| Ephedrine is widely used in clinical medicine because of its great pharmacological effects. Due to its mild reaction conditions and outstanding stereo chemical specificity, biotransformation reaction using microorganisms and enzymes is an ideal way for producing ephedrine and has great development potential, but a problem of which is the reaction needed coenzyme provide the reducing power. If the ephedrine producing strain has the ability of regenerating coenzyme, the cost of biotransformation would be decreased sharply. This thesis focuses on the problem of coenzyme regeneration during the biotransformation process in the early experiences of this laboratory. In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, B. subtilis was chosen as host and two recombinant strains were constructed which were mldh free expression strain and mldh integrated expression strain.B. subtilis was used as host and the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette in this thesis. First, mldh gene together with the expression cassette were inserted into plasmid pHY300plk and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600, a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD) was obtained. The results for whole-cell biotransformation from this recombinant B. subtilis showed that it could catalyze MAK (1-phenyl-1-keto-2-methylaminopropane) to d-pseudoephedrine without adding coenzyme in the presence of glucose. Further experiments showed that, under the conditions of 0.4 mg MAK and 18 mg glucose, 0.2 g 12 h-cultivated recombinant strain could have a yield of d-pseudoephedrine up to 97.5 mg/L and the conversion rate of MAK was 24.1%.After that, using B. subtilis amylase gene amyE as the integration site and kanamycin resistance gene kan from pUB110 as the selection marker, an integrated expression vector pUC19-5E-pmt-loxpkan-3E containing loxp sequences at both ends of kan gene was constructed; and by transformed into B. subtilis Wb600, a recombinant B. subtilis Wb600 (5E-pmt-loxpkan-3E) was obtained. Then, a recombinant enzyme free expression vector pHY300plk-PxylR-cre-trpsD was constructed and transformed into B. subtilis Wb600 (5E-pmt-loxpkan-3E), with which CRE recombinant enzyme gene cre from pSH47 could be expressed under the promoter PxylR from B. subtilis. Subsequently, with the induction of xylose, CRE recombinant enzyme could delete kan gene in chromosomal DNA of B. subtilis Wb600 (5E-pmt-loxpkan-3E). After several times of subculture, the plasmid pHY300plk-PxylR-cre-trpsD could be losted and an integrated strain B. subtilis Wb600 (5E-pmt-loxp-3E) was acquired. The results for whole-cell biotransformation from this integrated B. subtilis showed that it could catalyze MAK to d-pseudoephedrine without adding coenzyme. In further experiments, under the conditions of 0.3 mg MAK and 6 mg glucose, 0.2 g 12 h-cultivated integrated strain could have a yield of d-pseudoephedrine up to 47.2 mg/L and the conversion rate of MAK was 15.5%. This study successfully integrated express the heterologous gene mldh in B. subtilis stably and complete coenzyme regeneration, whose results was useful to explore biotransformation production of d-pseudoephedrine from recombinant B. subtilis. |
