Dataset Information

Multiomic studies of inhibitory effects of lignocellulose-derived inhibitors on xylose utilization in Zymomonas mobilis using chemical defined synthetic hydrolysates

ABSTRACT: Previously we found that modified Zymomonas mobilis is able to convert glucose to ethanol when fermenting highly concentrated hydrolysates such as 9% glucan-loading AFEX-pretreated corn stover hydrolysate (ACSH), but that xylose conversion after glucose depletion is greatly impaired. We hypothesized that impaired xylose conversion is caused by lignocellulose-derived inhibitors (LDIs) present in highly concentrated hydrolysates. To investigate the effects of LDIs on xylose utilization in Z. mobilis, we generated synthetic hydrolysates (SynHs) that contains nutrients and LDI at concentrations found in authentic 9% ACSH. Comparative fermentations of Z. mobilis 2032 using SynH with or without LDI were performed, and samples were collected for endproduct, transcriptomic, metabolomic, and proteomic analyses. Our data suggest that the overall flux of xylose metabolism is reduced in the presence of LDIs. However, the expression of most genes involved in glucose and xylose assimilation was not affected by LDIs nor did we observe blocks in glucose and xylose metabolic pathways. Several LDI-specific effects were observed including intracellular accumulation of mannose-1P and mannose-6P, down regulation of a Type I secretion system (ZMO0252-0255), and upregulation of sulfur metabolism genes and the RND family efflux pump system (ZMO0282_0283_0285). This efflux pump system is involved in detoxification. Together, our findings identify cellular responses to LDIs and possible causes of impaired xylose conversion that will enable future strain engineering of Z. mobilis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Zymomonas Mobilis

SUBMITTER: Evgenia Shishkova

LAB HEAD: Prof. Joshua J Coon

PROVIDER: PXD015007 | Pride | 2019-10-28

REPOSITORIES: Pride

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			Action	DRS
	ASH_2017_07_25_multi_omics_zymo_10_20170805111943.raw	Raw
	ASH_2017_07_25_multi_omics_zymo_11_20170805132340.raw	Raw
	ASH_2017_07_25_multi_omics_zymo_12_20170805152723.raw	Raw
	ASH_2017_07_25_multi_omics_zymo_13_20170805173103.raw	Raw
	ASH_2017_07_25_multi_omics_zymo_14_20170805193444.raw	Raw

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Publications

Multiomic Fermentation Using Chemically Defined Synthetic Hydrolyzates Revealed Multiple Effects of Lignocellulose-Derived Inhibitors on Cell Physiology and Xylose Utilization in <i>Zymomonas mobilis</i>.

Zhang Yaoping Y Vera Jessica M JM Xie Dan D Serate Jose J Pohlmann Edward E Russell Jason D JD Hebert Alexander S AS Coon Joshua J JJ Sato Trey K TK Landick Robert R

Frontiers in microbiology 20191107

Utilization of both C5 and C6 sugars to produce biofuels and bioproducts is a key goal for the development of integrated lignocellulosic biorefineries. Previously we found that although engineered <i>Zymomonas mobilis</i> 2032 was able to ferment glucose to ethanol when fermenting highly concentrated hydrolyzates such as 9% glucan-loading AFEX-pretreated corn stover hydrolyzate (9% ACSH), xylose conversion after glucose depletion was greatly impaired. We hypothesized that impaired xylose convers ...[more]

PMID: 31787963

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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 and enzymatic hydrolysis of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and eventually the production rate and yield. Acetate is one of the major inhibitors liberated from hemicelluloses during dilute acid pretreatment. An understanding of the toxic effects of acetate on the fermentation microorganism is critical to improving biofuel yields in the process. In addition, the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results: We have observed previously that some pretreatment inhibitors affect growth and performance in Zymomonas mobilis 8b differently when different sugars (e.g. glucose or xylose) are used as substrate. To investigate this phenomenon at the cellular level, microarray technology was used to investigate the acetate stress responses of Z. mobilis 8b when using single carbon sources of glucose or xylose, and mixed sugars of glucose and xylose. We designed a microarray based on the most up-to-date genome annotation for both coding sequences and intergenic regions. In the presence of acetate, 8b still can utilize all the glucose (though xylose utilization was inhibited) with similar ethanol yield although the growth, final biomass, and ethanol production rate were reduced. The presence of acetate caused genes related to biosynthesis, flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Our result indicates that Z. mobilis utilized different mechanism for xylose utilization compared to that of glucose, with even more dramatic results than those caused by treatment of the culture with the inhibitor acetate. Our study also suggests that redox imbalance caused by stressful conditions may trigger a metabolic reaction that leads to the accumulation of toxic intermediates such as xylitol, but Z. mobilis appears to be capable of managing its carbon and energy metabolism through the control of individual reactions to overcome the inhibition caused by stressful conditions. Several target gene candidates based on transcriptomic result have been selected for genetic manipulation and a TonB-dependent receptor knockout mutant was confirmed to have advantage on acetate tolerance. Conclusions: We have gained insights into the molecular responses of the model ethanologenic bacterium Z. mobilis to the inhibitor acetate when grown in different sugar sources. These insights will facilitate future metabolic modeling studies and help further strain metabolic engineering efforts for better xylose utilization and acetate tolerance. Two series of microarray studies using total RNA extracted from Zymomonas mobilis subsp mobilis 8b (an xylose-utilizing recombinant) were carried out to investigate the effect of carbon source and acetate on Z. mobilis. One study compared the acetate effect in either glucose or xylose at exponential phase and another study investigated the acetate effect in mixed sugar of glucose and xylose at three growth phases of exponential, transition, and stationary. Tthree biological replicates were used for each condition.

Project description:Background Lignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results A new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance. Conclusions Our results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.