温度对法夫酵母MK19虾青素合成调控机制的研究

The great demand for astaxanthin is still increasing due to its strong anti-oxidant property against reactive oxygen species (ROS), supporting the immunological system and protecting cells from ageing and diseases such as cancer, etc. The yeast Phaffia rhodozyma has many properties desirable for astaxanthin production,such as the rapid heterotrophic metabolism and high cell yield. However, the wildtype contains quite low astaxanthin (0.03 mg/g) and can only synthesize astaxanthin at low temperatures (17-21°C), resulting low production yield and long producing period. Cell growth of MK19，an astaxanthin over-producing mutant stran, was also inhibited at 28 °C,but the levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis, reached 2mg/g and 7mg/g respectively for MK19 after 48 h at 28 °C, ~2-fold of the levels at 21 or 25 °C. . These findings suggest that the isoprenoid biosynthetic(MVA) pathway is activated at 28 °C in MK19.Strengthening of the MVA pathway is a feasible metabolic engineering approach for the enhancement of astaxanthin synthesis in MK19 or similar producer strains..In this proposal,strain MK19 was chosen as a model. The transcriptomic patterns of strain MK19 at 21 and 28 °C will be compared to determine the candidate enzymes of the MVA that regulated by temperature, also the protein expressions, enzymes activities and metabolic flux will be compared to verifiy the key enzyme(s)in MVK pathway that regulated by temperature. The studies will contribute to understand the molecular regulatory mechanism of temperature on the astaxanthin synthesis in detail, explain the reasons that astaxanthin over accumulation at 28°C in MK19 and lay a good foundation for molecular breeding of astaxanthin overproducing strain of Phaffia rhodozyma.

虾青素极具抗氧化活性、可增强免疫力、预防癌症等，市场需求极大。法夫酵母代谢迅速，易高密度培养，是生产虾青素的理想菌株；但野生菌只能低温(17-21°C)合成虾青素、含量极低(0.03 mg/g)。中温型耐糖高产突变株MK19虾青素含量达野生型的30倍; 28°C下生长虽仍受抑制，但虾青素及支路终产物麦角甾醇含量较低温都增加1倍，表明MK19中两者及上游-甲羟戊酸合成途径被激活，但调控机理尚不清楚。本申请提出，以MK19为出发菌株，通过比较转录组学鉴定其21°C和28°C下基因转录水平差异，尤其是色素及甲羟戊酸途径的基因表达差异，通过蛋白表达、酶活水平及该通路代谢流的比较分析，明确受温度调控的关键酶基因；解释MK19在28°C下虾青素较低温大量积累的原因，初探温度对法夫酵母虾青素合成代谢调控的分子机理，为MK19进一步提高虾青素提供合适改造靶点及法夫酵母高产虾青素突变株的分子育种奠定基础。