【作者】 孙乃霞；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2006， 硕士

【摘要】 虾青素是一种高附加值的酮式类胡萝卜素,具有极强的淬灭单线态氧和清除自由基的能力,在食品、医药等方面有广阔的应用前景。红法夫酵母天然可产虾青素,且具有发酵周期短,易于实现高密度培养等特点而受到广泛关注。目前的研究热点主要是高产菌株的筛选和发酵条件的优化。本论文以野生型红法夫酵母As2.1557作为出发菌株,经过两轮紫外线诱变和两轮亚硝基胍诱变,以10-4-10-3 mol/L的β-紫罗酮作为抗性筛选剂,最终选育出一株虾青素高产菌株UV-N2-7。在未优化条件下,此突变株虾青素的含量和产量分别为933.8μg/gDCW和1.614 mg/L,较出发菌株分别提高了2.3倍和82%,且遗传性能稳定。本论文利用单因素实验研究了突变株UV-N2-7的摇瓶发酵条件。分别考察了发酵培养基中不同的碳源、氮源、培养基的初始pH值、以及种龄、接种量、装液量(溶氧)、发酵温度等因素对红法夫酵母生长以及虾青素合成的影响。并根据单因素发酵实验的结果,采用统计学方法优化红法夫酵母合成虾青素的发酵过程。首先利用Plackett-Burman设计法考察了培养基组分和发酵条件中的10个因素对菌体生长和虾青素合成的影响,从中筛选出三个影响虾青素产量的关键因素:碳源浓度、初始pH值和发酵温度。随后采用最陡爬坡实验确定最大响应区域,最后利用响应面分析得到虾青素合成的最佳培养基组成和培养条件为:蔗糖31.7 g/L,氮源(牛肉膏:(NH4)2SO4=1:1) 5 g/L, KH2PO4 2 g/L,MgSO4·7H2O 0.5 g/L,酵母抽提物0.2 g/L,NaCl 0.1 g/L,CaCl2·2H2O 0.1 g/L,初始pH 5.05,种龄24 h,接种量5%,装液量40 mL/500 mL三角瓶,发酵温度17.8℃,在该最优条件下发酵,虾青素的产量可达到6.338 mg/L,含量为1242μg/gDCW。本文运用代谢通量分析的方法,分析和对比了分批发酵时出发菌株和突变株的代谢通量分布情况。结果表明:突变株PP途径的通量较野生株有所减小,但是EMP途径和TCA循环的途径的通量增大;突变株用于生物质合成的通量略有减小;二者丙酮酸脱氢酶的反应效率均较低,约有32%的丙酮酸分泌到胞外。因此丙酮酸是红法夫酵母代谢过程中的关键节点,预计通过遗传改造和发酵控制增大丙酮酸到乙酰辅酶A的通量应可能会进一步提高虾青素的产量。更多还原

【Abstract】 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.更多还原