Dataset Information

Engineered 3-Ketosteroid 9?-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

ABSTRACT: 3-Ketosteroid 9?-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1N and KshA2N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1 N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1 N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2 N to be a redundant form of kshA1 N Through a combined modification of kshA1 N, kshA2 N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter-1 ADD from the transformation of 20 g liter-1 phytosterols in 168 h. Moreover, we improved a previously established 9?-hydroxy-AD-producing strain via the overexpression of a mutant KshA1N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter-1 9?-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9?-hydroxylase, KshA1N and KshA2N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

SUBMITTER: Liu HH

PROVIDER: S-EPMC6029100 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

Liu Hao-Hao HH Xu Li-Qin LQ Yao Kang K Xiong Liang-Bin LB Tao Xin-Yi XY Liu Min M Wang Feng-Qing FQ Wei Dong-Zhi DZ

Applied and environmental microbiology 20180702 14

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1<sub>N</sub> and KshA2<sub>N</sub>, were characterized and further engineered in a sterol-digesting strain, ...[more]

PMID: 29728384

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Project description: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.

Project description:Background9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a significant intermediate for the synthesis of glucocorticoid drugs. However, in the process of phytosterol biotransformation to manufacture 9-OHAD, product degradation, and by-products restrict 9-OHAD output. In this study, to construct a stable and high-yield 9-OHAD producer, we investigated a combined strategy of blocking Δ1‑dehydrogenation and regulating metabolic flux.ResultsFive 3-Ketosteroid-Δ1-dehydrogenases (KstD) were identified in Mycobacterium fortuitum ATCC 35855. KstD2 showed the highest catalytic activity on 3-ketosteroids, followed by KstD3, KstD1, KstD4, and KstD5, respectively. In particular, KstD2 had a much higher catalytic activity for C9 hydroxylated steroids than for C9 non-hydroxylated steroids, whereas KstD3 showed the opposite characteristics. The deletion of kstDs indicated that KstD2 and KstD3 were the main causes of 9-OHAD degradation. Compared with the wild type M. fortuitum ATCC 35855, MFΔkstD, the five kstDs deficient strain, realized stable accumulation of 9-OHAD, and its yield increased by 42.57%. The knockout of opccr or the overexpression of hsd4A alone could not reduce the metabolic flux of the C22 pathway, while the overexpression of hsd4A based on the knockout of opccr in MFΔkstD could remarkably reduce the contents of 9,21 ‑dihydroxy‑20‑methyl‑pregna‑4‑en‑3‑one (9-OHHP) by-products. The inactivation of FadE28-29 leads to a large accumulation of incomplete side-chain degradation products. Therefore, hsd4A and fadE28-29 were co-expressed in MFΔkstDΔopccr successfully eliminating the two by-products. Compared with MFΔkstD, the purity of 9-OHAD improved from 80.24 to 90.14%. Ultimately, 9‑OHAD production reached 12.21 g/L (83.74% molar yield) and the productivity of 9-OHAD was 0.0927 g/L/h from 20 g/L phytosterol.ConclusionsKstD2 and KstD3 are the main dehydrogenases that lead to 9-OHAD degradation. Hsd4A and Opccr are key enzymes regulating the metabolic flux of the C19- and C22-pathways. Overexpression of fadE28-29 can reduce the accumulation of incomplete degradation products of the side chains. According to the above findings, the MF-FA5020 transformant was successfully constructed to rapidly and stably accumulate 9-OHAD from phytosterols. These results contribute to the understanding of the diversity and complexity of steroid catabolism regulation in actinobacteria and provide a theoretical basis for further optimizing industrial microbial catalysts.

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

Engineered 3-Ketosteroid 9?-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

Publications

Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs.

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