Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 and acetate-tolerant mutant AcR at different time points of 130, 148, 166, and 190h post-inoculation with 10g/L sodium acetate (NaAc) treatment were carried out to investigate the expression differences between ZM4 and AcR. Two biological replicates for treatment or control condition.

Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment, hydrolysis, and subsequent conditioning of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and in many cases the production titer and rate. An understanding of the toxic effects of compounds found in hydrolysate on the fermentation microorganism is critical to improving biofuel yields in the process. One of the inhibitory compounds is furfural, liberated from hemicelluloses, which strongly inhibits the cell growth and ethanol production especially from xylose. Zymomonas mobilis is a capable ethanologenic bacterium with high ethanol productivity and high levels of ethanol tolerance. The development of robust biocatalyst to tolerate the lignocellulosic pretreatment inhibitors is one of the key elements for economic biofuel production. Results: In this study, the molecular responses of Z. mobilis to furfural, one major pretreatment inhibitor, were investigated using transcriptomic approaches of chip-based microarray. Furfural shock time course experiment with 3 g/L furfural supplemented when cells reach exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the short and long-term effect of furfural on 8b physiological and transcriptional profiles. The presence and supplementation of furfural negatively affect 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural is dynamic, complex and differences exist between short-term shock response and long-term stress response. However, the gene function categories are similar with most downregulated genes related to translation and biosynthesis, while the furfural-upregulated genes were mostly related to cellular processes of general stress response and energy metabolism. Conclusions: Similar to previous report that acetate inhibited the growth of Z. mobilis 8b in RM using glucose or xylose as carbon source, the existence or supplementation of another major hydrolysate inhibitor furfural also inhibited 8b growth with slowing the substrate utilization and ethanol production. The difference between carbon sources is more dramatic than that of the major hydrolysate inhibitors of both NH4OAc (GSE57553) and furfural (this study). Several gene targets have been selected for genetic studies with promising preliminary results.

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Zymomonas mobilis

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