermentation have been investigated (Dong et al 2013;Franden et al 2009Franden et al , 2013Gu et al 2015;Yi et al 2015). The results demonstrated that acetate, furfural, and phenolic aldehydes are three major inhibitors in the dilute acid-pretreated corn stover hydrolysate for Z. mobilis, and the inhibitory activity was strongly correlated to the hydrophobicity of these compounds.…”

“…For example, transcriptomic profiles of Z. mobilis wild-type strain ZM4 or its xyloseutilizing derivative 8b in response to furfural, acetate, or phenolic aldehyde inhibitors (4-hydroxybenzaldehyde, syringaldehyde, and vanillin) have been investigated (He et al 2012;Yang et al 2014a;Yi et al 2015). In addition, integrated proteomic and transcriptomic approaches were used to understand the molecular mechanism of an acetate-tolerant strain (Yang et al 2014c).…”

“…Based on these studies, many candidate genes responsible for the inhibitor tolerance in Z. mobilis have been identified, and recombinant strains with enhanced tolerance to lignocellulosic hydrolysates have been constructed by engineering these candidate genes into parental strains. For example, everal reductase-encoding genes (ZMO1116, ZMO1696, and ZMO1885) were identified to play key roles in response to phenolic aldehydes through transcriptomics study (Yi et al 2015). Overexpression of these genes increased the tolerance of Z. mobilis ZM4 against hydrolysate inhibitors, especially 4-hydroxybenzaldehyde and vanillin.…”

Evaluation of Cellulase Production by Zymomonas mobilis

“…"Z. mobilis cellulase," BioResources 12(1), 1165-1178. 1166 production of byproducts, lower biomass production, no requirement for controlled addition of oxygen during fermentation, and tolerance to toxic inhibitors and substrates (Rogers et al 2007;Yang et al 2014;Gu et al 2015;Yi et al, 2015). These characteristics make Z. mobilis a promising ethanologenic microorganism for large-scale production of bioethanol from sugar rich substrates or lignocellulosic materials; the technology can be further developed to provide a CBP microbe in the ethanol production using lignocellulosic material (Sarks et al 2016).…”