Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons

Abstract

Background3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modifications of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identified in Mycobacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplified their potential application for industrial steroid transformation.ResultsThe recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-dione (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specific activities of 22.40 and 19.19 U mg−1, respectively. However, the specific activities of recombinant KstD2 from Escherichia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had significantly lower specific activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) from 8 g L−1 AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the substrate, while the mRNA levels of kstD2 and kstD3 were bearly affected by the phytosterols. Therefore, we attempted to create a 4HP producing strain without kstD1, which could covert 20 g L−1 phytosterols to 14.18 g L−1 4HP.ConclusionsIn vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the findings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0916-9) contains supplementary material, which is available to authorized users.

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum

“…The biotransformation process is a rate-limiting step in the microbes producing steroid intermediates. It usually takes 120 to 144 h to realize a satisfactory conversion rate of the substrate to target steroid intermediates in the microbes [5,6,25]. However, it only takes about 48 to 72 h in most of other prokaryotic microorganisms [26][27][28].…”

Biosynthesis and Industrial Production of Androsteroids

“…Phytosterol biotransformation with M. neoaurum has been improved by manipulation of its methylcitrate cycle, a mechanism of detoxification of intracellular propionyl-CoA, a concomitant toxic byproduct [ 30 ]. Propionyl-CoA is the major by-product during β-oxidation of the phytosterol side chains and is toxic in high concentrations [ 31 ].…”

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

“…Phytosterols mainly exist in plants and are abundant in crops, such as beans and cereals, which are starting materials for steroid drugs production through mycobacterium transformation because of their low cost and environment-friendly attributes [4]. The gene-editing of the sterol side chain degradation metabolic pathway has made the industrial production of the steroidal pharmaceutical precursor by phytosterol biotransformation possible [5][6][7][8][9][10][11], but on the premise of improving production efficiency, reducing production costs is still the continuous pursuit of industrial production. Recently, studies on toxic metabolites, such as reactive oxygen species (ROS) in the production process of strains, have provided new ideas for the study of phytosterol conversion [12,13].…”