Engineering for Efficient NADPH Cofactor Supply in Cytorchrome P450 Monoxygenase Reaction
It is a general practice in metabolic engineering to amplify or disrupt enzymes to increase the productivity of target metabolite. Here it was rerouted the carbon metabolic flux from the EMP pathway to the PP pathway in E. coli to increase NADPH availability, and thereby increase the production in cytochrome P450 enzyme reaction which essentially requires NADPH reducing equivalent. In E. coli, the balance between NADH and NADPH can be maintained by two isoforms of pyridine nucleotide transhydrogenases, i.e. membrane-bound tranhydrogenase (pntAB) and soluble trnashydrogenasse (udhA). PntAB catalyses the transfer of reducing power from NADH to NADP+ in an energy-dependent manner when intracellular NADPH level is low, while Udh mainly catalyzes NADPH to NAD+ in an energy-independent manner when NADPH level is high. Here, NADPH concentration through metabolic engineering of the pentose phosphate pathway (PPP) was increased. The first strategy involved deleting the phosphoglucose isomerase (pgi) to increase flux through the (NADPH-producing) PPP resulting in improved redox potential for P450 enzymatic reaction. In the second strategy, NADPH level by expressing NADH kinase which can directly convert NADH to NADPH was also increased The third strategy is regeneration of NADP into NADPH using both glucose 6-phosphate dehydrogenase (GDH) and Transhydrogenase (STH) coupled with glycerol dehydrogenase (GLD).
The disruption of pgi and the over-exrpession of GDH/STH/GLH/NADK altered the intracellular redox equivalents levels, resulting in increased concentration of reduced form of NADPH. During cultivation at steady state, this mutant strain didn’t change oxidized form of NADP+, while greatly increased NADPH in cells more than five times resulting in the ratio shift to high redox potential (from 1.65 to 0.65). Using this Dpgi::GDH::STH::GLH::NADK strain as a biotransformation host, the selected CYP102D1 mutant L84A/F96V/A243S/L448A/T247R was investigated for their biotransformation efficiency. The resting cell assay result revealed that 500 μM of daidzein were converted into 350 μM of 3’-hydroxylated product with 70% of conversion which is 1.3 times higher than that by wild-type host system.