Screening And Characterization The Carotenoid Profiles Of High Yield Carotenoids Red Yeast Mutants

Carotenoids were widely used in nutritional supplements,antioxidants and other food industries.Production of carotenoids by microbial fermentation had a distinct advantage.Rhodosporidium toruloides is one of the most effective strains for carotenoids production.However,the low carotenoid content per cell impedes their developments for industrial production.In view of the above problems,The Agrobacterium tumefaciens-mediated transformation method was used to induce the mutation of red yeast,and the mutant library was constructed.The transformation efficiency was improved by optimizing the transformation conditions.Mutants with high ptoduction carotenoids were selected from mutant libraries and the insertion sites of the mutants were further analyzed and explained.The research results were as followes:Firstly,the method of Agrobacterium tumefaciens-mediated transformation was optimized,and factors which have effect on transformation efficiency were optimized respectively.The results indicated that,the AGL1 harbored plasmid PZPK-PGK-HYG-Tnos(we named A1),and co-cultivation of Agrobacterium tumefaciens and R.toruloides at a ratio of 1000:1 at the temperature of 26? incubate for 60 h in medium containing 0.1 mM acetosyringone resulted in the highest transformation efficiency(2369 per 105 yeast cells),which was 24 times the reported 100 transformants per 105 yeast cells.Seventeen mutants with different colors from wildtype were selected to be further research objects.Secondly,the fermentation experiments were carried out on mutants and carotenoids characterization and quantitation were anlysed by HPLC-MS.The result showed that,there were significant differences in the yield of carotenoids between the wild type and the mutants.Among them,the total amount of carotenoids in A1-15 was 13.6 times higher than that of wild type,and the yield of torularhodin was as high as 11,5 mg/g DCW,accounting for 88%of the total carotenoid content.The high proportion make it convenient to separate and purify.The production of ?-carotene in Al-3 was 2.1 mg/g DCW,which was 3.0 times more than the wild strain.Furthermore,the carotenoids produced by Al-3 only isomers y-carotene and?-carotene were detected.It is conducive to the subsequent separation and purification due to the high selectivity.Finally,inserted sites were analysed by genome walking.The results indicated that the insertions of T-DNA was randomly.There were 4 mutants with the same inserte sites,it is speculated that the insertion of T-DNA may have a certain preference.Of the 13 sites that have been obtained,11 have been noted.Subsequently,the effects of these sites on the metabolic pathways were analyzed.The results will provide a basis of engineering targets for further genetic engineering.