Heterologous Expression of Rhizopus Oryzae CYP509C12 Gene in Rhizopus Nigricans Enhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone

摘要

The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae ( RoCYP ) was introduced into R . nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R . nigricans . The Hsub2/subOsub2/sub levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans , providing an effective genetic method to construct the engineered strains for steroid industry.