Transformation of Zymononas mobilis by electroporation

Lam, Cuong K.; O'Mullan, Patrick; Eveleigh, D. E.

doi:10.1007/bf00192083

Cited by 9 publications

(9 citation statements)

References 25 publications

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“…In preliminary experiments by original protocols based on the reported papers , the transformation of ZM4 with pZB21‐mini, pZB21 and pZA22, and pBBR1MCS‐2 by electroporation was all successful, showing that those plasmids could replicate stably in Z. mobilis . At the same time, their transformation efficiencies were found to differ greatly from each other.…”

Section: Resultsmentioning

confidence: 98%

“…In our preliminary experiments by original protocols based on the reported papers , transformation of ZM4 with plasmids by electroporation was found to generally be more ineffective than CP4. In this work, in order to obtain a more universal and effective electrotransformation protocol of ZM4 by different kinds of plasmids, ZM4 was first transformed by several replicative plasmids, and the influences of many key electrical and biological parameters on the transformation efficiency were studied.…”

Section: Introductionmentioning

confidence: 86%

“…Among the diverse transformation protocols proposed for Z. mobilis , electroporation is the most convenient method and has been reported . But in most of these papers, the electroporation protocols were only briefly described as part of the methods section and constituted minor points .…”

Section: Introductionmentioning

confidence: 99%

“…Most of the above‐discussed papers were related to transformation of Z. mobilis with replicative plasmids by electroporation , with three papers related to chromosomal integration of Z. mobilis with integrating plasmids by electroporation . These three papers have all reported Z. mobilis ATCC29191 as the host strain.…”

Section: Introductionmentioning

confidence: 99%

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Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

Zou

Zhang

You

et al. 2012

Engineering in Life Sciences

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Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmidsThe Zymomonas mobilis ZM4 strain with excellent ethanol-producing capabilities was the first strain of Z. mobilis, which was sequenced. This strain is resistant to transformation, and no previous study has shown a detailed protocol for electrotransfer of ZM4 with foreign DNA. In this work, many electrical and biological parameters were selected and evaluated in order to optimize the electrotransformation of ZM4. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants/mg DNA were separately achieved with shuttle plasmid pZB21-mini (3082 bp), pZB21 (5930 bp), pZA22 (6994 bp) and broad-host-range vector pBBR1MCS-2 (5144 bp) all prepared from Escherichia coli JM110. The crucial factors affecting the transformation efficiency included the source of the plasmid (the best strain was ZM4), origin and size of the plasmids, growth phase of the cells (the most ideal phase was early log phase with OD 600 of 0.3-0.4), the electric field strength (generally 11.75 kV/cm-13.25 kV/cm) and the recovery time (3-24 h). Further, based upon the optimal transformation protocol mentioned above for replicative plasmids in ZM4, (i) the electrotransformation by recombinant plasmid pBBR1MCS-2-Pgap-FLP (6880 bp) was an immediate success with the transformation efficiency 10 2 transformants/mg DNA; (ii) the site-specific integration efficiencies (expressed in terms of ''per mg of DNA'') of 3-6 integrating transformants was obtained using the integrating plasmid pBR328-ldhR-cml-ldhL (7447 bp). This study will assist genetic and biotechnological research of ZM4 and other Z. mobilis strains by providing information about suitable vectors and a more universal and reliable procedure for introducing DNA into this strain.

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Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

Zou

Zhang

You

et al. 2012

Engineering in Life Sciences

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“…Only a few reports have been published on whether microgravity affects secondary metabolism of microorganisms 54. However, ground‐based studies were carried out in our laboratory to help us predict whether secondary metabolite biosynthesis occurs in space and whether any of its characteristics are altered.…”

Section: Discussionmentioning

confidence: 99%

Secondary metabolism in simulated microgravity

Demain

Fang

2001

The Chemical Record

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We have studied microbial secondary metabolism in a simulated microgravity (SMG) environment provided by NASA rotating-wall bioreactors (RWBs). These reactors were designed to simulate some aspects of actual microgravity that occur in space. Growth and product formation were observed in SMG in all cases studied, i.e., Bacillus brevis produced gramicidin S (GS), Streptomyces clavuligerus made beta-lactam antibiotics, Streptomyces hygroscopicus produced rapamycin, and Escherichia coli produced microcin B17 (MccB17). Of these processes, only GS production was unaffected by SMG; production of the other three products was inhibited. This was determined by comparison with performance in an RWB positioned in a different mode to provide a normal gravity (NG) environment. Carbon source repression by glycerol of the GS process, as observed in shaken flasks, was not observed in the RWBs, whether operated in the SMG or NG mode. The same phenomenon occurred in the case of MccB17 production, with respect to glucose repression. Thus, the negative effects of carbon source on GS and beta-lactam formation are presumably dependent on shear, turbulence, and/or vessel geometry, but not on gravity. Stimulatory effects of phosphate and the precursor L-lysine on beta-lactam antibiotic production, as observed in flasks, also occurred in SMG. An almost complete shift in the localization of produced MccB17 from cells to extracellular medium was observed when E. coli was grown in the RWB under SMG or NG. If a plastic bead was placed in the RWB, accumulation became cellular, as it is in shaken flasks, indicating that sheer stress favors a cellular location. In the case of rapamycin, the same type of shift was observed, but it was less dramatic, i.e., growth in the RWB under SMG shifted the distribution of produced rapamycin from 2/3 cellular:1/3 extracellular to 1/3 cellular:2/3 extracellular. Stress has been shown to induce or promote secondary metabolism in a number of other microbial systems. RWBs provide a low stress SMG environment, which, however, supports only poor production of MccB17, as compared to production in shaken flasks. We wondered whether the poor production in RWBs under SMG is due to the low level of stress, and whether increasing stress in the RWBs would raise the amount of MccB17 formed. We found that increasing shear stress by adding a single Teflon bead to the RWB improved MccB17 production. Although shear stress seems to have a marked positive effect on MccB17 production in SMG, addition of various concentrations of ethanol to RWBs (or to shaken flasks) failed to increase MccB17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the RWB were much more resistant to the growth- and production-inhibitory effects of ethanol than cells growing in shaken flasks. With respect to S. hygroscopicus, addition of Teflon beads to the RWB reversed the inhibition of growth, but rapamycin production was still markedly inhibited, and the distribution...

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Zymomonas mobilis: an alternative ethanol producer

Panesar

Marwaha

Kennedy

2006

J of Chemical Tech & Biotech

126

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Zymomonas mobilis is a unique bacterium in the microbial world, and offers a number of advantages over the existing ethanol-producing microorganisms. Being a prokaryote, it is more amenable to genetic manipulations. Thus, it has attracted great attention in the ethanol production world and efforts have been made to commercialize its application for the purpose. Despite the various efforts made worldwide, none of the processes using this microbe has been commercialized owing to certain bottlenecks. To circumvent the hindrances currently associated with a Zymomonas process, researchers have made various attempts to improve the technology using different techniques. This paper reviews the different substrates and the genetic improvement techniques with special emphasis on mutagenesis and recombinant DNA technology used for ethanol production by Zymomonas strains.

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Transformation of Zymononas mobilis by electroporation

Cited by 9 publications

References 25 publications

Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

Secondary metabolism in simulated microgravity

Zymomonas mobilis: an alternative ethanol producer

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