Breeding Of Thermotolerant Astaxanthin-producing Strains

Astaxanthin, a kind of carotenoids, is widely used in aquaculture, pharmaceuticals,cosmetics etc. with pigmentation effect, ultra-strong antioxidative activity and function ofanticancer, immuno-enhancement, skin aging preventing effect and so on. Phaffiarhodozyma is attracting widespread interest in producing astaxanthin as for its highproportion in carotenoids. However, the strain cannot produce astaxanthin normally above22℃, so a large amount of energy is consumed to control the fermentation temperature,especially in hot areas. To reduce energy cost, a thermotolerant astaxanthin-producingstrain is of great importance.In this study, protoplast fusion method was utilized to breed thermotolerant strain andgenome shuffling was used to improve its astaxanthin production.(1) The protoplasts were prepared from Phaffia rhodozyma, Saccharomycescerevisiae, Rhodotorula glutinis and Pichia pastoris. Protoplast fusion was used to obtainhybrids between P. rhodozyma and S. cerevisiae, R. glutinis or P. pastoris. We obtainedpigment-producing hybrids which could not grow at temperature above26℃by fusion ofP. rhodozyma and S. cerevisiae. Hybrids by fusion of P. rhodozyma and R. glutinis canproduce pigment and grow at30℃, but there was no or little astaxanthin tested by HPLCin pigment extract. Hybrids PP11which can produce astaxanthin as the main pigment andgrow at30℃were obtained by fusion of P. rhodozyma and P. pastoris, and it had theexpected characteristics.(2) The colonies of PP11were round and red with regular edge and wet smoothsurface. The ellipsoidal cells of PP11were similar to P. rhodozyma in shape, and close tothe sum of volumes of its parent strains P. rhodozyma and P. pastoris in size.(3) The optimum temperature for PP11to grow and produce astaxanthin was provedto be30℃. It was demonstrated in astaxanthin synthesis curve that the astaxanthinproduction was up to5.99mg/L after incubation for72h in flask, while the biomass andastaxanthin content were up to21.9g/L and273μg/g DCW respectively. Compared withP. rhodozyma, the biomass of PP11increased by76.5%, while the astaxanthin production and astaxanthin content were respectively reduced by47.08%and70%.(4) Genome shuffling was used to improve astaxanthin production of thermotolerantstrain. Several strains with3.53-4.71%higher astaxanthin production than PP11wereobtained through cooperation mutagenesis of ultraviolet and NTG, and then were used asoriginal strains for genome shuffling, followed by the screening of diphenylamine and96-well plate culture. After three round genome shuffling, two strains named PPG3-115and PPG3-211were obtained, whose astaxanthin production reached7.29mg/L and7.32mg/L respectively,32.55%and33.09%comparing with PP11respectively.