| γ-decalactone is a food additive, which is mainly used for preparation of cream, strawberries, peaches, citrus and coconut and other spices. Being a lactone-like aroma compound, it has a very rich aroma, and is recognized internationally as a safe food additive and medicine additives.γ-Decalactone obtaining from the microbial fermentation is also regarded as "natural". It was reported that, a non-conventional yeast-Yarrowia lipolytica producedγ-decalactone in a higher yield than other yeasts.In this paper, Yarrowia lipolytica AS2.1045 was used as the starting strain, for the construction of genetically modified strain to the microbial production ofγ-decalactone. The main contents and conclusions were as follows:In order to get high-yieldγ-decalactone and genetic markers, an auxotrophic Yarrowia lipolytica strain was constructed by genetic modification. In this study, URA3, a key gene for uracil synthetase of Yarrowia lipolytica, was disrupted using the method of gene homologous recombination, then transformants were screened by uracil auxotrophic medium(SD-URA) in addition to a certain concentration of 5-FOA and uracil. The results showed that the uracil auxotrophic strain was able to grow at the SD-URA medium containing 5-FOA and uracil while the wild-type strains didn't have the ability, and a rapid method for obtaining auxotrophic Yarrowia lipolytica strain was established.The auxotrophic strains and wild-type strains of uracil-containing medium optimization of fermentation conditions were studied and the best fermentation conditions of auxotrophic strain forγ-decalactone production were obtained as the following: castor-oil plant sesame oil 5%, peptone 0.5%, Tween -80 0.4%, yeast extract 0.25%, MgSO4 ? 7H2O 0.3%, NaCl 0.5%, uracil adding amount 5ng/μl, and then contrast to wild-type strain under the best conditions and the nutrition-deficient strain fermentation yield is very highly. In the 58h or so, the original strain ofγ-decalactone production reached the maximum yield of 199.3 mg/L, while the auxotrophic strains reached the maximum amount of 1032.9 mg/L at a fermentation period of 63h.In the work, a highγ-decalactone yield recombinant yeast strain Yarrowia lipolytica TA1 was constructed by knocking out the key POX3 gene encoding for short chain acyl-CoA oxidases which has a negative impact in the production ofγ-decalactone. The genetically modified strain TA1 was made by introducing the copper-resistance gene CRF1 from yeast Y. lipolytica as a selection marker by homologous recombination.The fermentation conditions of the genetically modified strain TA1 whose POX3 gene was knocked out were optimized. The following factors such as carbon and nitrogen sources, fermentation temperature, pH, inoculum amount, medium volume were studied in a single factor manner. Then the opitimization of the four factors such as carbon source, fermentation temperature, pH, inoculation were carried out by orthogonal experimental of L9(34). The best fermentation conditions were obtained as the following: castor oil 5%; yeast extract 0.7%; peptone 0.25%; MgSO4 ? 7H2O 0.3%; NaCl 0.5%; pH 7.0; Tween-80 0.4%; inoculum 8% (strain concentration 1.6×107 /mL); 115mL medium in a 250 mL flask; temperature of 28℃; rotation speed 150 r/min.After being cultured in shake-flask in the optimum fermentation conditions at 28℃for 100h, TA1 reached aγ-decalactone yield of 630.9 mg/L (highest at 58h), being 2.9 times as high as that of wild type of Yarrowia lipolytica As2.1045 (199.7mg/l, highest at 57h). The variation of the yield ofγ-decalatone was slight after 50 generations, so the heredity stability of mutant strain proven to be good. No heterologous DNA sequences or drug-resistance genes were introduced into TA1. So, the gene manipulations in this work properly solved the addressed problems in commercial fragrance manufacture. |
PDF Full Text Request