低氮促进红发夫酵母合成虾青素机理的研究

The Mechanism of Low Nitrogen Promoting Astaxanthin Biosynthesis in Phaffia rhodozyma

培养基中初始氮(硫酸铵)浓度对红发夫酵母虾青素合成有明显的影响,低氮能够促进红发夫酵母合成虾青素。运用代谢通量分析的方法,定量分析不同氮浓度下红发夫酵母的中心碳代谢表明:生成乙酰辅酶A的反应的通量与合成虾青素反应的通量正相关,并且这两个通量都随培养基中初始氮浓度的降低而升高。这说明乙酰辅酶A是红发夫酵母合成虾青素的一个限制因素。进一步测定红发夫酵母丙酮酸代谢相关酶的活性表明:参与丙酮酸脱氢酶旁路的两种酶———乙醛脱氢酶和乙酰辅酶A合成酶在红发夫酵母中的活性极低;然而,柠檬酸裂解酶的活性较高,并且与红发夫酵母虾青素的合成明显呈正相关。该结果说明,柠檬酸裂解反应决定着红发夫酵母细胞质中乙酰辅酶A的供给,从而显著影响着其虾青素的合成。低氮条件能够提高柠檬酸裂解酶的活性和限制蛋白质的合成,从而增加乙酰辅酶A的供给,促进红发夫酵母合成虾青素。

The initial nitrogen （ammonium sulfate） concentration remarkably affected astaxanthin biosynthesis in Phaffia rhodozyma. Low nitrogen concentration promoted the biosynthesis of astaxanthin of P. rbodozyma. Metabolic flux analysis was utilized to characterize the influences of nitrogen concentration on the central metabolism of P. rhodozyrna. The flux of acetyl-CoA production was positively correlated with the flux of astaxanthin biosynthesis and both of these two fluxes increased with the decrease of the nitrogen concentration. This suggested that acetyl- CoA was a limiting factor for astaxanthin biosynthesis in this yeast. Further more, the enzyme assays were employed to investigate the pyruvate metabolism of P. rhodozyrna in detail. The activities of acetaldehyde dehydrogenase and acetyl-CoA synthetase, two enzymes functioning in pyruvate dehydrogenase bypass, were extremely low. In contrast, the activity of ATP： citrate lyase was much higher. Furthermore, the activity of ATP： citrate lyase was positively correlated with the biosynthesis of astaxanthin in P. rhodozyma. These results showed that ATP： citrate lyase reaction determines the supply of acetyl-CoA, the precursor for astaxanthin biosynthesis, in cytosol of this yeast. Low nitrogen concentration improved the activity of ATP： citrate lyase and limited the synthesis of protein, thereby increasing the supply of acetyl-CoA and promoting the biosynthesis of astaxanthin in P. rhodozyma.