Abstract
Mycobacterium neoaurum is a saprophytic, soil-dwelling bacterium. The strain NRRL B-3805 converts phytosterols to androst-4-ene-3,17-dione (androstenedione; AD), a precursor of multiple C19 steroids of importance to industry. NRRL B-3805 itself is able to convert AD to other steroid products, including testosterone (Ts) and androst-1,4-diene-3,17-dione (androstadienedione; ADD). However to improve this strain for industrial use, genetic modification is a priority. In this chapter, we describe a range of genetic techniques that can be used for M. neoaurum NRRL B-3805. Methods for transformation, expression, and gene knockouts are presented as well as plasmid maintenance and stability.
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Acknowledgments
We are grateful to Beatrix Suess for donating the plasmid pGUS_ermE_E* and to the Biotechnology and Biological Sciences Research Council, UK, for funding (project BB/L003619).
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Loraine, J.K., Smith, M.C.M. (2017). Genetic Techniques for Manipulation of the Phytosterol Biotransformation Strain Mycobacterium neoaurum NRRL B-3805. In: Barredo, JL., Herráiz, I. (eds) Microbial Steroids. Methods in Molecular Biology, vol 1645. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7183-1_7
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DOI: https://doi.org/10.1007/978-1-4939-7183-1_7
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