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Redetermination of 5?-androstane-3,17-dione.

ABSTRACT: The structure of the title compound, C(19)H(28)O(2), has been redermined at 295?(2)?K, with much improved precision. The structure and mol-ecular packing of the title compound was first reported by Coiro et al. [Acta Cryst. (1973). B29, 1404-1409] by means of potential-energy calculations. The cell parameters in this study differ considerably in space group C2. It is a derivative of testosterone and consists of a cyclo-penta-none ring (A) fused to to successive cyclo-hexane (B and C) and cyclo-hexa-none (D) rings. The three cyclo-hexa-none rings are in slightly distorted boat configurations and the cyclo-penta-none ring is a distorted half-chair. The crystal packing is stabilized by weak inter-molecular C-H?O inter-actions involving O atoms from each of the cyclo-hexa-none and cyclo-penta-none rings and H atoms from each of their respective rings.

SUBMITTER: Jasinski JP

PROVIDER: S-EPMC2979654 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Redetermination of 5α-androstane-3,17-dione.

Jasinski Jerry P JP Butcher Ray J RJ Hakim Al-Arique Q N M QN Yathirajan H S HS Narayana B B

Acta crystallographica. Section E, Structure reports online 20100529 Pt 6

The structure of the title compound, C(19)H(28)O(2), has been redermined at 295 (2) K, with much improved precision. The structure and mol-ecular packing of the title compound was first reported by Coiro et al. [Acta Cryst. (1973). B29, 1404-1409] by means of potential-energy calculations. The cell parameters in this study differ considerably in space group C2. It is a derivative of testosterone and consists of a cyclo-penta-none ring (A) fused to to successive cyclo-hexane (B and C) and cyclo-h ...[more]

PMID: 21579576

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Project description:Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space-time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space-time yield of 4.22 g·L-1·day-1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. KEY POINTS: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration.

Project description:4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Dataset Information

Redetermination of 5?-androstane-3,17-dione.

Publications

Redetermination of 5α-androstane-3,17-dione.

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