1 INTRODUCTION
9α-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) is a C17 steroid
intermediate that can be further synthesized into steroids, such as sex
hormones and corticosteroids.[1,2] At present,
steroid intermediates are usually prepared by microorganisms, mainly
mycobacteria, to generate the required steroid intermediates by side
chain degradation and nucleus oxidation of
sterols.[3] Phytosterols (PS) are wastes separated
from soybean processing and oil refining.
It is used as the substrate to
produce steroid intermediates to increase the added value of
agricultural products. However, due to the low bioavailability of PS,
the toxicity of steroids to cells, and the low viability of existing
strains, its transformation efficiency is not ideal. Thus, how to
improve the transformation efficiency of PS remains a research hotspot
in the field of steroids.[4,5]
Currently, there are many approaches to improve the biosynthesis
efficiency of steroid intermediates. The most common method to optimize
the reaction system was to enhance the solubility of PS and thus improve
the conversion efficiency. The resting cell-cyclodextrin system was
established to solubilize PS and reduce the toxicity of PS to
cells.[6,7] Gao et al. reported an increase in
9-OH-AD production by PS bioconversion through the resting
cell-cyclodextrin system.[8] In addition, the use
of genetic engineering technology to improve the production capacity ofmycobacteria is also an effective way to enhance the biosynthesis
efficiency of steroid intermediates. Yao et al. improved 9-OH-AD
production by overexpressing kshA that facilitated the 9-position
hydroxylation in Mycobacterium neoaurum. [9]Sun et al. increased the yield of 9-OH-AD in recombinant strains by
1.45-fold by overexpressing four key genes, hsd, hsd4A ,kshA1 and kshB [10], that enhance the
side-chain degradation. Chang et al. increased the production of AD from
3.2 g/L to 4.5 g/L through the overexpression of
Hsd4A.[11] Liu reported that the yields of
9-OHPDC-M were remarkably improved by individually overexpressing the
genes hsd4A and kshA1. [12]
However, there are still problems affecting the biosynthesis efficiency
of steroid intermediates, especially the accumulation of
by-products.[13] In the production of 9-OH-AD bymycobacteria , there are mainly two types of by-products
accumulated, one is the incomplete degradation of the side chain,
including the by-product 9,22-dihydroxy-23,24-bisnorster-4-en-3-one
(DHBC) and 9,24-dihydroxycholester-4-en-3-one (DHC). The other is the
complete degradation of side chain, such as androsten-4-ene-3,17-dione
(AD), resulting in a lower purity and yield of the main product 9-OH-AD.
In this study, 9-OH-AD was prepared by the biotransformation of PS in
the resting cell-cyclodextrin system with Mycobacterium neoaurum .
Two key genes kshA1 and hsd4A , which may be related to
by-product accumulation, were selected to verify their functions in the
degradation pathway of phytosterols. The genetically engineered strain
facilitated for balancing metabolic flux of the side-chain degradation
and 9-position hydroxylation in Mycobacterium neoaurum , which
significantly improved the yield and purity of 9-OH-AD.