Study On Strain Screening And Breeding Of High Biotin-Producing

Biotin, a water-soluble B-group of Vitamins, played an importantrole in protein and fatty acid metabolism, which could not be synthesizedby our bodies. Because of its significicant biological function, biotin hadbeen used in many fields in the past years. In this paper, strain breedingof high biotin-producing had been studied in detail, including thescreening of biotin-producing strains, classification of biotin-producingstrains,the mutation breeding of the biotin-producing strain bycooperation of UV and LiCl,the detection of biotin by HPLC andconstruction of genetic engineering microorganism of highbiotin-producing strain.According to nutritional requirement of Corynebacteriumammoniagenes, through pre-screening and re-screening, at last obtainedtwo high yield and stable biotin-producing strains. The productivity ofone strain named FD reached191.6μg/L.To know more closely at this strain, based on analysis of thephenotype, physiologic and biochemical specificity and16S rRNA genesequence, it is confirmed that the two soil bacteria strains FD and DJ①belong to Pseudomonas sp.. After UV and LiCl compound mutation, a mutant was obtained fromculture of biotin analogue, its productivity reached207μg/L. The contentof biotin in fermentation liquor was determined by HPLC in this study.The linear regression equation was Y=9566.99695X-20893.30388，R2=0.9992, there was a good linear relationship between absorptive peakarea (y) and the content of biotin (x) in the range of15~200μg/mL.The experiment had constructed recombinant expression vectorpBBR1MCS-5bioB, and then it was transformed into biotin-producingPseudomonas FD by the method of triparental conjugative transfer.Therefore, the gene engineering strain was obtained. The result ofRT-PCR proved that the bioB gene was transcribed in PseudomonasFD-bioB successfully. The HPLC detection results showed that the biotinyield of engineering strain was929.24μg/L, which was higher than wildstrain by3.85times.