Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis

Reynen, Michael; Reipen, Ina; Sahm, Hermann; Sprenger, Georg A.

doi:10.1007/bf00265073

Cited by 28 publications

(18 citation statements)

References 33 publications

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“…The MIC was defined as the lowest concentration of antibiotic that resulted in lack of visible growth on GYx plates after 48 h at 30 ° C. Transformants were selected by spread plating on GYx plates with Ap (600 ~tg/ml) and/ or Km (350 gg/ml). Z. mobilis expression vectors pPTZ-3, and pPTZ-7 (Reynen et al 1990) were also used with tetracycline (Tc) as a selective marker. E. coli transformants were selected on LB plates containing Ap (50 gg/ml) and/or Km (25 gg/ml) or Tc (15 rtg/ml).…”

Section: Methodsmentioning

confidence: 99%

Transformation of Zymononas mobilis by electroporation

Lam

O'Mullan

Eveleigh

1993

Appl Microbiol Biotechnol

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Zymomonas mobilis is being considered for the industrial production of ethyl alcohol. Expansion of its substrate range of glucose, fructose and sucrose could be advantageous, but genetic manipulation of Z. mobilis is restricted as it is resistant to transformation. We present a protocol using electroporation for high efficiency transformation of this bacterium. Optimal parameters included cooled cells (0-4 ° C), use of 10% glycerol as an osmotic support medium, a pulse in the 12 kV/cm range for 10 ms and outgrowth on GYx medium prior to selection. The routine efficiency achieved was greater than 1.0× 107/~tg DNA, a major increase over other transformation methods in which yields ranged from 100-2000/~g DNA.

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Section: Methodsmentioning

confidence: 99%

Transformation of Zymononas mobilis by electroporation

Lam

O'Mullan

Eveleigh

1993

Appl Microbiol Biotechnol

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“…They were introduced into Z. mobilis CP4 on a broad host range expression vector (pPTZ om3 derived from pPTZ3; Reynen et al 1990). The construction of vectors pZY 225 and pZY 228 is depicted in Fig.…”

Section: Expression Of Xylose Catabolic Genes In Z Mobilismentioning

confidence: 99%

“…2. The xylAB genes were thus under the control of the strong and constitutive pdc promoter of Z. mobilis (Reynen et al 1990). …”

Section: Expression Of Xylose Catabolic Genes In Z Mobilismentioning

confidence: 99%

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

Feldmann

Sahm

Sprenger

1992

Appl Microbiol Biotechnol

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The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. I n cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640mU/mg), phosphoribose isomerase (1600mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action Of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO2 and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly.

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“…PDC is one of the most abundant proteins in Z. mobilis. Previous studies showed that pdc promoter was a strong promoter in Z. mobilis (Reynen et al 1990). In previous work and our study, it was supposed that this promoter could also promote gene expression in E. coli.…”

Section: Construction Of the Cell-surface Expression Plasmidsmentioning

confidence: 98%

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

Hong

Zhang

2008

Biotechnol Lett

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A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.

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Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis

Cited by 28 publications

References 33 publications

Transformation of Zymononas mobilis by electroporation

Transformation of Zymononas mobilis by electroporation

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

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