Heterologous Expression Research Of Aspergillus Ochraceus Steroid 11α-Hydrolyase Gene

Aspergillus ochraceus is used industrially to convert 11α-hydroxy-16α, 17α-epoxy progesterone to 1α-hydroxy-16α, 17α-epoxy progesterone, which is a key glucocorticoid intermediate. The enzyme involved in the steroid 11α-hydroxylation reaction is a cytochrome P450 enzyme. The results showed that AOH recombinant yeast cells could convert 16α,17α-epoxy progesterone to form product 11α-16α, 17α-epoxy progesterone,indicating the functional expression of AOH gene in Saccharomyces cerevisiae. The relationship between the structure and function of the enzyme is of great significance because of the industrial value of the A. ochraceus 11α-hydroxylase. Although the AOH gene was successfully expressed in yeast cells, its level of expression was low and thus it was difficult to obtain sufficient amounts of purified 11α-hydroxylase for crystallization and protein structure analysis.The TMHMM Server software predicts that the N-terminus of the AOH gene contains a transmembrane region of 33 amino acids. In order to increase the soluble expression of 11α-hydroxylase, the 33 amino acids in the transmembrane region were truncated by 20, 25 and 33 amino acids respectively. AOH-33, AOH-25 and AOH-20 recombinant yeast cells could successfully transform the steroid substrate 16a, 17a-epoxy progesterone, but conversion levels were lower than that of full-length AOH gene recombinant yeast cells. To increase the expression level of steroid 11α-hydroxylase, we further tried the Escherichiacoli expression system. The AOH-20, AOH-25 and AOH-33 expression vectors of E. coli were constructed and the corresponding recombinant bacterial strains were obtained. Since the steroid 11α-hydroxylase must work with the P450 reductase to 11α-hydroxy late the steroid substrate, the AOR gene expression vector was generated and the corresponding E.coli recombinant strain was obtained. The steroid 11α hydroxylation activity of the recombinant E. coli strain was assessed by using steroid substrate 16α, 17α-epoxy progesterone, and the accumulation of 11α-hydroxy product was not observed. SDS-PAGE analysis showed that AOH reductase was expressed in the form of inclusion body, while AOH and the truncated AOH-33, AOH-25 and AOH-20 were not expressed in E. coli cells.