Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Abstract

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

“…Such product formation with the support of an NADPH system also showed the roles played by alternative redox partners and reducing equivalents with regard to the catalytic efficiency of CYP154C8 and its product distributions. In addition, it was also established that the appropriate choice of reducing equivalents and surrogate redox partners might play an important role with regard to catalytic efficiency and product distributions for CYP enzymes generally . The multistep oxidation of steroids in the presence of CYP154C8 was not previously observed for any bacterial source of CYPs, although cytochrome P450 BioI (CYP107H1) and MycG from bacterial sources have been reported to catalyze the multistep oxidation of fatty acids and mycinamycin IV, respectively .…”

“…Although both the NADPH‐ and the NADH‐dependent systems efficiently supported the activity of CYP154C8, they never induced formation of 6β‐hydroxylated product from the substrates progesterone and 11‐oxoprogesterone or from any other substrates, thus suggesting the roles for redox partners in protein–protein interactions for modulating the specificity of CYPs. In addition, the functional groups, such as hydroxy or carbonyl groups, in the substrate can also influence the selectivity of hydroxylation, although such switching in the selectivity of different CYPs might not be consistent . The factors underlying the B‐ring hydroxylation in the presence of CYP154C8 supported by (diacetoxyiodo)benzene and H 2 O 2 are unclear.…”

“…It is generally believed that the choice of surrogate partners or their mode of action does not affect the type and selectivity of the reactions catalyzed by CYPs . However, alternative redox partners might influence the catalytic efficiency and the product distribution …”

Biosynthesis of the Nylon 12 Monomer, ω‐Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω‐TA Enzymes

“…All possible candidates of CYP153As were obtained through a Pattern Hit Initiated BLAST (PHI‐BLASTP) search (performed via blastp, http://blast.ncbi.nlm.nih.gov) using the reported Marinobacter aquaeolei VT8 CYP153A33 (MaqCYP153A) and Mycobacterium marinum M CYP153A16 (MmaCYP153A) . Among the candidates, CYP153A from Mycobacterium parascrofulaceum (GI: EFG78917.1; KCTC collection no 9979) was chosen as the novel target for the omega hydroxylation of fatty acids.…”

“…Moreover, the use of FFAs instead of ester‐forms is more suitable for the biotransformation due to their higher solubility, and ω‐hydoxylation of FFAs by P450 will reduce the reaction steps compared to unsaturated fatty acid‐based bioprocesses. The versatile cytochrome (CYP) P450, enzymes are capable of catalyzing intricate reactions such as regio‐ and stereo‐selective oxidation of non‐activated FFA's CH bonds to the corresponding hydroxy (COH) products . However, the selective terminal hydroxylation of FFAs remains a challenge.…”