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.