Strains, plasmids and primers
All strains used in this study are described below (Table 1). Escherichia coli DH5α (TIANGEN Biotech. Co., Ltd., Shanghai, China) was used for plasmid amplification. The wild type M. neoaurum ATCC 25795 (Mn) was purchased from American Type Culture Collection (ATCC). The C19 steroidal intermediate 9-OHAD producer MnΔkstD1ΔkstD2ΔkstD3 (WI) was constructed by Kang Yao [5]. The C22 steroidal intermediate 4-HBC-producing strain MnΔkshAΔhsd4AΔkstD1ΔkstD2ΔkstD3 (WIII) was constructed by Li-Qin Xu [6]. Others were all derived from the above three M. neoaurum strains. Common plasmids (Additional file 1: Table S1) and primers (Additional file 1: Table S2) were used for constructing the mutants.
Media and culture conditions
Media and culture conditions were the same as the previously described conditions [28, 29]. E. coli DH5α was inoculated at 37 °C in 5 mL of Luria-Bertani (LB) medium. Kanamycin (50 mg/L) or hygromycin (100 mg/L) was added to the culture medium as required. Mycobacterial strains were firstly cultivated in 5 mL of LB until OD600 was between 1.2 and 1.8. Then, according to an inoculum volume ratio of 1:10 (v/v), the cell suspension was inoculated into 30 mL of MYC/01 medium (20.0 g/L glycerol, 2.0 g/L citric acid, 2.0 g/L NH4NO3, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, and 0.05 g/L ammonium ferric citrate, pH 7.5) in 250-mL flasks to obtain the mycobacterial seed suspension (OD600 =1.2-1.8).
For phenotypic identification, according to an inoculum volume ratio of 1:10 (v/v), the cultivated cells were then transferred into 30 mL of minimal medium (MM) (2.0 g/L NH4NO3, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, and 0.05 g/L ammonium ferric citrate) with 1 g/L glycerol or 1 g/L cholesterol (purity > 95.0%, Adamas Reagent, Ltd., Shanghai, China). Cells were harvested by the centrifugation at 4,000 g for 10 min.
For the bioconversion in growth cells, according to an inoculum volume ratio of 1:10 (v/v), the cultivated seed cells were inoculated into 30 mL of MYC/02 medium (10.0 g/L glucose, 2.0 g/L citric acid, 2.0 g/L NH4NO3, 0.5 g/L MgSO4·7H2O, and 0.05 g/L ferric ammonium citrate, pH 7.5) with 5 g/L phytosterols (purity > 95.0%, every 100 g of phytosterol contained 47.5 g of β-sitosterol, 26.4 g of campesterol, 17.7 g of stigmasterol, 3.6 g of brassicasterol and 4.8 g of undetermined components) (Zhejiang Davi Pharmaceutical Co., Ltd., Zhejiang, China) [29]. Cholesterol (100.0 g/L) and phytosterol (100.0 g/L) was emulsified in Tween 80 (5% w/v) aqueous solution at 121 °C for 60 min before use. The shake flask experiments of M. neoaurum strain were carried out at 30 °C and 200 rpm.
For resting cell conversion, according to an inoculum volume ratio of 1:10 (v/v), the cultivated cells were transferred into 150 mL of MYC/02 medium in 1000-mL shake flasks for the growth at 30 °C and 200 rpm. The cells were harvested by the centrifugation at 8,000 g for 15 min, washed with 20 mM KH2PO4, and diluted into 200 g/L of cell suspensions. The subsequent conversion step was performed in 250-mL flasks containing 100 g/L mycobacterial cells, 20 g/L phytosterols and 80 g/L hydroxypropyl-β-cyclodextrin (HP-β-CD, RSC Chemical Industries Co., Ltd., Jiangsu, China) in at 30 °C and 200 rpm [30]. Standard 9-OHAD (99%) was purchased from J&K Scientific Ltd. (Beijing, China). Standard reference 4-HBC (97%) was purified and identified by ourselves [6].
Construction of genetically modified strains
Target gene-deleted strains were obtained through allelic homologous recombination in mycobacteria as previously described [31]. p2NIL and pGOAL19 were used for the construction of the homologous recombination plasmids (Additional file 1: Table S1). The knockout-plasmids p19-gene was transferred into mycobacterial cells via electroporation.
To complement the deficient-gene function, the complete gene sequence was firstly amplified from the wild type strain and then inserted into the pMV261 to create a recombinant pMV261-gene plasmid, which could be used to overexpress the carried gene in multiple copies. Moreover, the expression cassette of the target gene containing a heat shock promoter hsp60 was obtained from the recombinant p261-gene through double-digestion and then integrated into the pMV306 to create a complemental plasmid p306-gene. The constructed plasmid p306-gene could be integrated into chromosomal DNA in single copy to complement the disrupted gene function.
Analysis of cell permeability and steroid uptake performance
The permeability change of cell envelope was estimated by measuring the fluorescence intensity of cells labeled by fluorescein diacetate (FDA, Aladdin Reagents (Shanghai) Co., Ltd., Shanghai, China) according to previous procedures with some minor amendments [32]. The cells were suspended in 4.5 mL of phosphate buffer (cell density reached 106 cells/mL), mixed with 0.5 mL of FDA acetone solution (2 mg/mL) and then vibrated at 32 °C for 10 min before the detection with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Labsystems Inc., PA, USA). Maximum excitation wavelength for the detection was 485 nm, and the emission wavelength was 538 nm.
The quantity of cholest-4-en-3-one (purity > 95.0%, Shanghai TITAN Scientific Co., Ltd., China) entering mycobacterial cells per unit time was determined to check for the cell permeability change. This steroid was emulsified in Tween 80 (5% w/v) aqueous solution at 121 °C for 60 min in advance for use. The cultivated cells were inoculated into 30 mL of MYC/02 medium with 1.0 g/L cholest-4-en-3-one. After 12-h growth, 5 mL of culture solution was sampled, centrifuged at 12,000 g for 10 min, washed with 1.0 mL of ddH2O for two times, and then washed with 1.0 mL of the mixture of petroleum ether and ethyl acetate (6:4, v/v) to remove the cholest-4-en-3-one from the media. The cells (50 mg, wet weight) were then suspended in 1.0 mL of the mixture of acetonitrile and ddH2O (7:3, v/v). Then, 0.8 g of glass beads were added in the suspension. The cells were destroyed with FastPrep-24 instrument (MP Biomedicals, CA, USA) and centrifuged at 12,000 g for 10 min. Cholest-4-en-3-one entering cells could be released and dissolved in acetonitrile. The extracts were analyzed with a reversed-phase C18-column (250 mm × 4.6 mm) at 254 nm with the Agilent 1100 series HPLC system. The mixture of methanol and water (8:2, v/v) was used as the mobile phase.
Analysis of mycolic acid methyl esters (MAMEs)
The MAMEs were extracted and analyzed as previously described [16, 28, 33]. Briefly, 50 mg (in wet weight) of mycobacterial cells were collected at 12,000 g for 10 min. After adding 0.5 mL of the mixture of methanol and chloroform (2:1, v/v), the homogenized mixture was incubated at 60 °C for 2 h and centrifuged at 12,000 g for 10 min. The polar lipids including TMM and TDM were dissolved in the supernatant.
Next, 500 μL of 10% tetrabutylammonium hydroxide (Sigma-Aldrich LLC., MO, USA) was added to the above defatted cells or 50 mg of whole cells and heated at 100 °C overnight. After cooling, 500 μL of ddH2O, 250 μL of dichloromethane, and 62.5 μL of iodomethane (Sigma-Aldrich LLC., MO, USA) were added into the mixture. Then, the diluted mixture was stirred for 30 min and centrifuged at 12,000 g for 10 min to remove the upper layer. The lower organic layer was washed with 1.0 mL of 1 M hydrochloric acid, followed by 1.0 mL of ddH2O. The reaction solution was dried under a stream of nitrogen. The residue was dissolved in a mixture of toluene (0.2 mL) and acetonitrile (0.1 mL), followed by the addition of acetonitrile (0.2 mL) for 1-h incubation at 4 °C. The MAMEs were centrifuged at 12,000 g for 10 min and then re-suspended in 200 μL of dichlormethane.
The extracted mycolic acids were analyzed by silica gel TLC plates in a solvent system (chloroform: methanol, 90:10, v/v). The keto-MA spots on preparative silica gel TLC were purified for MALDI-TOF-MS (Xevo G2, Waters, Ltd., MA) analysis as described [15].
Sterol bioconversion and the extraction and analysis of steroidal intermediates
Both vegetative cells and resting cells were determined to assess the sterol conversion capability [28, 30]. Firstly, the vegetative cell biotransformation medium (0.5 mL) was extracted with the same volume of ethyl acetate. Then the sample containing steroidal intermediates from resting cell transformation system was extracted with ten times of volume of ethyl acetate.
A gas chromatography (GC) system 7820A (Agilent Technologies, CA, USA) was used for the quantitative determination of cholesterol and phytosterols. The ethyl acetate extracts (5 μL) were injected into a DB-5 column (30 m × 0.25 mm (i.d.) × 0.25 μm film thickness, Agilent Technologies, CA, USA). The oven temperature was programmed as follows: 200 °C for 2 min, 200 °C to 280 °C within 4 min, 280 °C for 2 min, 280 °C to 305 °C within 1.5 min, and 305 °C for 10 min. Inlet and flame-ionization detector temperatures were maintained at 320 °C. Nitrogen carrier gas flow was 2 mL/min at 50 °C.
A 1100 series high-performance liquid chromatography system (HPLC) (Agilent Technologies, CA, USA) was employed to analyze the extracts containing steroidal intermediates. The prepared samples were analyzed with a reversed-phase C18-column (250 mm × 4.6 mm) (Agilent Technologies, CA, USA) at 254 nm. The mixture of methanol and water (8:2, v/v) was used as the mobile phase.