Improving the Production of 9,21-di​hydroxy-​20-​methyl-pregna-​4-​en-​3-​one from Phytosterols in Mycobacterium Neoaurum by Modifying Multiple Genes and Improving the Intracellular Environment

BackgroundSteroid drugs are particularly important for disease prevention and clinical treatment. However, traditional chemical methods are rarely implemented during the whole synthetic process to generate steroid intermediates due to the intricate steroid structure. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9-OH-4-HP is a potential steroid drug precursor 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.ResultsA 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 could accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols. 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 double-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, and its selectivity reached 94.9%. Subsequently, the catalase katE from Mycobacterium and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the selectivity of 9-OH-4-HP improved to 97%.Conclusionhsd4A and fadA5 are key enzymes in the C19 pathway for phytosterol side chain degradation. Deletion of hsd4A and fadA5 could almost entirely block the C19 pathway. Improving the intracellular environment of Mycobacterium during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Rate of steroid double-bond reduction catalysed by the human steroid 5β-reductase (AKR1D1) is sensitive to steroid structure: implications for steroid metabolism and bile acid synthesis

AKR1D1 displayed differential kinetic behaviours with its various substrates. Results provide insights into the functions of AKR1D1 in hepatic steroid hormone catabolism, the peripheral formation of active 5β-reduced steroids and bile acid synthesis.

Brassinosteroid phytohormones: structure, bioactivity and applications

Brassinosteroids are a new class of plant hormones with a polyoxygenated steroid structure showing pronounced plant growth regulatory activity. This review covers their natural occurrence, biological and chromatographic methods for their detection, biosynthesis and metabolism, biological activity, structure-activity relationships and prospective agricultural uses.