Construction And Use In Biotin Systhase Gene Expression Of An Expressional Shuttle-Vector PYG43

Bacillus subtilis, harmless to humans and animals, is the best-characterized Gram-positive bacterium known today on genetic, biochemical and physiological level, and can secret a large of industrial important enzymes into the medium. In order to exploit and apply in Bacillus subtilis, several promoters were cloned and compared on the transcriptional activity. Then an efficient expressional and E.coli-Bacillus shuttle vector was constructed with promoter P43 and was assayed on secretion by endo- β -1,4-glucanase activity, and then was primarily applied into the expression of biotin synthase gene.Fifty-seven Bacillus cereus promoter sequences were cloned through random shooting and blue-white screen on X-gal plates. Four of them (C1, C2, C4, C5) can achieve transcription efficiently in E.coli, whith β -galactosidase activity are 1129U, 780U, 1136Uand 1522U, and are limited by glucose. Only two of them(C1,C4) transformed into B.subtilis show β -galactosidase activity. The sequenced result indicates that they contain full promoter region and Shine-Dalgarno sequence.Promoter P43 and Promoter Pamy was obtained by PCR and analyzed on transcriptional activity by 3 -galactosidase activity. The activity by P43 transcription in E.coli and in B. subtilis is similar, which the activity emerges in the early exponential stage, and increases quickly during this stage and then slowly in the plateau stage. The rich nutrition can improve the transcription and expression of P43. The activity by Pamy transcription is just observed (1000U) under induction by starch, otherwise it is near to the host strain.Plasmid pYG43, an E.coli-B.subtilis shuttle vector with a promoter P43, was constructed. Then the expressional activity was assayed by endo-β -1,4-glucanase gene inserted at P43 downstream. B. subtilis 1A747 with pYG43- bglC can product endo-β -1,4-glucanase near 60U/ml into culture medium. The enzyme activity is enhanced 5~7 times than the host strain. The vector is stable and efficient in B.subtilis, and the transcription is efficient and is earlier than the plateau of proteolysis enzymes.pYG43 was applied into the expression of the biotin synthase gene (blo B),which increases the product of biotin to 200mg/L. The result confirms the efficiency of pYG43 and implies the effect of biotin synthase on biotin biosythesis primarily.