利用代谢工程阻断β-氧化获得烷烃高转化率的研究

Long chain dicarboxylic acid (DCA) is an excellent base material for the production of perfumes and plastics. Recently, more and more DCAs are produced by Candida tropicalis, which can utilize n-alkanes as a carbon source and energy source. The characteristic of Candida tropicalis has been studied and some mutants of it have been used for the industrial production of DCA. However, the transferring of fatty acids forβ-oxidation could reduce the rate of DCA production. The blocking of β-oxidation in Candida tropicalis by metabolic engineering was invesitigated in this project. It was confirmed that the carnitine palmitoyltransferase (CPT) and the carnitine acetyltransferase (CAT) were important enzymes for transferring the fatty acid in Candida tropicalis. To increase DCA production, an integrative DNA transformation plasmid was constructed, in which CAT gene was disrupted by inserting hygromycin B resistance gene into CAT gene to block its expression and act as selectable marker. Homologous recombination was accomplished by transforming Candida tropicalis with this plasmid. The activity dermination for CAT and CPT was established, and the kinetic relationship between the activity of CAT and CPT amd the rate of DCA production was studied in pilot. We also studied the characteristics of the recombined strain, the effect of carnitine acetyltransferase disruption on DCA production. It was showed that disruption of CAT was available to increase DCA production.

通过代谢工程改变或阻断热带假丝酵母中的COT酶，使产物DCA不再转运至微体而被?氧化所消耗，从而达到提高转化率和高产的目的。同时通过对COT酶作用机制及其基因的芯浚徊浇沂就樘峄砑唉?氧化和α、ω-氧化机理。本研究在具有重要理论意义耐保步夜突Чひ抵猩锔咝录际醪档慕⒑头⒄固峁┯τ美砺刍