| This study explored strategies for developing carotenoid producing Lactobacillus casei using genetic engineering, in a hope that carotenoid biosynthesis could be realized in probiotic strains of lactobacilli . First, an E. coli-lactobacillus shuttle vector pCSVMS was constructed based on the plasmid pCAR16 containing the carotenoid biosynthetic pathway (crt) genes of Erwinia uredovora. This was accomplished by incorporating a strong gram-positive promoter region of lactococcus expression vector pMG36e and a gram-positive origin of replication of pAMβ1 derived vector pIL253 plasmid, into pCAR16 to create pCSVMS. After transformation, the resulting vector both successfully expressed carotenoid biosynthesis in E. coli and shuttled between E. coli and Lactobacillus casei, while maintaining structural stability. Since carotenoid biosynthesis was not expressed in Lactobacillus casei harboring pCSVMS, genetic modification of the vector pCSVMS was attempted in order to achieve carotenoid production in Lactobacillus casei through site-directed mutagenesis of the original ribosome binding site of the crt gene cluster. To further explore ways to achieve carotenoid production in Lactobacillus casei, another shuttle vector, pCMS was constructed using the polymerase chain reaction to introduce gram-positive plasmid derived ribosome binding sites in front of each subsequent crt gene in the cluster. To promote transcription, the promoter and ribosome binding site from the Lactococcus lactis subsp. lactis derived vector pMG36e, was inserted upstream of the crt gene cluster. The former sequences were positioned such that translation of the first crt gene (crtE) of the cluster was coupled to translation of a short open reading frame terminating within the crtE initiation site. The recombinant plasmid pCMS was electroporated into Lactobacillus casei after addition of a gram-positive origin of replication from pIL253 and transformants were obtained. Restriction analysis of the plasmids prepared from the recombinant strain of Lactobacillus casei confirmed that the shuttle vector pCMS was structurally intact and stable in Lactobacillus casei although carotenoid production was not yet achieved. |
