Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB , were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII- pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII- pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII- pbpB might contribute to the positive effect on sterol utilization. The productivity of 4-HBC was enhanced by 28% in the WIII- pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.
Background The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9 α -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 kasB -deficient 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 9 α -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.
Understanding the mycobacteria degradation of sterol may facilitate strategies for the directed biotransformation of steroidal drug intermediates. Due to the long and complicated catabolic process, several key steps that involved in the C-C bond cleavage of steroidal side-chain remain unknown. In the present study, an aldolase SalA from Mycobacterium neoaurum was identified that was related to the synthesis of 3-oxo-4-pregnene-20-carboxyl aldehyde and 20-hydroxymethyl pregn-4-ene-3-one (4-HBC). As a member of the thiolase superfamily, the aldolase SalA-SalB is composed of a thiolase-like protein (SalA) and a DUF35 domain (SalB). As a result, the gene salA was determined that had special significance for the synthesis of 4-HBC in sterol metabolism by performing knockout and replenishment. Based on insights provided by the docking model and mutants analysis, the hypothesis of the catalytic mechanism was proposed. Furthermore, the deletion of salA absolutely eliminated the by-product 4-HBC and meanwhile the molar yield of ADD was increased by 10 %. The dominant role of SalA in the C24 steroidal side-chain cleavage as confirmed in this study provided a further clarification on the phytosterol degrading pathway in Mycobacterium neoaurum , which is of significant importance for the directed biotransformation between C19- and C22-steroids.
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