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Background  Lactic 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.    Results  Best transformation efficiencies were obtained using the strain  L. plantarum  CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 10 9  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.    Conclusions  Transformation 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.

microbial cell factories

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