Fig. 2: Engineering the electron transfer system of BtDHCR7.

A The catalysis of 7-dehydrocholesterol (Dhc) by 7-dehydrocholesterol reductase (DHCR7) involved two steps: substrate capture (white region) and substrate binding (yellow region). Recognition sites (white region) are involved in substrate binding; F430 and F434 serve as binding residues. In the catalytic pocket, Y317 donates electrons for catalysis. Y55, F56, F430, F434, and Y317 (marked in red) together form the electron transfer chain. B Residues in the electron transport chain were identified by alanine scanning or by substituting tyrosine with phenylalanine. Y317 of codon-optimized DHCR7 (BtDHCR7) was mutated to Phe and lost activity. C Rationally engineering BtDHCR7 was divided into four steps: 1. Engineering the L67 and L426 of DHCR7’s substrate-recognizing domain; 2. Engineering the W280 and Y290 of DHCR7’s substrate-binding domain; 3. Engineering the A49 and I53 of DHCR7’s NADPH-binding domain; 4. Engineering the F430 and F434 of DHCR7’s electron transfer chain. ETE of BtDHCR7 was step 3 and 4. The two blue arrows represent the capture and transfer of electrons from NADPH to C7 = C8, respectively. The yellow arrow represents DHC entering the catalytic pocket (step 2) after being captured by DHCR7 (step 2). D Rationally engineering BtDHCR7 by engineering substrate-recognizing domain and substrate-binding domain. W280 and Y290 were subjected to saturation mutagenesis, and the mutants with the highest Cho production were shown. L67V-L426A-BtDHCR7 was named M1. L67V-L426A-W280Y-Y290W-BtDHCR7 was named M2. The final OD₆₀₀ value and Cho yield of fermentation are provided. (E) Rationally engineering BtDHCR7 by engineering NADPH-binding domain and electron transfer chain. I53T-M2 was named as M3. I53T-F430Y-F434Y-M2 was named M4. The final OD₆₀₀ value and Cho yield of fermentation are provided. F Electron transfer chain 2 was another electronic transfer chain in M4. G Western blot analysis of BtDHCR7 and its variants. 1: WT-BtDHCR7, 2: L67V-BtDHCR7, 3: M1, 4: M2, 5: I53W-M2, 6: M3, 7: F430Y-M3, 8: M4. H Engineering the electron transfer chain of MuBtDHCR7 demonstrated the electron transfer chain 1 was the primary chain. Values are shown as mean ± s. d. from three biological replicates. Source data are provided as a Source Data file.