Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Abstract

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway

“…The productivity of these strains could be further improved by the use of mycobacterial resting cells [ 41 – 44 ]. Very recently, an engineered strain of M. neoaurum DSM 44074 could efficiently convert phytosterols to 9OH-BA [ 45 ]. These investigations have revealed that the dual competing pathways between the overwhelming C19 steroid pathway and the C22 steroid pathway are involved in phytosterol sidechain degradation [ 22 , 24 , 45 – 48 ].…”

“…Very recently, an engineered strain of M. neoaurum DSM 44074 could efficiently convert phytosterols to 9OH-BA [ 45 ]. These investigations have revealed that the dual competing pathways between the overwhelming C19 steroid pathway and the C22 steroid pathway are involved in phytosterol sidechain degradation [ 22 , 24 , 45 – 48 ]. Some key specific genes, such as hsd4A [ 14 ], fadA5 [ 13 , 14 ], Sal [ 49 ], hsaG [ 50 ] and OpccR [ 13 ], were involved in controlling metabolic flux between the two pathways (Scheme 1 ).…”

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

“…Additionally, kstD3 has no catalytic capacity for 9-OHAD, but has high dehydrogenation activity for saturated A-ring and produces 1,4-diene structure. Similarly, KstDs with diversi ed functions has been found in many steroid producers, such as M. neoaurum ATCC25795 [18], M. neoaurum DSM1381 [9], A. simplex CGMCC14539 [42], Mycobacterium smegmatis [32,34], etc. This residual KstD activity becomes more complex due to the redundancy of KstD enzymes and the rich substrate spectrum characteristics.…”

“…The change of NADH/NAD + level could lead to the opposite catalytic function. Furthermore, the lack of activity Hsd4A could also lead part of the steroids ow to the C22 pathway, and nally, 9-OHHP is generated [21,42]. Moreover, a new biosynthetic mechanism of 4-HP was deciphered in the steroids degradation pathway of M. neoaurum CCTCC AB2019054, the C-terminal domain of mnOpccr converts 3-OPC-CoA to 3-OPA, and the Nterminal domain further restores 3-OPA to 4-HP [22].…”

“…Compared with other steroid transformation strains [36][37][38], ATCC 35855 has the advantages of short growth cycle and fast degradation of phytosterols (Fig. 5 AB).…”