Fusing Engineered CYP109E1 and a New Reductase Domain for 25(OH)VD₃ Biosynthesis

Area of Science:

• Biotechnology
• Enzyme Engineering
• Metabolic Engineering

Background:

• 25-hydroxyvitamin D3 (25(OH)VD3) is a crucial nutritional supplement.
• Direct enzymatic C25 hydroxylation of vitamin D3 (VD3) offers a sustainable production method.
• Limitations in cytochrome P450 catalytic activity and electron transfer hinder efficient VD3 production.

Purpose of the Study:

• To enhance the production of 25(OH)VD3 through protein engineering and fusion protein construction.
• To overcome the limitations of low catalytic activity and electron transfer efficiency in VD3 hydroxylation.
• To establish a foundation for the industrial-scale biosynthesis of 25(OH)VD3.

Main Methods:

• Structure-guided semirational design was used to engineer CYP109E1 into a mutant form (CYP109E1M2).
• A novel reductase domain, BmRD, was identified and fused with CYP109E1M2.
• Fusion protein optimization involved truncating the N-terminus of BmRD, creating Chimera-2.
• Engineered Escherichia coli strain 03-2 was utilized for fermentation under optimized conditions.

Main Results:

• The engineered CYP109E1M2 mutant showed a 2-fold increase in 25(OH)VD3 production compared to the wild-type.
• The fusion protein Chimera-2, with a truncated BmRD, further increased 25(OH)VD3 production by 38.5%.
• The optimized engineered E. coli strain 03-2 achieved a final 25(OH)VD3 concentration of 491.3 mg/L, with 49.0% conversion and a space-time yield of 61.4 mg/(L·h).

Conclusions:

• Protein engineering of P450 enzymes and fusion protein strategies can significantly improve biocatalytic efficiency.
• The developed fusion protein system and engineered E. coli strain provide a robust platform for high-yield 25(OH)VD3 biosynthesis.
• This study lays the groundwork for the industrial application of enzymatic 25(OH)VD3 production, offering a sustainable alternative to traditional methods.