9α-Hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid pharmaceutical intermediate which can be produced by the conversion of soybean phytosterols in mycobacteria. However, the unsatisfactory productivity and conversion efficiency of engineered mycobacterial strains hinder their industrial applications. Here, a sigma factor D (sigD) was investigated due to its dramatic downregulation during the conversion of phytosterols to 9-OHAD. It was determined as a negative regulator in the metabolism of phytosterols, and the deletion of sigD in a 9-OHAD-producing strain significantly enhanced the titer of 9-OHAD by 18.9%. Furthermore, a high yielding strain was constructed by the combined modifications of sigD and choM2, a key gene in the phytosterol metabolism pathway. After the modifications, the productivity of 9-OHAD reached 0.071 g/L/h (10.27 g/L from 20 g/L phytosterol), which was 22.5% higher than the original productivity of 0.058 g/L/h (8.37 g/L from 20 g/L phytosterol) in the industrial resting cell biotransformation system.

BackgroundThe strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC.Results MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system.ConclusionsIncreasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0705-x) contains supplementary material, which is available to authorized users.

Background: The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. Results: The deletion of the nonessential kasB, encoding a β-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasBdeficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. Conclusions: The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.

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 and KshA2, were characterized and further engineered in a sterol-digesting strain, ATCC 25795 to construct androstenone-producing strains. Thereinto, 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 led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined to be a redundant form of Through a combined modification of , , and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g l ADD from the transformation of 20 g l phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1 that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g l 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g l phytosterol in 120 h. Steroidal drugs are widely used for anti-inflammation, anti-tumor, endocrine regulation, 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 stems from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1 and KshA2, in 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.

Integrated transcriptome and proteome studies were performed to investigate sterol biotransformation in wild-type Mycobacterium neoaurum ATCC 25795 ( Mn) and the mutant strains producing steroid intermediates. Transcriptome and proteome studies indicated that several metabolic activities were noticeably dynamic, including cholesterol degradation, central carbon metabolism, cell envelope biosynthesis, glycerol metabolism, and transport. Interestingly, a poor overall correlation between mRNA and translation profiles was found, which might contribute to the metabolic adaptation in cholesterol catabolism. A gene cluster covering 111 genes was discovered to encode for cholesterol catabolism in Mn. Generally, transcription and/or translation of the genes in KstR1 regulon was upregulated, and the induction of genes in KstR2 regulon was not as significant as that of KstR1 regulon. Several induced genes showing potential roles for cholesterol catabolism were found. Further identification of these genes and investigation of the correlation among key metabolic activities could help for the development of efficient steroid-producing strains.

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Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.

Ergothioneine (EGT) represents valuable protective functions for humans, but EGT from the diet cannot meet daily requirements. Although the heterologous synthesis of EGT had been achieved, it is still a challenge to obtain stable and highyield EGT-producing cell factories. Here, after the co-overexpression of the EGT synthetic gene cluster and hisG, hisC, and allB1 in Mycolicibacterium neoaurum, the natural EGT titer was increased by 7.2-folds. However, the degradation problem of EGT in large-scale fermentation needs to be urgently solved. A putative lyase gene Mn_3042 was inactivated, thus inhibiting the product degradation and increasing the EGT titer by 21%. Moreover, the enhancement of S-adenosyl-L-methionine regeneration further increased EGT titer by 28%. After optimization of fed-batch fermentation, the yield of EGT was boosted to 1.56 g/L with a productivity of 7.2 mg/L/h. This study provides a systematic engineering strategy for developing EGT-producing cell factories.