合成8二甲基异戊烯基柚皮素的人工酿酒酵母菌株构建

Metabolic Engineering of Saccharomyces cerevisiae for Production of 8-Dimenthylally Naringenin

8二甲基异戊烯基柚皮素(8DN)作为生产黄酮类药物淫羊藿苷的重要前体,在医药合成领域具有重大应用潜力。由于其合成路径及相关基因的复杂性,目前主要通过饲喂8DN的直接前体(柚皮素、异黄腐酚等)的方式合成8DN,而在生物体内全合成8DN的研究工作还未见报道。为了实现8DN在酿酒酵母体内的生物全合成,通过组合筛选8DN前体物柚皮素合成所需的多种外源基因(TAL、4CL、CHS、CHI),获得30株柚皮素生产菌,发现不同来源的基因组合使柚皮素产量的具有明显差异(0.37~22.33mg/L)。并且利用Delta位点将较优的基因组合整合至酵母基因组,实现了稳定的柚皮素高产菌株(Sy BE_Sc02050031)构建。在此基础上进一步导入带有苦参来源的异戊烯基转移酶基因(N8DT)多拷贝质粒,实现8DN合成的完整反应过程,8DN的摇瓶发酵产量达到36.7μg/L。另外,通过关键限速酶N8DT的序列优化策略,发现截断定位信号肽序列的N8DT明显提高了从柚皮素到8DN这一关键反应的催化效果,8DN的产量提高到52.6μg/L(144.2%)。首次在酿酒酵母中成功构建高产8DN的生物全合成路径,为在微生物体内合成其他黄酮类天然产物提供了参考,具有重要的指导意义。

8-dimenthylallynaringenin（8DN） is the important precursor for the flavonoids medicine Icaritin,which has wide applications in pharmaceutical area. Due to the complexity of its biosynthetic pathway and related genes,main attentions were paied to biotransfering by procursor（ like naringenin or isoxanthohumol） feeding. To date,no report about de novo biosynthesis of 8DN has been uncovered. In present study,in order to realize 8DN production in Saccharomyces cerevisiae,four key genes involved in naringenin biosynthesis,TAL,4CL,CHS and CHI from different species,were combinatorially screened,obtaining 30 naringenin producing strains,in which significant production variation ranged from 0. 37mg/L to 22. 34mg/L. Furthermore,the best combination of TAL,4CL,CHS and CHI genes were integrated into chromosome for better genetic stability by delta integration,generating strain Sy BE_ Sc02050031. Subsequently,prenyltransferase gene（N8DT） from Sophora flavescens incorporated with multicopy plasmid was introduced into Sy BE_Sc02050031（ gaining strain Sy BE_Sc02050032）and a titer of 8DN at 36. 7μg/L in shaking flask was observed accordingly,indicating de novo biosynthesis of 8DN in yeast was successfully achieved. In addition,truncation tailoring strategy was explored to improve the catalysis function of N8 DT,leading to 8DN production（ up to 52. 6μg/L） increasing by 44% compared to that in strain Sy BE_Sc02050032. de novo biosynthesis of 8DN in microbes is firstly accomplished,which provides a good reference for microbial production of other natural flavonoids.