Isoprenoid biosynthesis has a significant requirement for the co-factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasing the efficiency of enzymes that generate NADPH. We first established a robust genetic screen that allowed us to screen improved variants. The pentose phosphate pathway enzyme, glucose 6-phosphate dehydrogenase (G6PD), was chosen for further improvement. Different gene fusions of G6PD with the downstream enzyme in the pentose phosphate pathway, 6-phosphogluconolactonase (6PGL), were created. The linker-less G6PD-6PGL fusion displayed the highest activity, and although it had slightly lower activity than the WT enzyme, the affinity for G6P was higher and showed higher yields of the diterpenoid sclareol in vivo. A second gene fusion approach was to fuse G6PD to truncated HMG-CoA reductase, the rate-limiting step and also the major NADPH consumer in the pathway. Both domains were functional, and the fusion also yielded higher sclareol levels. We simultaneously carried out a rational mutagenesis approach with G6PD, which led to the identification of two mutants of G6PD, N403D and S238QI239F, that showed 15–25% higher activity in vitro. The diterpene sclareol yields were also increased in the strains overexpressing these mutants relative to WT G6PD, and these will be very beneficial in synthetic biology applications.

中文翻译：

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葡萄糖 6-磷酸脱氢酶变体增加了酵母类异戊二烯生产的 NADPH 池

类异戊二烯生物合成对辅因子 NADPH 有很大的需求。因此，在合成生物学中提高 NADPH 水平对于提高类异戊二烯产量至关重要。之前的努力主要集中在将通量转移到磷酸戊糖途径或过量产生产生 NADPH 的酶。在这项研究中，我们的重点是提高产生 NADPH 的酶的效率。我们首先建立了强大的遗传筛选，使我们能够筛选改良的变体。选择戊糖磷酸途径酶、葡萄糖 6-磷酸脱氢酶 (G6PD) 进行进一步改进。创建了 G6PD 与戊糖磷酸途径下游酶 6-磷酸葡萄糖酸内酯酶 (6PGL) 的不同基因融合。无接头的 G6PD-6PGL 融合体显示出最高的活性，尽管其活性略低于 WT 酶，但对 G6P 的亲和力更高，并且在体内显示出更高的二萜香紫苏醇产率。第二种基因融合方法是将 G6PD 与截短的 HMG-CoA 还原酶融合，这是该途径中的限速步骤，也是 NADPH 的主要消耗者。两个结构域均具有功能，并且融合还产生了更高的香紫苏醇水平。我们同时对 G6PD 进行了合理的诱变方法，从而鉴定了 G6PD 的两个突变体 N403D 和 S238QI239F，其体外活性提高了 15-25% 。相对于 WT G6PD，过表达这些突变体的菌株中二萜香紫苏醇的产量也有所增加，这在合成生物学应用中将非常有益。