Efficient Synthesis of Steroidal Intermediates with a C17 Side Chain from Phytosterols by Genetically Modified Mycolicibacterium neoaurum NRRL B-3805 Strain.

22-Hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) and 3-oxo-4,17-pregnadiene-20-carboxylic acid methyl ester (PDCE) are useful precursors for the synthesis of steroidal active pharmaceutical ingredients. In this study, we identify the sterol metabolism-related genes, which encode the aldolases (Ltp2 and Thl) and carboxylic acid reductases (CAR) in Mycolicibacterium neoaurum NRRL B-3805 (B3805), by analysis of the metabolites from phytosterols biotransformation. Based on these results, a genetically modified strain is constructed by disrupting the kstD, ltp2, and hsd4A genes and overexpressing the aldolase gene (thl) in the strain B3805. This recombinant strain (B3805V) is able to transform 5 g L^-1 phytosterols to 2.0 g L^-1 4-HBC without detectable AD by-product. Additionally, by disrupting the ltp2 and car genes, a strain (strain B3805VI) is obtained to transform phytosterols to PDCE with 1.44 g L^-1 titer. The PDCE concentration is further increased by about 42% to 2.1 g L^-1 without 4-HBC by-product by deleting thl gene (strain B3805VII). On the preparative scale, the strain B3805VII transforms 10 g L^-1 of phytosterols into PDCE with 5.1 g L^-1. This study presents one-step bioproduction of pharmaceutically important 4-HBC and PDCE with high yield and purity from bio-renewable phytosterols, which are readily available as a by-product from the plant oil industry.