【作者】 张怀渊； 赵建新； 陈永泉； 张灏； 陈卫； 宋元达；

【Author】 Huaiyuan Zhang Jianxin Zhao Yongquan Chen Hao Zhang Wei Chen Yuanda Song (School of Food Science and Technology,Jiangnan University,Wuxi 214122)

【机构】 江南大学食品学院；

【摘要】 本研究主要探索在氮限制培养基（碳源为甘油,氮源为酒石酸铵和酵母提取物）中耶氏解脂酵母油脂积累的机制耶氏解脂酵母在氮限制培养基中发酵培养,并测定发酵过程中几种与油脂积累代谢相关的酶活性变化,同时检测分泌到细胞外柠檬酸和异柠檬酸含量。根据NCBI公布的耶氏解脂酵母苹果酸酶基因（YALI0E18634g）,将其克隆至原核表达载体pET28a（+）,并转入大肠杆菌BL21（DE3）,用IPTG诱导重组苹果酸酶表达Ni-NTA亲和层析柱纯化得到苹果酸酶重组蛋白质,测定重组苹果酸酶对辅酶NAD和NADP的K_m值。结果显示N源耗尽时,AMP脱氨酶活性增加而NAD依赖型异柠檬酸脱氢酶活性降低,这可能是酵母细胞油脂积累的开始。细胞内能提供NADPH的三种酶（苹果酸酶、葡萄糖-6-磷酸脱氢酶、NADP依赖型异柠檬酸脱氢酶）的活性变化并没有和油脂积累相关联。ATP柠檬酸裂解酶活性变化与油脂积累变化趋势一致,可能是耶氏解脂酵母油脂积累的关键酶。最终酵母细胞内油脂含量占细胞干重的18.1%,且同时分泌大量的柠檬酸和异柠檬酸,浓度分别达到51.79 g/1和14.31g/l。原核表达得到的重组苹果酸酶对NAD的K_m为1.57 mM,V_max为714.29nmol/min*mg,而重组苹果酸酶虽能利用NADP,但其对NADP的亲和力很低,无法计算K_m值,揭示苹果酸酶（YALI0E18634p）并不能提供还原力NADPH,可能不是耶氏解脂酵母油脂积累的关键酶。更多还原

【Abstract】 The aim of this study is to explore the mechanism of lipid accumulation in Yarrowia lipolitica, when the yeast was cultivated in N-limited medium（The carbon source is glycerol,the nitrogen source is ammonium tartrate and yeast extract）.The enzyme assays of malic enzyme,glucose-6-phosphate dehydrogenase,NADP-isocitrate dehydrogenase,AMP deaminase,NAD-isocitrate dehydrogenase and ATP citrate lyase were measured,at the same time,the secreted citrate and isocitrate by yeast cells into medium was also determined by HPLC.Accoding to the malic enzyme gene cDNA(YALI0E18634g Sequences in NCBI.The malic enzyme gene was cloned to pET28a（+ ）,and transformed into E.coli BL21（DE3）. Expression of malic enzyme was induced by IPTG,After the recombination protein was purified through Ni-NTA affinity chromatography,the enzyme assays of fusion malic enzyme were accomplished and calculated the K_m for coenzyme NAD or NADP.As the cells ran out of available nitrogen in the medium, AMP deaminase showed a sharp increase in acitivity while the activity of NAD-ICDH was opposite to AMPD,suggesting the onset on lipid accumulation.It was observed that acitivaty of malic enzyme, glucose-6-phosphate dehydrogenase,NADP-isocitrate dehydrogenase,used for producing NADPH,were active throughout the fermentation,were not concomitant with the change of lipid accumulation,and thus may not be the regulating enzyme involved in lipid accumulation.However the change of the activity of ACL was in accordance with the change of lipid accumulation,suggesting that ATP citrate lyase might be the key enzyme involved in lipid accumulation in Yarrowia lipolytica.Finally the lipid content is up to 18.1%.the concertration of citrate and isocitrate in medium was respectively 51.79 g/l and 14.31 g/l.The recombinant malic enzyme had high affinity for NAD with a K_m of 1.57 mM and V_max of 714.29 nmol/min*mg.However,the recombinant malic enzyme had extremely low affinity for NADP,indicating that malic enzyme（YALI0E18634p）con not provide the NADPH for lipid accumulation,might not be the key enzyme in lipid accumulation in Yarrowia lipolytica.更多还原