Construction Of Recombinant Bacillus Subtilis Strains For Efficient Synthesis Of The Biotin Precursor: Pimelic Acid

Biotin (vitamin H) is a one of the vitamin B family essential for animal to maintain the normal physiological function. Biotin is extensive used in animal husbandry, foodstuff and medicine field. The biotin we need mostly had to import from Japan or American every year, expensively. So it is necessary to research the synthesis of biotin. For the high environmental pollution of the chemosynthesis, recently, it was one of hot spots of high-technologic competition that biotin was produced by fermentation technology. Bacillus subtilis was an excellent host for gene expression and secretion, so it became the preferred strains for the production of biotin.As one of the precursors of biotin, pimelic acid plays an important role in biosynthesis pathways of biotin in B. subtilis. Fermentations with added pimelic acid could improve the production of biotin, however, linked with high cost and environmental burden which limited its application, and it became a bottleneck in the fermentation of biotin desiderated to resolve. Construction of a recombinant strains to improve the biosynthesis of pimelic acid in B. subtilis was only method to solve the problem. People studied a lot in the biosynthesis gene of pimelic acid, there seems to be a consensus that the cistron sequence bioI-orf2 in B. subtilis bio opreon were invloved in pimelic acid formation. The unknown gene, orf3 was juxtaposed with the cistron, it was responsible for the transport of the pimelic acid and biotin, probably.There are two factors influence the expression efficiency of the gene: the strength of the promoter and the copies number of gene. For high instability and low expression efficiency of plasmid in B. subtilis, integration of cloned genes into the chromosome is a strategy used to avoid those problems. In this experiment, we reported the integratioin of a cistron sequence including bioI-orf2-orf3 into the B. subtilis PY79 chromosome by homologous recombinant and those genes were put under the control of a strong maltose promoter Pglv and linked with CmR. We amplified the copy number of the cistron region to improve the synthesis efficiency of pimelic acid.CmR genes (~1.0 kbp) and the cistrons bioI-orf2-orf3 (~2.8 kbp) PCR products were amplified from plasmid pGJ103 and B. subtilis 168 chromosome DNA using the primers Cobio1-up/Cobio2-down and the primers Cm-up/Cm-down respectively. The purified PCR products were subcloned into pGEM-T vector and pBluskm vector to generate pLHX1 and pLHX2, respectively. pYG57 was constructed by ligating a 2.8 kbp EcoRI-XbaI fragment from pLHX2, containing the [bioI-orf2-orf3] cassette and 300 bp ApaI-EcoRI fragment from pGJ113 contained Pglv into corresponding sites of pGEM-T. pLHX5 was constructed by cloning the CmR from pLHX1 into pE3 vector. The purified XbaI-ApaI fragment of pYG57 was inserted into pLHX5 to generate integration vector pLHX8.All ligation products were transformed into E. coli DH5αcompetence cells for amplification by electrotransformation. Whereas the bioI-orf2-orf3 cistron in pLHX8 is homologous to B. subtilis PY79 bio operon, pLHX8 was transformed into B. subtilis PY79 via a single crossover event that formed BpLHX8. After PCR and high CmR (50μg/ml) screen, the copy-number of cistrons in the right colonies were increased to 4~5 copy determined by Southern blot. Reverse phase-high performance liquid chromatography was used to determine the production of pimelic acid in integrative recombinant strains.The results showed that the integration and increase of copy number of the Pglv-bioI-orf2-orf3 cistron sequence in B. subtilis chromosome increase the pimelic acid production. It had been validated the bioI-orf2-orf3 played an important role in biotin biosynthesis in B. subtilis which supplied a reseach strategy and theory foundation for increase of biotin production and low cost.