Dataset Information

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

ABSTRACT: Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TSS-seq), termination sequencing (term-seq), ribosome profiling, and label-free shotgun proteomic mass spectrometry, across different growth conditions and used these data 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. We developed statistical methods for annotating transcript 5' and 3' ends, enabling the identification of 3,940 TSSs and their corresponding promoters and 2,091 transcription termination sites, which were distinguished from RNA processing sites by the lack of an adjacent RNA 5' end. Our results revealed that Z. mobilis ?A -35 and -10 promoter elements closely resemble canonical Escherichia coli -35 and -10 elements, with one notable exception: the Z. mobilis -10 element lacks the highly conserved -7 thymine observed in E. coli and other previously characterized ?A promoters. The ?A promoters of another alphaproteobacterium, Caulobacter crescentus, similarly lack the conservation of -7 thymine in their -10 elements. Our results anchor the development of Z. mobilis as a platform for synthetic biology and establish strategies for empirical genome annotation that can complement purely computational methods.IMPORTANCE Efforts to rationally engineer synthetic pathways in Zymomonas mobilis are impeded by a lack of knowledge and tools for predictable and quantitative programming of gene regulation at the transcriptional, posttranscriptional, and posttranslational levels. With the detailed functional characterization of the Z. mobilis genome presented in this work, we provide crucial knowledge for the development of synthetic genetic parts tailored to Z. mobilis This information is vital as researchers continue to develop Z. mobilis for synthetic biology applications. Our methods and statistical analyses also provide ways to rapidly advance the understanding of poorly characterized bacteria via empirical data that enable the experimental validation of sequence-based prediction for genome characterization and annotation.

SUBMITTER: Vera JM

PROVIDER: S-EPMC7566282 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

Vera Jessica M JM Ghosh Indro Neil IN Zhang Yaoping Y Hebert Alex S AS Coon Joshua J JJ Landick Robert R

mSystems 20200721 4

Zymomonas mobilis is an ethanologenic alphaproteobacterium with promise for the industrial conversion of renewable plant biomass into fuels and chemical bioproducts. Limited functional annotation of the Z. mobilis genome is a current barrier to both fundamental studies of Z. mobilis and its development as a synthetic biology chassis. To gain insight, we collected sample-matched multiomics data, including RNA sequencing (RNA-seq), transcription start site (TSS) sequencing (TS ...[more]

PMID: 32694125

Similar Datasets

Project description:BackgroundZymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production.ResultsIn this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R 2 values more than 0.7 and 0.9, respectively.ConclusionsThis study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.

Project description:BACKGROUND: Zymomonas mobilis ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of Z. mobilis is required to systematically analyze in silico the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions. RESULTS: The genome-scale metabolic model of Z. mobilis ZM4, ZmoMBEL601, was reconstructed based on its annotated genes, literature, physiological and biochemical databases. The metabolic model comprises 579 metabolites and 601 metabolic reactions (571 biochemical conversion and 30 transport reactions), built upon extensive search of existing knowledge. Physiological features of Z. mobilis were then examined using constraints-based flux analysis in detail as follows. First, the physiological changes of Z. mobilis as it shifts from anaerobic to aerobic environments (i.e. aerobic shift) were investigated. Then the intensities of flux-sum, which is the cluster of either all ingoing or outgoing fluxes through a metabolite, and the maximum in silico yields of ethanol for Z. mobilis and Escherichia coli were compared and analyzed. Furthermore, the substrate utilization range of Z. mobilis was expanded to include pentose sugar metabolism by introducing metabolic pathways to allow Z. mobilis to utilize pentose sugars. Finally, double gene knock-out simulations were performed to design a strategy for efficiently producing succinic acid as another example of application of the genome-scale metabolic model of Z. mobilis. CONCLUSION: The genome-scale metabolic model reconstructed in this study was able to successfully represent the metabolic characteristics of Z. mobilis under various conditions as validated by experiments and literature information. This reconstructed metabolic model will allow better understanding of Z. mobilis metabolism and consequently designing metabolic engineering strategies for various biotechnological applications.

Project description:UNLABELLED: BACKGROUND:High tolerance to ethanol is a desirable characteristics for ethanologenic strains used in industrial ethanol fermentation. A deeper understanding of the molecular mechanisms underlying ethanologenic strains tolerance of ethanol stress may guide the design of rational strategies to increase process performance in industrial alcoholic production. Many extensive studies have been performed in Saccharomyces cerevisiae and Escherichia coli. However, the physiological basis and genetic mechanisms involved in ethanol tolerance for Zymomonas mobilis are poorly understood on genomic level. To identify the genes required for tolerance to ethanol, microarray technology was used to investigate the transcriptome profiling of the ethanologenic Z. mobilis in response to ethanol stress. RESULTS:We successfully identified 127 genes which were differentially expressed in response to ethanol. Ethanol up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. These genes were classified as being involved in a wide range of cellular processes including carbohydrate metabolism, cell wall/membrane biogenesis, respiratory chain, terpenoid biosynthesis, DNA replication, DNA recombination, DNA repair, transport, transcriptional regulation, some universal stress response, etc. CONCLUSION:In this study, genome-wide transcriptional responses to ethanol were investigated for the first time in Z. mobilis using microarray analysis.Our results revealed that ethanol had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to ethanol. Although the molecular mechanism involved in tolerance and adaptation of ethanologenic strains to ethanol is still unclear, this research has provided insights into molecular response to ethanol in Z. mobilis. These data will also be helpful to construct more ethanol resistant strains for cellulosic ethanol production in the future.

Dataset Information

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

Publications

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets