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

【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.