Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1- dehydrogenase deficiency and multiple gene regulation in Mycobacterium fortuitum

Abstract

Background 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) refers to a significant intermediate for synthesis of glucocorticoid drugs. Microbial transformation of phytosterols to manufacture 9-OHAD has obvious advantages in terms of lead time, cost control, environmental friendliness, etc. However, the product degradation and the formation of by-products restrict their application. In this study, a combined strategy of blocking Δ1‑dehydrogenation and regulating metabolic flow was investigated to construct a stable high-yield 9-OHAD producer. Results Five 3-Ketosteroid-Δ1-dehydrogenase (KstD) were identified in ATCC 35855 and each of them was functionally confirmed by heterologous expression, transcriptional response and knockout analysis. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4 and KstD5. In particular, KstD2 has a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 are the main assailants of 9-OHAD degradation. Compared with ATCC 35855, MFΔkstD, the five KstDs deficient train, realized the stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of Opccr or the overexpression of Hsd4A alone could not reduce the metabolic flow of C22 pathway, while the overexpression of Hsd4A based on the knockout of Opccr in MFΔkstD could remarkably reduce the contents of 9-OHHP by-products. The inactivation of FadE28-29 leads to a large amount of accumulation of incomplete side chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminated two by-products. Compared with MFΔkstD, the purity of 9-OHAD was improved from 80.24% to 90.14%. Ultimately, 9‑OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h. Conclusions Five putative KstDs have been identified and functionally confirmed in ATCC 35855. KstD2 and KstD3 were the main murderers of 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of C19 and C22-pathways. The overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of side chains. According to the findings above, MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to understanding the diversity and complexity of steroid catabolism regulation in actinobacteria and providing a theoretical basis for further optimizing industrial microbial catalyst.