3.3 Verification of CYP5139G1 catalytic function by in vitro enzymatic reaction
To verify the C-28 oxidase activity of CYP5139G1, we used in vitro microsome reaction system as previously (Wang et al., 2018). The microsomal fractions from yeast YL-T3 expressing CYP5139G1 (strain CPR-CYP5139G1, Table S3) were incubated with HLDOA in the presence of NADPH at 30°C for 2 h. LC-MS analysis of the reaction mixture revealed that the CYP5139G1 containing microsome produced a new peak with the same retention time as the purified compound A (DHLDOA). No reaction was observed in boiled microsome sample or in that with void plasmid (strain CPR-EV, Table S3) (Fig. 4a). The LC-MS fragmentation pattern of the new peak included fragments with m/z ratios of 437 [M-2H2O+H]+ and 455 [M-H2O+H]+, which were consistent with those of the purified DHLDOA (Fig. 4b). The results demonstrated that CYP5139G1 catalyzed the conversion of HLDOA to DHLDOA.
We did not observe any hydroxylated products of HLDO and HLDA in the strain CYP5150L8-P45 (Table S3). In order to understand whether CYP5139G1 could catalyze C-28 hydroxylation of HLDOA derivatives - HLDO and HLDA, we used CYP5139G1 containing microsome incubated with HLDO and HLDA, respectively. As a result, no HLDO or HLDA oxidation was observed compared with the boiled CYP5139G1 harboring microsome and the void plasmid control (data not shown). Those evidences indicated CYP5139G1 had a strong substrate specificity to HLDOA.
Taking all the above facts together, we could conclude that CYP5139G1 was a HLDOA C-28 hydroxylase. By introducing CYP5139G1 into the yeast chassis, the reconstructed GA biosynthetic pathway from glucose was extended from HLDOA to DHLDOA. Here, both a novel GA and a new hydroxylase were found (Fig. 5).