利用大肠杆菌工程菌株生物转化脂肪酸的初步研究

Study on Engineering Strain of E. Coli Biotransformation Fatty Acid

羟基脂肪酸(Hydroxyfatty Acid,HFA)是一种有多种用途和广阔应用前景的多碳化学品。细胞色素P450单加氧酶(P450 P450 BM3)可高特异性的催化脂肪酸的ω碳位发生羟基化反应而生成羟基脂肪酸,该研究利用工程大肠杆菌成功表达了来源于巨大芽孢杆菌(Bacillus megaterium)的单加氧酶基因P450 BM3,实现了底物脂肪酸的成功转化。最终获得了羟基十二酸和羟基十四酸的最高产量分别为593.3 mg/L和547.3 mg/L,同时外源脂肪酸的转化率分别为59.7%和52.3%。对3种产物ω1、ω2、ω3-HFA进行定量分析发现3种产物的产量基本相同,表明单加氧酶对脂肪酸的羟基化位置没有特定偏爱性。最后通过优化表达载体的拷贝数来进一步优化单加氧酶基因P450 BM3的表达水平,最终利用中拷贝数质粒载体pACYCDuet-1表达单加氧酶基因P450 BM3获得了547.3 mg/L的羟基十四酸,代表了目前羟基十四酸生产的一个较高水平。

Hydroxyfatty acid （HFA）is a versatile and broad application prospect carbon chemical. Cytochrome P450 BM3 can hydroxylates fatty acids into hydroxyfatty acids（HFAs） at the co- positions with high enantioselectivity. The study used engineering E. coli successfully expressed monooxygenase gene from Bacillus megaterium （P450 BM3 ） and converted the substrate fatty acid into hydroxyfatty acid. The highest production of hydroxy dodecanoic acid and hydroxy tetradecanoic acid we finally obtained were 593.3 mg/L and 547.3 mg/L, meanwhile the conversion rate of fatty acid were respectively 59.7% and 52.3%. The production of ω1 ,ω2 ,ω3 - HFA revealed that theP450 BM3 had no substrate preference on fatty acid hydroxylate position. Finally, we further improved the express level of P450 BM3 by optimizes the plasmid copy number. The plasmid pACYCDudt - 1 with medium copy number expressed that P450 BM3 had obtained the production of 547.3 mg/L, which was a higher hydroxy tetradecanoic acid production reported in the world.