[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-030] Structure-guided engineering of 6-phosphogluconate dehydrogenase from Yarrowia lipolytica for enhanced NADPH production

○Yujin Kim¹, Seungmi Stella Kim¹, Hyeoncheol Francis Son^1,2,3,4 (1. School of Biological Sciences and Technology, Graduate School Chonnam National University (Korea), 2. School of Biological Sciences and Technology, Chonnam National University (Korea), 3. Institute of Synthetic Biology for Carbon Neutralization, Chonnam National University (Korea), 4. Institute of Systems Biology and Life Science Informatics, Chonnam National University (Korea))

Keywords:

6-phosphogluconate dehydrogenase,structure-based enzyme engineering,NADPH supply,Yarrowia lipolytica

Cofactor imbalance is a major bottleneck in the microbial production of high-value compounds, as it can reduce product yield and biosynthetic efficiency. Imbalance between NADH and NADPH limits carbon flux toward reductive biosynthetic pathways by restricting the availability of reducing power. NADPH is an essential reducing cofactor for fatty acid synthesis; the biosynthesis of one C16 fatty acid requires a total of 16 molecules of NADPH. Therefore, sufficient NADPH supply is important for efficient biochemical production. To increase NADPH supply, several strategies have been used: activation of the pentose phosphate pathway (PPP), conversion of NADH to NADPH, cofactor specificity control, and metabolic and enzyme engineering.
In the PPP, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are two key enzymes responsible for NADPH production. G6PDH catalyzes the first step of the PPP, converting D-glucose 6-phosphate and NADP⁺ into 6-phospho-D-glucose-1,5-lactone and NADPH. In contrast, 6PGDH catalyzes the conversion of 6-phospho-D-gluconate and NADP⁺ into D-ribulose 5-phosphate and NADPH.
Yarrowia lipolytica is a non-model, GRAS-status yeast that is widely used as an industrial host to produce various biochemicals and lipids. As an oleaginous microorganism, Y. lipolytica requires a large amount of NADPH for lipid biosynthesis, making intracellular NADPH supply an important factor for its production performance.
In this study, we identified 6PGDH from Y. lipolytica (Yl6PGDH) and determined its three-dimensional structure by X-ray crystallography. By analyzing the complex structure with substrate and cofactor, we analyzed the catalytic mechanism of Yl6PGDH. Based on multiple sequence alignment (MSA) and structural analysis, we designed nine mutation candidates. After confirming the activity of the wild-type Yl6PGDH by measuring NADPH production at different 6PG concentrations, we identified a final mutant that exhibited 1.59-fold higher NADPH-producing activity than the wild-type Yl6PGDH. We also analyzed the reason for the improved activity based on structural changes around the tunnel entrance. A residue located at the tunnel entrance may partially limit substrate access to the active site or product release by forming strong electrostatic interactions with 6PG through its positively charged side chain. In contrast, substitution to a non-polar side chain may remove the positive charge without significantly changing the side-chain length, which could relax the electrostatic environment at the tunnel entrance and improve substrate movement and turnover.
These results provide structural insight into how local changes around the active site can modulate enzyme activity and enhance NADPH production. This study provides a valuable insight for developing strategies to improve NADPH supply in industrial microorganisms and may contribute to the optimization of lipid and high-value biochemical production processes.