Study On Astaxanthin-overproducing Strain Breeding, Fermentation Condition Optimization And Metabolic Flux Analysis Of Phaffia Rhodozyma

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. Nowadays many researches are focused on breeding high-producing strains and optimizing culture conditions.In this paper, wildtype strain As2.1557 was treated with ultraviolet radiation and N-methyl-N'-nitro-N-nitrosoguanidine each for two rounds in series, a mutant named UV-N2-7 was selected with the selective pressure of 10-4-10-3 mol/Lβ-ionone. Under the unoptimized fermentation condition, the concentration and content of astaxanthin were 1.614 mg/L and 933.8μg/gDCW, which are 0.82 and 2.3 folds higher than the parent As2.1557, respectively. The mutant is genetically stable.The effects of fermentation conditions on astaxanthin formation were investigated by single factor experiments, including carbon source, nitrogen source, initial pH, seed age, inoculum concentration, medium volume and temperature. Then we evaluated the effects of 10 factors during fermentation by Plackett-Burman design and picked out three most important ones that affected the astaxanthin productivity the most. They are sucrose concentration, initial pH and temperature, which were further optimized followed by steepest ascent and response surface methodology. The obtained optimal condition was: sucrose 31.7 g/L, nitrogen source 5 g/L , KH2PO4 2 g/L,MgSO4·7H2O 0.5 g/L,yeast extract 0.2 g/L,NaCl 0.1 g/L,CaCl2·2H2O 0.1 g/L,initial pH 5.05,temperature 17.8℃,seed age 24 h,inoculum concentration 5%,medium volume 40 mL/500 mL. Under this condition, strain UV-N2-7 could produce astaxanthin of 6.338 mg/L and 1242μg/gDCW by shaking-flask fermentation.We used metabolic flux analysis (MFA) to probe the characters of wild strain and the mutant at mid-log-phase. Comparing the flux distribution of the mutant with the wildtype strain, we found that the pentose phosphate flux was lower, while the EMP and TCA cycle flux were higher; the requirement of biomass was less in mutant. Pyruvate dehydrogenase is inefficient and about 32% pyruvate is excreted. The production of astaxanthin could be improved from the genetic manipulation and fermentation control by enhance the conversion from pyruvate to AcCoA.