【摘要】 以野生型红法夫酵母As2.1557为出发菌株,依次进行两轮紫外线诱变和亚硝基胍诱变,并以10-4与10-3mol/Lβ-紫罗酮作为筛选剂,得到突变株UV-N2-7,其虾青素产量和含量分别较出发菌株提高81.7%和2.25倍。采用Plackett-Burman设计法及响应面分析法对发酵培养基的组分及发酵条件进行了优化,突变株的虾青素产量由从初始的2.674 mg/L提高到了6.338 mg/L。建立了红法夫酵母的代谢网络,并对比了突变株和野生株间歇培养条件下对数生长期的代谢通量分布。结果表明:突变株与野生株相比,PP途径通量有所减小,而EMP途径和TCA循环有所增强,突变株用于菌体生长的通量有所减小;二者的丙酮酸脱氢酶的活性均较低,分泌至胞外的丙酮酸的代谢通量约为32%。因此预计通过遗传改造和发酵控制提高丙酮酸脱氢酶的活性可能会进一步提高虾青素的产率。更多还原

【Abstract】 Phaffia rhodozyma 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/L β-ionone.The astaxanthin concentration and content of the mutant are 0.817 and 2.25 times higher than the parent,respectively.Plackett-Burman design,steepest ascent and response surface design were used to optimize the components and conditions of fermentation.The concentration of astaxanthin produced by UV-N2-7 was found to be 6.338 mg/L under the optimized conditions.Metabolic network of Phaffia rhodozyma in batch fermentation was constructed,including pentose phosphate pathway,glycolytic pathway,TCA cycle,metabolite requirements for biomass and astaxanthin synthesis.Comparing the flux of the mutant with the wild strain,the following conclusions can be drawn:the relative pentose phosphate flux was lower,while the glycolytic pathway and TCA cycle flux were higher.The precursor requirements were less in mutant.Pyruvate dehydrogenase is inefficient and about 32% pyruvate is excreted.The production of astaxanthin can be improved from the genetic manipulation and fermentation control by enhance the activity of pyruvate dehydrogenase.更多还原