Production of 1, 12- dodecanedioic acid by recombinant microbial cytochrome P450 in pichia pastoris and saccharomyces cerevisiae

Abstract

1,12-Dodecanedioic acid (DDA) is a primary compound which is used as an intermediate precursor for production of valuable chemical products. Biosynthesis of 1,12-DDA by recombinant (r) microorganisms can be used as an alternative method to compensate several disadvantages from chemical synthesis. The purpose of this study was to clone a CYP52A17 from Candida tropicalis to produce cytochrome P450 which can terminally oxidize fatty acids to hydroxy-fatty acids and further to dicarboxylic acids (ω-oxidation). The wild type rCYP52A17 and its engineered L261S/L490S (leucine changed to encode for serine) form (rCYP52A17mut) were expressed in Pichia pastoris and Saccharomyces cerevisiae which coexpressing the yeast NADPH cytochrome P450 reductase in order to produce 12-hydroxydodecanoic acid (HDDA) and, potentially, 1,12-DDA from lauric acid. The P450 contents of microsomes from P. pastoris/pPICZA-CYP52A17 and P. pastoris/pPICZA-CYP52A17mut were 0.2 nmol/mg of protein. P. pastoris/pPICZA-CYP52A17 and CYP52A17mut presented a few the oxidation ability. The recombinant P. pastoris/pPICZA-CYP52A17mut accumulated the highest level of 12-HDDA (0.8 µM) within 24 h. The recombinant S. cerevisiae expressing rCYP52A17 showed higher P450 contents than the others. The P450 content of microsomes from S. cerevisiae BY(2R)/pYeDP60-CYP52A17 and S. cerevisiae BY(2R)/pYeDP60-CYP52A17mut were 0.13 and 0.28 nmol/mg of protein, where lauric acid was oxidized to provide approximately 4 and 12 µM of 12-HDDA, respectively. Moreover, biotransformation of lauric acid by S. cerevisiae BY(2R)/pYeDP60-CYP52A17mut showed the highest level of HDDA (45.8 µM) at 24 h, which was oxidized to yield 20.8 µM 1,12-DDA at 72 h. The optimal lauric concentration of biotransformation was 500 µM, while the levels of HDDA and 1,12-DDA production in bioreactor were almost similar to the shake flask cultivation. The recombinant yeast cells which initially cultured in YPGE can be able to produce the highest 1,12- DDA from coconut milk wastewater in 24 h. From this study, S. cerevisiae BY(2R)/pYeDP60-CYP52A17mut demonstrated as a promising source to produce potential 1,12-DDA and can be applied for industrial applications.