Identification and characterization of ethanol‐inducible promoters of <i>Zymomonas mobilis</i> based on omics data and dual reporter–gene system

Yang, Yongqiang; Rong, Ziyue; Song, Haoyue; Yang, Xiuxiu; Li, Mian; Yang, Shihui

doi:10.1002/bab.1838

Cited by 9 publications

(8 citation statements)

References 37 publications

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“…21 The molecular mechanism for the self-flocculation of Z. mobilis has been revealed, 22,23 which can be employed to develop the self-flocculating phenotype for recombinant strains with different genetic backgrounds. Moreover, exogenous and native CRISPR-Cas genome-editing toolkits and biological part identification and characterization systems have been established in Z. mobilis, [24][25][26][27][28][29] and various heterologous metabolic pathways have been constructed to produce products such as lactate, 2,3-butanediol (2,3-BDO), isobutanol, and ethylene. 20,[30][31][32] The heterologous PHB pathway has also been engineered into Z. mobilis driven by a strong native promoter Ppdc with 0.7% (DCW) PHB being produced.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering ofZymomonas mobilisfor continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Yang

Wang

et al. 2022

Green Chem.

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Section: Introductionmentioning

confidence: 99%

Metabolic engineering ofZymomonas mobilisfor continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Yang

Wang

et al. 2022

Green Chem.

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“…In addition, the genome sequence, functional re-annotation, and substantial systems biology studies and metabolic models of Z. mobilis have been reported [12,15,[17][18][19][20][21][22][23][24]. Recent development of genome editing such as exogenous and native CRISPR-cas toolkits as well as methods for biological part identification and characterization [25][26][27][28][29][30][31] will facilitate heterologous pathway engineering and unravel the underlying mechanisms for balanced production and robustness [32] in Z. mobilis.…”

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confidence: 99%

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Qiu

Shen

Yan

et al. 2020

Biotechnol Biofuels

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Background: Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. Results: In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. Conclusions: This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

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“…We generated a hand-curated set of 14 promoter sequences that spanned an ∼100-fold range in relatively constant expression of the downstream gene, were the single or predominant TSS for the gene, and were ≤200 nucleotides upstream of the gene ( Table 3 ). This constant-expression promoter catalog expands the small but growing set of genetic parts enabling the use of the Z. mobilis chassis for synthetic biology ( 33 , 42 ) and illustrates the power of multiomics data in identifying promoters.…”

Section: Resultsmentioning

confidence: 83%

“…In expanding the number of empirically defined TSSs and cognate promoters in Z. mobilis from a few ( 33 , 42 , 58 ) to more than a thousand (see Data Set S1 in the supplemental material) and applying sequence analyses, our results revealed the surprising lack of conservation of T −7 in the −10 element of housekeeping promoters in Z. mobilis as well as, in retrospect, C. crescentus ( Fig. 5 ).…”

Section: Discussionmentioning

confidence: 88%

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

et al. 2020

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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 σA −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 σA promoters. The σA 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.

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Identification and characterization of ethanol‐inducible promoters of Zymomonas mobilis based on omics data and dual reporter–gene system

Cited by 9 publications

References 37 publications

Metabolic engineering ofZymomonas mobilisfor continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Metabolic engineering ofZymomonas mobilisfor continuous co-production of bioethanol and poly-3-hydroxybutyrate (PHB)

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

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

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