3.3 Effects of co-expression of hsd4A and kshA1 on PS conversion to 9-OH-AD
To optimize the distribution of metabolic flux in PS biotransformation, KshA1 that promotes the 9-position hydroxylation of steroid nucleus and Hsd4A that enhances the complete degradation of steroid side chain were co-expressed in Mycobacterium neoaurum NwIB-HK86 W. Furthermore, it was reported that the distance of genes from promoters in the same operon affects their expression intensity.[20,21]The expression levels were higher for gene locations close to the origin of replication compared to locations close to the replication terminus. Based on these results, the mutants co-expressing endogenous geneskshA1 and hsd4A were constructed in two patterns as shown in Fig. 3 and named M. neoaurum HK86 A-K and HK86 K-A. PS transformation using mutants HK86 A-K and HK86 K-A could balance the activity of KshA and Hsd4A. The PS transformation results using two mutants were shown in Table 3.
Compared with the PS transformation of strain HK86 A, HK86A-K showed no remarkable difference in the yield, purity of 9-OH-AD, and the proportion of other by-products, indicating that the augment of KshA activity in the co-expression mutant did not contribute to improving the 9-position hydroxylation level. Considering the pivotal role of Hsd4A in enhancing the side-chain degradation of PS, it was speculated that the KshA activity did not match the Hsd4A activity in the co-expression mutant HK86 A-K.
The transformation results of HK86 K-A were significantly different from HK86 A-K (Table 3). Compared with HK86-A-K, the content of byproduct AD was significantly reduced from 11.73% to 3.61%, and the 9-position hydroxyl products, 9-OH-AD, DHBC, and DHC were increased correspondingly. The yield and purity of 9-OH-AD using HK86 K-A were further increased to 26.04 g/L and 85.61%, respectively, despite the slight decrease of total complete degradation products (AD and 9-OH-AD). These results indicated that the expression level of kshA1 that was closer to the promoter was higher when co-expressed with hsd4A in the same operon. In mutant HK86 K-A, the decreased expression level of hsd4A could better balance the activities of Hsd4A and KshA1, then effectively increasing the proportion of 9-position hydroxyl products in metabolites and meanwhile enhancing the side-chain degradation to decrease the incomplete side-chain degradation byproducts.
3.4 Decreasing the PS conversion rate to balancethe metabolic flux of the side-chain degradation and 9-position hydroxylation
Deduced from the results abovementioned, the decreased PS conversion rate led to the metabolic flux decrease of the side-chain degradation and could better balance the reaction of 9-position hydroxylation. According to our previous work, the ratio of the PS and CD in the reaction system affects the PS conversion rate. The results of PS transformation in different ratios of the PS to CD were shown in Table 4. The PS conversion rate decreased as expected with the ratio of the PS to CD changed from 1:4 to 1:2 and the production of 9-OH-AD peaked at 26.04, 27.89, and 28.27 g/L respectively on the 4, 5, and 6th day of bioconversion. In the meantime, the purity of 9-OH-AD increased and the by-products decreased correspondently. The optimized result was obtained when the ratio of PS to CD was 1:2. If the ratio lowed to 1:1, the reaction rate was severely impeded and the nucleus degradation aggravated.