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.