Glucose 6‐phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production

• Published in: FEBS open bio 2024-03, Vol.14 (3), p.410-425
• Main Authors: Adusumilli, Sri Harsha, Alikkam Veetil, Anuthariq, Choudhury, Chinmayee, Chattopadhyaya, Banani, Behera, Diptimayee, Bachhawat, Anand Kumar
• Format: Article
• Language: English
• Subjects: 6-Phosphogluconolactonase; Cloning; Dehydrogenases; Diterpenes; Energy Metabolism; Enzymes; Gene fusion; Genes; Genetic screening; Genetic Screens; Glucose; glucose 6‐phosphate dehydrogenase; Glucosephosphate dehydrogenase; isoprenoids; Mutagenesis; Mutants; NADP - metabolism; NADPH; NADPH dehydrogenase; Pentose phosphate pathway; Phosphates; Plasmids; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; sclareol; Terpenes; Yeast; ZWF1
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1002/2211-5463.13755

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. This study focuses on enhancing isoprenoid yields by increasing NADPH levels. Gene fusions of S. cerevisiae glucose 6‐phosphate dehydrogenase (ScG6PD) with 6‐phosphogluconolactonase (6PGL) and truncated HMG‐CoA reductase were created, and overexpressing them yielded higher sclareol (diterpenoid). Rational mutagenesis of ScG6PD identified mutants (N403D and S238QI239F) with 15–25% higher activity and overexpression of the N403D mutant also showed increased sclareol yields.