“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

Abstract

BackgroundLactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.ResultsBest transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.ConclusionsTransformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

The Impact of Non-electrical Factors on Electrical Gene Transfer

“…The disadvantage of this product is that it is highly methylated. This methylated DNA inhibits the transformation efficiency via electroporation to lactic acid bacteria, the bacteria that was used to produce therapeutic recombinant proteins, by a factor of 1,000(23). Unfortunately, such a simple test has not been found in either cell culture or in vivo tissues in a mammalian system but remains a project worthy of attention.…”

“…It has been shown that the mechanisms for enhanced transformation via electroporation in lactic acid bacteria were eliminating methylation-specific restriction enzyme activity and reducing DNA degradation(23). This explanation cannot exclude the possibility of an increased level of gene expression from the unmethylated DNA.…”

“…Many investigators could not repeat the gene transfer efficiency data from a nucleofection system because they use much larger DNA but not the company-provide GFP plasmid DNA. Other DNA properties that significantly affect the efficiency of gene transfer via electroporation include methylation, restriction enzyme sites, the drug selection gene, composition, and replication(22,23). Each of these will be reviewed here.…”

Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH

“…Lactobacillus plantarum CD033 is a lactic acid bacterium 39 (LAB) that was isolated from grass silage and was previously 40 characterized regarding its use as a possible expression host 41 (Spath et al, 2012a(Spath et al, ,2012b. The species L. plantarum belongs to 42 the mammalian oral-and gastrointestinal natural inhabitants 43 (Kleerebezem et al, 2003), is responsible for fermentation of 44 meat and milk products as well as for preservation of feed raw 45 material such as grass and cereal grains (Wanker et al, 1995;46 Siezen et al, 2010).…”

“…Since LAB have a long history in food and Transformation of E. coli JM109 cells was carried out by 122 electroporation according to Sambrook and Russel (2001). 123 Transformation of L. plantarum was performed by electropora-124 tion as described elsewhere (Spath et al, 2012a). followed by overnight capillary blotting (Southern, 1975).…”