Impact of hfq and sigE on the tolerance of Zymomonas mobilis ZM4 to furfural and acetic acid stresses

Abstract

Zymomonas mobilis, as an ethanologenic microorganism with many desirable industrial features, faces crucial obstacles in the lignocellulosic ethanol production process. A significant hindrance occurs during the pretreatment procedure that not only produces fermentable sugars but also releases severe toxic compounds. As diverse parts of regulation networks are involved in different aspects of complicated tolerance to inhibitors, we developed ZM4hfq and ZM4-sigE strains, in which hfq and sigE genes were overexpressed, respectively. ZM4-hfq is a transcription regulator and ZM4-sigE is a transcription factor that are involved in multiple stress responses. In the present work, by overexpressing these two genes, we evaluated their impact on the Z. mobilis tolerance to furfural, acetic acid, and sugarcane bagasse hydrolysates. Both recombinant strains showed increased growth rates and ethanol production levels compared to the parental strain. Under a high concentration of furfural, the growth rate of ZM4-hfq was more inhibited compared to ZM4-sigE. More precisely, fermentation performance of ZM4-hfq revealed that the yield of ethanol production was less than that of ZM4-sigE, because more unused sugar had remained in the medium. In the case of acetic acid, ZM4-sigE was the superior strain and produced four and twofold more ethanol compared to the parental strain and ZM4-hfq, respectively. Comparison of inhibitor tolerance between single and multiple toxic inhibitors in the fermentation of sugarcane bagasse hydrolysate by ZM4-sigE strain showed similar results. In addition, ethanol production performance was considerably higher in ZM4-sigE as well. Finally, the results of the qPCR analysis suggested that under both furfural and acetic acid treatment experiments, overproduction of both hfq and sigE improves the Z. mobilis tolerance and its ethanol production capability. Overall, our study showed the vital role of the regulatory elements to overcome the obstacles in lignocellulosic biomass-derived ethanol and provide a platform for further improvement by directed evolution or systems metabolic engineering tools.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis

“…As shown in Figure 1D, all glucose was consumed by ZM4-hfq to achieve a maximum ethanol titer of 25.82 g/L within 24 h, almost 6 h faster than that of control, while 6.83 g/L residual glucose was still detected at 36 h by strain ZM4-Δhfq. All these results indicated that hfq in Frontiers in Bioengineering and Biotechnology frontiersin.org Z. mobilis contributes to the ethanol tolerance, just as its positive roles responding to other stresses such as furfural and acetic acid (Yang et al, 2010;Nouri et al, 2020).…”

Recent advances in metabolic engineering of microorganisms for advancing lignocellulose-derived biofuels

“…Recent efforts in Z. mobilis engineering have also enabled several strains to tolerate furfural and acetic acids, which are the predominant toxic inhibitors during lignocellulosic ethanol production [ 92 , 93 ]. Nouri et al [ 94 ] developed recombinant strains with improved tolerance against the multiple inhibitors by overexpressing the genes hfq (a transcription regulator) and sigE (a transcription factor). Furthermore, strain engineered to express sigE also showed 2-fold and 4-fold higher ethanol yields than the hfq-overexpressing strain and parental strain, respectively.…”

Functional Gene Identification and Corresponding Tolerant Mechanism of High Furfural-Tolerant Zymomonas mobilis Strain F211

“…(2) the genes related to macromolecule synthesis are upregulated to resist furfural stress (Miller et al, 2009;Yang et al, 2020); and (3) transcription factors regulate the expression of multiple genes in response to stress (He et al, 2012;Nouri et al, 2020). However, the defined roles of these corresponding genes are not yet known.…”

“…Under furfural stress, the growth and survival of Z. mobilis were inhibited, and the specific production rate of ethanol was correspondingly reduced ( Franden et al, 2009 ; Nouri et al, 2020 ). In addition, furfural also inhibits the synthesis of various proteins, causes DNA damage, and leads to the differential expression of genes involved in membrane and cell wall biogenesis, transcriptional regulators, and energy metabolism ( He et al, 2012 ; Yang et al, 2020 ).…”

“…In addition, furfural also inhibits the synthesis of various proteins, causes DNA damage, and leads to the differential expression of genes involved in membrane and cell wall biogenesis, transcriptional regulators, and energy metabolism ( He et al, 2012 ; Yang et al, 2020 ). Zymomonas mobilis may respond to stress in the following ways: (1) conversion of furfural to furfuryl alcohol reduces toxicity ( Wang et al, 2017 ); (2) the genes related to macromolecule synthesis are upregulated to resist furfural stress ( Miller et al, 2009 ; Yang et al, 2020 ); and (3) transcription factors regulate the expression of multiple genes in response to stress ( He et al, 2012 ; Nouri et al, 2020 ). However, the defined roles of these corresponding genes are not yet known.…”