Metabolic Remodeling during Nitrogen Fixation in Zymomonas
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Zymomonas Mobilis Subsp. Mobilis Zm4 = Atcc 31821
SUBMITTER: 
Katherine Overmyer
LAB HEAD: Joshua J Coon
PROVIDER: PXD028526 | Pride | 2021-10-07
REPOSITORIES: Pride
Dataset's files
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| 20200228_EAT_G00007460.raw | Raw | |||
| 20200228_EAT_G00007461.raw | Raw | |||
| 20200228_EAT_G00007462.raw | Raw | |||
| 20200228_EAT_G00007464.raw | Raw | |||
| 20200228_EAT_G00007467.raw | Raw |
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Project description:Protein phosphorylation is an important post translational modification that plays a major role in cellular regulatory processes in both eukaryotes and prokaryotes. In recent years, aided by advancements in mass spectrometry techniques, there has been a growing interest in studying protein phosphorylation in prokaryotic model organisms. There are, however, only a limited number of phosphoproteomics reports on non-model organisms. Here, using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins and 172 unique phosphopeptides across these three growth conditions. The phosphoproteins identified belonged to major pathways, including glycolysis, TCA cycle, protein biosynthesis, electron transport, and nitrogen fixation. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across anaerobic, aerobic, and N2-fixing growth conditions. For example, two different serine residues of KDPG aldolase, an Entner-Doudoroff pathway enzyme, were differentially phosphorylated under aerobic and N2-fixing conditions. On the other hand, the final enzyme in the ethanol fermentation pathway, alcohol dehydrogenase, showed phosphorylation on its Ser99 only under N2-fixing condition while its Ser126 was only phosphorylated under anaerobic condition. Moreover, nitrogenases and nitrogen regulatory proteins were differentially phosphorylated at multiple sites under aerobic and N2-fixing conditions. Altogether, this study provides new knowledge regarding potential phosphorylation regulatory sites of specific proteins in pathways relevant to ethanol production and overall physiology and establishes new ground for future engineering of Z. mobilis for advanced biofuel production.
2019-08-08 | PXD014065 | Pride
Project description:Limited functional annotation of the Z. mobilis genome is a current barrier to both basic studies of Z. mobilis and its development as a synthetic-biology chassis. To gain insight, we collected sample-matched multiomics data including RNA-seq, transcription start site sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry across different growth conditions to improve annotation and assign functional sites in the Z. mobilis genome. Proteomics and ribosome profiling informed revisions of protein-coding genes, which included 44 start codon changes and 42 added proteins.
2020-07-10 | PXD016962 | Pride
Project description: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.
2019-10-28 | PXD015007 | Pride
Project description:We engineered Z. mobilis ZM4 to express a recombinant glycosyl hydrolase (GH) from Caulobacter crescentus and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and secretion of GH. These changes were found to be reversible, i.e. not a genetic adaptation, but could be preserved upon transfer to fresh cellobiose medium. After adaptation to cellobiose, our GH expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, adaptation by growth in sucrose medium also enabled immediate growth of Z. mobilis GH3 on cellobiose. Proteomics analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid secretion of GH into the medium. Changes in the cell envelope may additionally enable entry of cellobiose into the cell periplasm or surface display of GHs.
2020-07-29 | PXD015770 | Pride
Project description:Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial biofuel production. Here, we examine the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis, likely caused by oxidative damage to the iron-sulfur co-factors of the final two enzymes of the pathway. This bottleneck was resolved with minimal changes in expression level of enzymes in the pathway but pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e. flavodoxin reductase and the suf operon). We also detected prominent changes in glucose utilization under aerobic conditions. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake after 24 hours of aerobic growth. Our results suggest that under aerobic conditions, electrons from the oxidation of glucose to gluconate are delivered to the electron transport chain to minimize oxidative damage by reducing reactive oxygen species such as H2O2. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome c peroxidase, and a cytochrome bd terminal oxidase.
2019-01-18 | PXD011545 | Pride
Project description:We herein conducted high throughput analyses of proteome and protein acetylation in Z. mobilis under N2-fixing conditions, and established its first acetylome.
2021-11-03 | PXD027418 | Pride
Project description:Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 at different time points of 6, 10, 13.5, and 26h post-inoculation with 6% (v/v) treatment compred to that of control without ethanol supplementation. Two biological replicates for treatment and control condition.
2013-09-04 | E-GEOD-21165 | biostudies-arrayexpress
Project description:Investigation of the expression profiling of the ethanologenic Zymomonas mobilis in response to ethanol stress. A six chip study using total RNA recovered from three separate wild-type cultures of Zymomonas mobilis ATCC31821 and three separate cultures of a triple treated with 5% ethanol. Each chip measures the expression level of 1800 genes from Zymomonas mobilis ATCC31821 and the associated plasmids, with three-fold technical redundancy.
2013-12-31 | E-GEOD-39558 | biostudies-arrayexpress
Project description:Investigation of the expression profiling of the ethanologenic Zymomonas mobilis in response to furfural stress. A six chip study using total RNA recovered from three separate wild-type cultures of Zymomonas mobilis ATCC31821 and three separate cultures of a triple treated with 1.0 g/l furfural. Each chip measures the expression level of 1800 genes from Zymomonas mobilis ATCC31821 and the associated plasmids, with three-fold technical redundancy.
2012-05-08 | E-GEOD-37848 | biostudies-arrayexpress
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.
2014-05-08 | E-GEOD-25443 | biostudies-arrayexpress