挖掘与调控乙酸胁迫响应基因提高重组酿酒酵母合成番茄红素水平  

Mining and regulating acetic acid stress-responsive genes to improve lycopene synthesis in recombinant Saccharomyces cerevisiae

作　　者：李佳蓉 林静远 李正宇 段长青[1,2] 燕国梁[1,2,3] LI Jiarong;LIN Jingyuan;LI Zhengyu;DUAN Changqing;YAN Guoliang(Centre for Viticulture and Enology,College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China;Key Laboratory of Viticulture and Enology,Ministry of Agriculture and Rural Affairs,College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China;Key Laboratory of Food Bioengineering(China National Light Industry),College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)

机构地区：[1]中国农业大学食品科学与营养工程学院葡萄与葡萄酒研究中心,北京100083 [2]中国农业大学食品科学与营养工程学院农业农村部葡萄酒加工重点实验室,北京100083 [3]中国农业大学食品科学与营养工程学院中国轻工业食品生物工程重点实验室,北京100083

出　　处：《微生物学通报》2023年第7期2781-2797,共17页Microbiology China

基　　金：国家自然科学基金(31972058);国家重点研发计划(2022YFD2101401)。

摘　　要：【背景】乙酰辅酶A是酿酒酵母异源合成番茄红素的重要中间产物,胞质中乙酰辅酶A主要来自乙酰辅酶A合成酶催化乙酸合成。【目的】通过外源添加乙酸盐结合调控乙酸胁迫应答基因增加胞内乙酰辅酶A含量,改善细胞生长,促进番茄红素合成。【方法】在合成番茄红素的重组酵母菌中过表达乙酰辅酶A合成酶编码基因(acs2),在发酵过程中添加10g/L乙酸盐,结合转录组学分析挖掘乙酸胁迫响应基因,进行单一和组合调控。【结果】添加乙酸盐后,重组菌Y02中番茄红素含量增加了19.14%,但细胞生长受到抑制,转录组学结果表明adk2、fap7、hem13、elo3、pdc5、set5、pmt5、hst4、clb2和swe1表达水平增加,因此构建了单基因和双基因过表达菌株,其中Y02-set5-hst4菌在添加乙酸盐后细胞生长得到了显著改善,同时胞内乙酰辅酶A浓度提高了78.21%,番茄红素含量和产量达到12.62 mg/g-DCW和108.67 mg/L,与对照菌Y02相比分别提高了42.76%和67.13%。同时该菌中甲羟戊酸途径中关键基因erg12、erg20和hmg1的表达量与对照菌相比分别上调了1.70、1.44和1.96倍。【结论】在过表达acs2的基础上,过表达set5和hst4能够增加酵母对乙酸胁迫的耐受能力,并提高乙酰辅酶A合成水平和甲羟戊酸途径代谢通量,促进番茄红素的合成,研究结果可为其他类异戊二烯产物的代谢工程研究提供有益的借鉴。[Background]Acetyl-CoA is an important intermediate for lycopene synthesis in Saccharomyces cerevisiae,and acetyl-CoA in cytosol is mainly derived from acetic acid catalyzed by acetyl-CoA synthase.[Objective]To improve cell growth and lycopene production by increasing the content of intracellular acetyl-CoA through adding acetate combined with regulation of acetic acid stress-responsive gene.[Methods]Lycopene recombinant yeast strains overexpressing acetyl-CoA synthase(acs2)were added with 10 g/L acetate during the fermentation process.The transcriptomic analysis was combined to excavate acetic acid stress-responsive genes for single and combined regulation.[Results]After adding acetate,the lycopene content of the recombinant Y02 strain increased by 19.14%,but cell growth was suppressed.The results of the transcriptional analysis indicated that the expression levels of adk2,fap7,hem13,elo3,pdc5,set5,pmt5,hst4,clb2,and swe1 were increased significantly.Therefore,a single-gene and dual-gene were overexpressed in the Y02 strain.It was found that the growth of Y02-set5-hst4 was significantly improved in the presence of acetate.At the same time,the intracellular acetyl-CoA concentration was increased by 78.21%,and the lycopene content and yield reached 12.62 mg/g-DCW and 108.67 mg/L,respectively,which were increased by 42.76%and 67.13%,respectively,as compared with the control strain Y02.In addition,the expression levels of key genes erg12,erg20,and hmg1 in the mevalonate pathway were increased by 1.70,1.44,and 1.96 folds,respectively,as compared with the control strain.[Conclusion]On the basis of overexpression of acs2,the overexpression of set5 and hst4 can improve the tolerance of yeast to acetic acid stress and increase the synthesis level of acetyl-CoA and the metabolic flux of mevalonate pathway,thus promoting the synthesis of lycopene.The results of this study provide valuable references for the metabolic engineering of other isoprenoid products.

关 键 词：酿酒酵母 番茄红素 乙酰辅酶A 乙酸胁迫 响应基因 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]