Breeding Of Phaffia Rhodozyma Astaxanthin-overproducing Strain By Genome Shuffling

Astaxanthin is a high-value additive carotenoid with different physiological functions. Phaffia rhodozyma has the natural ability of producing astaxanthin and has some advantages just like shorter period of fermentation, easy to culture in a high density. The research is aimed to improve the Phaffia rhodozyma strain by genome shuffling, and the results are as follows.Wildtype strain As 2.1557 was treated with ultraviolet radiation and NTG, three mutants were selected with the selective pressure of 75μmol/L DPA. The best of the three, the concentration of astaxanthin was 1.188 mg/L, which was 0.48 higher than that of the parent As 2.1557. The mutant is genetically stable.The conditions for protoplast preparation, regeneration and f usion were studied. The effects of seed age, the lysozyme concentration, the lysozyme treating time and temperature on preparation and regeneration of protoplast were studied. At the optimal lysozyme treating coditions obtained through response surface methodology, the product of protoplast preparation and regeneration was above 60%. The effects of the PEG concentration, the Ca2+ concentration, the fusion time and temperature on protoplast fusion were studied, and obtained the optimal conditions of protoplast fusion. The protoplasts were inactivated by UV and heat respectively and then fusion between biparental inactivated protoplasts was carried out.The three strains we obtained after complex mutation and another mutant preserved in our lab were selected as parent strains for genome shuffling. After 2 cycles of recursive protoplast fusion, a shuffled strain named P. rhodozyma F2-11 was selected, the concentration of astaxanthin in it was 1.950mg/L, which was 1.43 higher than that of the parent As 2.1557.Last, we used metabolic flux analysis (MFA) to probe the characters of wild strain, the mutant and the shuffled strain. Comparing the flux distribution of the three strains, we found that the pentose phosphate flux of shuffled strain was lower than the mutant, while the EMP and TCA cycle flux were higher; the requirement of biomass was also a little higher than that of the mutant. Pyruvate dehydrogenase was still inefficient and about 32% pyruvate was excreted. The enzyme assays proved that the activities of pyruvate dehydrogenase in shuffled strains was only a little higher, but there's much improve on the activities of ATP: citrate lyase which is affinitive with the acetyl coenzyme A (a precursor of astaxanthin). That somehow illuminated the mechanism that the shuffled strains could over-prouduce astaxanthin.