强化类球红细菌辅因子NADPH再生以提高法尼醇的产量

Reinforcement of Rhodobacter sphaeroides cofactor NADPH to increase the production of farnesol

摘要:

法尼醇 (Farnesol, FOH) 是由焦磷酸异戊烯基 (IPP) 和焦磷酸二甲基烯丙基 (DMAPP) 合成的法尼酰基焦磷酸盐 (FPP) 去焦磷酸化作用生成的。在类球红细菌中IPP和DMAPP是由MEP途径生成，而完整的MEP途径需要消耗大量的辅因子NADPH，增加胞内NADPH的量有可能强化FOH的合成。文中从增加NADPH的生成和降低NADPH的消耗这两个策略出发，分别干扰了编码6-磷酸葡萄糖异构酶基因 (pgi) 和谷氨酸脱氢酶基因 (gdhA) 的表达，同时强化了磷酸戊糖途径中6-葡萄糖磷酸脱氢酶基因 (zwf) 和6-葡萄糖酸磷酸脱氢酶基因 (gnd) 的表达。实验结果表明，经改造的菌株NADPH含量显著增加，干扰菌株中菌株RSpgii的产量较高，为3.91 mg/g，在过表达的菌株中同时过表达zwf和gnd基因的重组菌株 (RSzg) 的FOH产量提高到了3.43 mg/g。为了获得FOH产量更高的菌株，以RSpgii为出发菌株，分别与zwf和gnd组合调控，获得的菌株RSzgpi的产量达到了最高量为4.48 mg/g，是出发菌株RS-GY2产率的2.24倍。

Abstract:

Farnesol (FOH) is produced by dephosphorylation of farnesyl diphosphate (FPP) derived from two universal building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In Rhodobacter sphaeroides these building blocks are generated by MEP pathway, however, many of the biosynthetic reactions and biotransformations in the MEP pathway are limited by low availability of NADPH. Improvement of the amount of intracellular NADPH may enhance the synthesis of FOH. In this study, we utilized the strategies of increasing the production of NADPH and decreasing the consumption of NADPH. The expression of glucose 6-phosphate isomerase (pgi) and glutamate dehydrogenase (gdhA) were inhibited by RNA interference, respectively, and overexpression of 6-glucose phosphate dehydrogenase (zwf) and 6-glucose phosphate dehydrogenase (gnd) in the pentose phosphate pathway were carried out. The results showed that the content of NADPH in the recombinant strains increased significantly, the highest FOH production of RSpgii in the RNA interfered strain was 3.91 mg/g, and the FOH production increased to 3.43 mg/g after zwf gene and gnd gene has been overexpressed. In order to obtain strains with higher FOH production, we used RSpgii as the starting strain, and zwf, gnd and co-overexpressed zwf + gnd gene were overexpressed in RSpgii, respectively. The highest FOH production of the strain RSzgpi reached to 4.48 mg/g which was 2.24 times that of the starting strain RS-GY2.