3.2 Hsd4A enhances the complete degradation of steroid side chain
Hsd4A is a bifunctional enzyme with 17β-hydroxysteroid dehydrogenation and hydroxyacyl-CoA dehydrogenation activity. The transcription ofhsd4A gene was highly upregulated along with other genes involved in sterol side chain degradation in R. jostii RHA1, so it is speculated that this gene is essential for sterol side chain degradation.[15,16] Zhao reported that Hsd4A is responsible for the dehydrogenation of 22-hydroxy-3-oxo-4-ene-24-carboxy-CoA in the second cycle of oxidation of the sterol side chain similar to fatty acids β-oxidation.[17] Furthermore, knock-out ofhsd4A in Mycobacterium can accumulate the steroid intermediate HBC.[18] Hence, its function in sterol side chain degradation was investigated by overexpressinghsd4A in HK86 W.
The cell growth of HK86 A showed no significant difference from that of the original strain HK86 W. The yield and purity of 9-OH-AD were increased from 22.18 g/L and 77.13% to 25.16 g/L and 82.45% (Table 2). The principal component analysis showed that the proportion of complete degradation products (9-OH-AD and AD) was increased and the proportion of incomplete degradation products (DHBC and DHC) was decreased in HK86 A. Among them, the proportion of AD increased from 3.36% to 11.86% and DHBC decreased from 11.96% to 0.93%, respectively. These results indicated that the pathway of complete degradation of the side chain was enhanced in HK86 A and hsd4A was a key gene for complete degradation of the steroid side chain. However, further investigation found that the increase in AD proportion is much higher than that of 9-OH-AD. It was reported that the physiological substrates of KshAB are CoA thioester intermediates of sterol side chain degradation in Mycobacteria and the AD is not the favorable substrate of KshAB (Fig. 2). [19] It was deduced that the metabolic pathway of AD and 9-OH-AD is competitive and 9-position hydroxylase (KshA1) activity was limited when the pathway of side chain degradation was enhanced, leading to much more AD accumulation. To transfer the metabolic flux to the target product 9-OH-AD and decrease the generation of byproduct AD, an appropriate catabolic division of steroid was needed to balance the side-chain degradation and 9-position hydroxylation.