恶臭假单胞菌F1表达P450酶催化柠檬烯生物合成紫苏醇

Biosynthesis of perillyl alcohol using limonene catalyzed by cytochrome P450 in Pseudomonas putida F1

紫苏醇是一种天然存在的单萜类化合物,在医药、日化、食品等行业具有广阔应用前景,但利用柠檬烯生物合成中的过程中存在选择性不强、底物对宿主菌株有毒性等问题。为此该文选择恶臭假单胞菌F1这种对环境具有高耐受度的菌株,构建在大肠杆菌和恶臭假单胞菌中皆可扩增并表达目的基因的广宿主载体,过表达选择性羟化酶CymAa、CymAb,异源表达特异性氧化柠檬烯生成紫苏醇的P450酶CYP153A6,并在表达P450酶的基础上对柠檬烯底物添加浓度进行了调整,与大肠杆菌BL21(DE3)一同进行柠檬烯生物合成紫苏醇实验,并使用紫外可见分光光度计和GC-MS分别测量菌株生长状况和紫苏醇产量。最终证明了恶臭假单胞菌F1在柠檬烯浓度为10 mmol/L内的环境中具有高耐受度,且添加1 mmol/L柠檬烯底物发酵48 h后获得最高产量75.6 mg/L,最高转化率达到49.66%,缩小了生物合成紫苏醇的底物添加范围,并获得对柠檬烯具有高耐受度的发酵宿主。

Perillyl alcohol is a kind of natural monocyclic terpene with wide applications in pharmaceutical,cosmetics and food industries.However,the process in the biosynthesis of perillyl alcohol using limonene suffers from low-selective and toxicity of substrate to the host strain.For this reason,we chose Pseudomonas putida F1,a strain with high environmental tolerance.We constructed a broad-host vector that self-amplifies and expresses the target gene in both E.coli and P.putida.CymAa and CymAb were overexpressed,selective hydroxylases origin from P.putida F1.The cytochrome P450(CYP153A6),which specifically oxidize limonene to perillyl alcohol,was heterologously expressed and the concentration of limonene substrate added was adjusted for the biosynthesis of perillyl alcohol using both E.coli BL21(DE3)and P.putida F1.Strain growth and perillyl alcohol yield were measured by UV-Vis spectrophotometer and GC-MS,respectively.Finally,we demonstrated that P.putida F1 was highly tolerant to limonene concentrations up to 10 mmol/L.The highest yield of 75.6 mg/L was obtained after 48 h of fermentation with the addition of 1 mmol/L limonene substrate,with a maximum conversion rate of 49.66%.This study narrows the range of limonene and provides a high tolerance fermentation host strain addition for biosynthesis of perillyl alcohol.