English

• The demand and market for steroidal drugs are huge and everincreasing largely due to their wide spectrum of indications such as anti-inflammation, anti-tumor, endocrine regulation, fertility management. The steroids market sales in 2011 were estimated in ＄8 billion [1]. The widely distributed sterols, including cholesterol, phytosterols [2], phytosterol-containing rice germ and wheat germ ethanolic extracts [3], and ergosterol, can be biotransformed into high-value-added intermediates, such as 4- androstene-3, 17-dione (4-AD) and androsta-1, 4-diene-3, 17-dione (ADD) by non-pathogenic mycobacteria, two main important intermediates for most steroidal drugs that are difficult to produce chemically [4-6]. Soil-dwelling Mycobacteria such as Mycobacterium fortuitum [7, 8] and M. neoaurum was well-characterized AD producer [1, 9-11]. To be commercially more competitive, it is most desirable to engineer and screen mutants [12] or recombinants which can produce higher yield of ADD or bisnoraldehyde (BA). Since gene manipulation for steroid medicines production is much more easier now, rapid, efficient strain selection technology is usually a bottleneck, especially for microbial transformation. Multiple factors are involved in the yield and distribution of biotransformed products of sterols in industrial applications, such as the phytosterol transport system for the uptake and transport of sterols [1], 3-ketosteroid-9α-hydroxylase catalyzing the transformation of 4-androstene-3, 17-dione (AD) into 9α-hydroxy-4- androstene-3, 17-dione (9-OH-AD) [13], KstD [14] (3-ketosteroid δ(1)-dehydrogenase) and Ksh [15] (3-ketosteroid-9α-hydroxylase) genes involved in the nucleus degradation during microbial biotransformation of phytosterol [16], multi-catabolic enzymes degrading the sterol side-chain [17], regulator such as sigma factor D (sigD) [18], the media such as cyclodextrin [19], and aqueous phytosterol microdispersion [20]. While targeted genetic mutants construction usually sufferring from limited knowledge on the regulation of bioconversion, large scale random transposon-based mutants library construction and high yield mutants screen are preferred but necessitate high throughput, streamlined methodology instead of current thin-layer chromatography, gas-liquid chromatography (GLC), GLC-mass spectrometry, or HPLC-based measurement [21], which is time-consuming, expensive and laborintensive.

  The sterols bioconversion products by M. neoaurum is a mixture containing BA, 4-AD and ADD. The productivity in the biotransformation of steroids was determined by multiple factors such as conversion rate, conversion ratio, or substrate concentration [17]. For a new method viable for expedient, high throughput screen the mutant with desired yield of targeted product, either 4- AD, or ADD, it is essential to evaluate the relative content of each components in the mixture. To this end, the standard curve (Fig. 1) was firstly established. As shown in Fig. 1, the OD reads of AD + 4AD vary with their concentration. The range used in the standard curve was included in subsequent mixture. Though response surface methodology was a powerful and efficient method to optimize the parameters of phytosterol biotransformation process [22], in this study, uniform design method [23, 24] popular in such studies was employed. For the 3 major components of the sterol bioconversion, we mimicked 5 concentration ratio for each component, namely 0, 0.3, 0.5, 0.8, and 1 (as shown in Table 1). Therefore, U5 (5³) uniform design table can be referred. Each of the 3 components was dissolved in DMSO and the volume for each well was calculated and added. SpectraMax®190 (MD) was used to assay the OD value at wavelength 254 nm of each well in a 96-well plate, since both AD and ADD have characteristic absorption peak at 254 nm, 200 μL samples were pippetted to each well for measurement.

  Figure 1

  

  Figure 1.  The standard absorption curve of AD + 4AD.

  DownLoad: Full-Size Img PowerPoint

  Table 1

  

  Table 1.  U5(5³) uniform design formula for the 3 components with different ratio in the mixture.

  DownLoad: CSV

  The data showed that the 96-well plate based method can discriminate the mixture of the 3 components with different ratio, and the detection time is less than 5 min for 96-well (as shown in Fig. 2). While the conventional HPLC-based method will take much more time for each compound and can not be used high throughput. In brief, this method can be applied to primary screen of mutants library for desired strains.

  Figure 2

  

  Figure 2.  The optimal densities of the mimic mixtures of the 3 components with 5 different ratios. The dot represents one experiment. The figure shows the result of 3 replicates. 96-well plate represents a cost-effective method to replace the HPLCbased characterization of the yield of sterol derivatives.

  DownLoad: Full-Size Img PowerPoint

  To validate this, M. neoaurum strain was cultured and the fermentation product using phytosterol was subjected to 96-well detection. The following media were used. LB medium (NaCl 10 g/L, yeast extract 5 g/L, tryptone 10 g/L) for conventional culture. MYC/ 01 seed culture media (glycerol 20 g/L, citric acid 2 g/L, Ferric ammonium citrate 0.05 g/L, K₂HPO₄ 0.5 g/L, MgSO₄·7H₂O 0.5 g/L, NH₄NO₃ 2g/L), pH 7.5, 121 ℃ heat sterilization for 20 min and preserved for future use. MYC/02 fermentation media (citric acid 2 g/L, Ferric ammonium citrate 0.05 g/L, K₂HPO₄ 0.5 g/L, MgSO₄·7H₂O 0.5 g/L, NH₄NO₃ 2 g/L, phytosterol 2 g/L), pH 7.5, 121 ℃ high-pressure steam sterilization for 20 min and for future use. The M. neoaurum was cultured overnight in the LB medium and preserved as inocula. Inoculation of 1% bacteria into a new LB medium from the inocula. M. neoaurum cultured in LB to OD₆₀₀ = 1.5 was inoculated with 1% strains into MYC/02 fermentation Media containing 2 g/L phytosterol to detect the substrate transformation capacity and metabolic product analysis. The bacteria was cultured in Erlenmeyer flask in a shaker for 5–6 days and sampled every 24 h to monitor the yield. The value of the OD reflects the relative content of the mixture. There is significant difference of OD as the function of fermentation time (Fig. 3). The data showed that the method can discriminate the yield of phytosterol bioconversion. Further works are needed to quantify the relationship between the OD value and the content of targeted biotransformed compounds. The basis underlying the up and down of the values in Fig. 2 remains to be determined, one explanation might be the effect of other similar compounds in the bioconversion process. This method can also be adapted to measure other useful byproduct [9]. Microorganisms, selected wild-type or engineered strains, are increasingly effective tools of steroid modifications for the production of high-valued steroidal drugs and their precursors for the pharmaceutical industry, and applied at an industrial level [25]. The steroid-transforming strains in combination with our novel approach provide a highly effective and versatile platform example in the field of microbial bioconversions.

  Figure 3

  

  Figure 3.  The optimal densities of the phytosterol fermentation products converted by Mycobacterium neoaurum. Each dot represents a replicate, the figure showed the results of three replicates.

  DownLoad: Full-Size Img PowerPoint

  In conclusion, this study provided an efficient high throughput method to screen mutants with expected quantitative traits.

  Acknowledgments

  The standard chemicals of 4-androstene-3, 17-dione, androsta-1, 4-diene-3, 17-dione, and bisnoraldehyde, and the Mycobacterium neoaurum strain were gifts from Gongtong Pharmaceutics, Hubei.

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• 

  Figure 1  The standard absorption curve of AD + 4AD.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  The optimal densities of the mimic mixtures of the 3 components with 5 different ratios. The dot represents one experiment. The figure shows the result of 3 replicates. 96-well plate represents a cost-effective method to replace the HPLCbased characterization of the yield of sterol derivatives.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  The optimal densities of the phytosterol fermentation products converted by Mycobacterium neoaurum. Each dot represents a replicate, the figure showed the results of three replicates.

  下载: 全尺寸图片 幻灯片

  Table 1.  U5(5³) uniform design formula for the 3 components with different ratio in the mixture.

  下载: 导出CSV
•