Over-expression of xylulokinase in a xylose-metabolising recombinant strain of<i>Zymomonas mobilis</i>

Jeon, Young Jae; Svenson, Charles J.; Rogers, Peter L.

doi:10.1016/j.femsle.2005.01.025

Cited by 42 publications

(6 citation statements)

References 29 publications

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“…2 a, b, though CCR was alleviated in Z. mobilis FR2 fermentation, its xylose utilization rate was still lower than its glucose utilization rate, and there was still a slight amount of xylose that remained (approximately 2.5 g/L) at the endpoint. These results agreed with other Z. mobilis strains, including C25, 39,676/pZB4L [ 27 ], ZM4/Ac R (pZB5, pJX1) [ 28 ], A3 [ 24 ], and AD50 [ 26 ], yet no exact reason of the incomplete xylose utilization has been identified thus far. In Z. mobilis , xylose is transported through a glucose facilitated diffusion protein [ 26 ], which is a native glucose transporter and has low affinity to xylose.…”

Section: Resultssupporting

confidence: 87%

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Sun

Zhang

et al. 2021

Biotechnol Biofuels

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Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

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Section: Resultssupporting

confidence: 87%

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Sun

Zhang

et al. 2021

Biotechnol Biofuels

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“…derivatives of pBBR1MCS, RSF1010 and the incW R plasmid Sa), and have been used for a variety of heterologous gene expression applications. However, they are prone to structural (genetic) instability, and their relatively large size constrains gene cloning strategies [12-15]. Consequently, the most common approach for heterologous gene expression in Z. mobilis has involved E. coli – Z. mobilis shuttle vectors; which incorporate replicons from E. coli plasmids, as well as those from native plasmids isolated from various Z. mobilis strains [12,13,16-22].…”

Section: Introductionmentioning

confidence: 99%

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Chen

Lacap-Bugler

et al. 2014

BMC Microbiol

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BackgroundThe ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications.ResultsA quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli – Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified.ConclusionsWe show that a shuttle vector-based protein affinity ‘pull-down’ approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

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“…Electro-competent cells were prepared following a standard protocol [35]. Plasmid DNA or Gibson assembly reactions (~100 ng) was used to transform of E. coli competent cells (~10 9 cells in 50 μl 10% v/v glycerol in water).…”

Section: Transformations Of E Coli and Z Mobilis Cellsmentioning

confidence: 99%

Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose

et al. 2020

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Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli , Saccharomyces cerevisiae , and Zymomonas mobilis . Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides ( e . g ., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z . mobilis , we engineered a strain ( Z . mobilis GH3) that expresses a glycosyl hydrolase (GH) with β-glucosidase activity from a related α-proteobacterial species, 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 extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells 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, pre-growth of Z . mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z . mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z . mobilis to sucrose that promotes sucrase production.

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Over-expression of xylulokinase in a xylose-metabolising recombinant strain ofZymomonas mobilis

Cited by 42 publications

References 29 publications

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

Heterologous expression of a glycosyl hydrolase and cellular reprogramming enable Zymomonas mobilis growth on cellobiose

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