The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors

Abstract

BackgroundZymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.ResultsWe generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.ConclusionsWe have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

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

“…Overexpression of these genes increased the tolerance of Z. mobilis ZM4 against hydrolysate inhibitors, especially 4-hydroxybenzaldehyde and vanillin. Transcriptomic studies also led to the identification of a transcriptional regulator hfq (ZMO0347), which was differentially regulated in response to various lignocellulosic hydrolysate inhibitors in Z. mobilis, and its knockout mutant reduced resistance to acetate, vanillin, furfural, and HMF (Yang et al 2009b(Yang et al , 2010b.…”

Discovery of Ethanol-Responsive Small RNAs in Zymomonas mobilis

“…To validate sRNA expression from the pool of all candidates, we performed large-scale Northern blotting. Cells were grown anaerobically and collected for RNA extraction in stationary phase given that Z. mobilis Hfq has been shown to be more abundantly expressed in anaerobic, stationary phase than in aerobic, stationary phase (26). As Hfq is known as a global sRNA regulator (39), we reasoned that there was a higher chance to identify (and experimentally validate) sRNAs under this condition.…”

“…Hfq is a conserved bacterial Sm-like family of RNAbinding proteins, particularly in Gram-negative bacteria, that can bind sRNAs and their target mRNAs to direct functionality (24,25). In addition, Hfq has been shown to play an important role in tolerance to multiple biomass pretreatment inhibitors such as acetate, vanillin, and furfural (26) in Z. mobilis. Collectively, these findings supported our initial hypothesis regarding the possibility that sRNAs play important mechanistic roles under differential oxygen (and thereby ethanol) conditions in this bacterium.…”

Markerless Gene Deletion System for Sphingomonads

“…In addition, most strains were naturally sensitive to kanamycin and resistant to carbenicillin (Table 1), indicating that pAK405 and the gene deletion strategy via conjugal transfer as outlined above can also be used for these organisms. Finally, pAK405 or the general strategy of generating streptomycin-sensitive rpsL alleles as described here might also be applicable to closely related bacteria not belonging to the sphingomonads, such as Erythrobacter, Citromicrobium, or Zymomonas species, which are naturally streptomycin resistant and whose rpsL alleles encode 33,34). …”