Sucrose utilization by Zymomonas mobilis: formation of a levan.
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ABSTRACT:
SUBMITTER: Dawes EA
PROVIDER: S-EPMC1264922 | biostudies-other | 1966 Mar
REPOSITORIES: biostudies-other
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Project description:BackgroundEfficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.ResultsHeterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.ConclusionsThis study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.
| S-EPMC8645129 | biostudies-literature
Project description:Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3-pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3-5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.
| S-EPMC7272483 | biostudies-literature
Project description:1. Enzymic evidence supporting the operation of the Entner-Doudoroff pathway in the anaerobic conversion of glucose into ethanol and carbon dioxide by Zymomonas mobilis is presented. 2. Cell extracts catalysed the formation of equimolar amounts of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate. Evidence that 3-deoxy-2-oxo-6-phosphogluconate is an intermediate in this conversion was obtained. 3. Cell extracts of the organism contained the following enzymes: glucose 6-phosphate dehydrogenase (active with NAD and NADP), ethanol dehydrogenase (active with NAD), glyceraldehyde 3-phosphate dehydrogenase (active with NAD), hexokinase, gluconokinase, glucose dehydrogenase and pyruvate decarboxylase. Extracts also catalysed the overall conversion of glycerate 3-phosphate into pyruvate in the presence of ADP. 4. Gluconate dehydrogenase, fructose 1,6-diphosphate aldolase and NAD-NADP transhydrogenase were not detected. 5. It is suggested that NAD is the physiological electron carrier in the balanced oxidation-reduction involved in ethanol formation.
| S-EPMC1264921 | biostudies-other
Project description:Zymomonas mobilis subsp. mobilis is an efficient ethanol producer with application for industrial production of biofuel. To supplement existing Z. mobilis genomic resources and to facilitate genomic research, we used Oxford Nanopore and Illumina sequencing to assemble the complete genome of the beer spoilage isolate Z. mobilis subsp. mobilis strain NRRL B-1960.
| S-EPMC5532819 | biostudies-literature
Project description:Zymomonas mobilis subsp. mobilis is one of the most rigorous ethanol-producing organisms known to date, considered by many to be the prokaryotic alternative to yeast. The two most applied Z. mobilis subsp. mobilis strains, ZM4 and CP4, derive from Recife, Brazil, and have been isolated from sugarcane fermentations. Of these, ZM4 was the first Z. mobilis representative strain to be sequenced and analyzed. Here, we report the finishing of the genome sequence of strain CP4, which is highly similar but not identical to that of ZM4.
| S-EPMC3886940 | biostudies-literature
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.
| S-EPMC3495753 | biostudies-literature
Project description:Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.
| S-EPMC4094786 | biostudies-literature
Project description:Zymomonas mobilis ZM4 produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. However, the genetic and physiological basis of the ZM4 response to various industrially-relevant stresses is poorly understood. In this study, the dynamics of ZM4 oxygen stress responses were elucidated by characterizing the transcriptomic and metabolomic profiles of aerobic and anaerobic fermentations using whole-genome microarray analysis and gas chromatography-mass spectrometry. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Under aerobic conditions, much less ethanol was observed with other end-products produced, including acetate, lactate, and acetoin. In the early exponential phase, no genes were detected as being significantly differentially expressed between aerobic and anaerobic conditions via microarray analysis. However, microarray analysis of the stationary phase cultures revealed that 166 genes were significantly differentially expressed by more than two-fold. Quantitative-PCR validated the expression values for seventeen genes from different categories of stationary phase microarray data. Transcripts for Entner-Doudoroff pathway genes and gene pdc, encoding a key enzyme leading to the ethanol production, were at least 30-fold more abundant under anaerobic conditions at stationary phase based on quantitative-PCR results. Expression of stress response genes was found to be greater under oxygen stress conditions. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 under anaerobic conditions contained lower levels of amino acids, glucose and ED pathway intermediates, whereas metabolites such as ribitol, trehalose, myristic acid, glyceric acid, glucose 6-phosphate, mannose 6-phosphate, and 4-hydroxybutanoic acid were more abundant than under aerobic conditions. Transcriptomic and metabolomic data were consistent with faster glucose consumption and greater ethanol production under anaerobic conditions and suggested gene targets for deletion and improved fermentation. Keywords: Time course/ Stress Response
2008-10-10 | GSE10302 | GEO
Project description:Zymomonas mobilis is an ethanologenic bacterium that has been studied for use in biofuel production. Of the sequenced Zymomonas strains, ATCC 29191 has been described as the phenotypic centrotype of Zymomonas mobilis subsp. mobilis, the taxon that harbors the highest ethanol-producing Z. mobilis strains. ATCC 29191 was isolated in Kinshasa, Congo, from palm wine fermentations. This strain is reported to be a robust levan producer, while in recent years it has been employed in studies addressing Z. mobilis respiration. Here we announce the finishing and annotation of the ATCC 29191 genome, which comprises one chromosome and three plasmids.
| S-EPMC3486092 | biostudies-literature
Project description:Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.
| S-EPMC3165638 | biostudies-literature