Abstract
This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire–MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC–MS analysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang F-Q, Yao K, Wei D (2011) From soybean Phytosterols to steroid hormones. In: Soybean and health
Xiong L-B, Liu H-H, Song X-W, Meng X-G, Liu X-Z, Ji Y-Q, Wang F-Q, Wei D-Z (2020) Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum. J Biotechnol 323:341–346
Martínez-Cámara S, Bahíllo E, Barredo J-L, Rodríguez-Sáiz M (2017) Scale-up of Phytosterols bioconversion into androstenedione. Methods Mol Biol Clifton NJ 1645:199–210
Donova MV, Nikolayeva VM, Dovbnya DV, Gulevskaya SA, Suzina NE (2007) Methyl-beta-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria. Microbiol Read Engl 153:1981–1992
Perez C, Falero A, Llanes N, Hung BR, Hervé ME, Palmero A, Martí E (2003) Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria. J Ind Microbiol Biotechnol 30:623–626
Egorova OV, Gulevskaya SA, Puntus IF, Filonov AE, Donova MV (2002) Production of androstenedione using mutants of mycobacterium sp. J Chem Technol Biotechnol 77:141–147
Marques MPC, Fernandes P (2017) β-Sitosterol bioconversion to androstenedione in microtiter plates. Methods Mol Biol Clifton NJ 1645:167–176
Fischer M, Sawers RG (2013) A universally applicable and rapid method for measuring the growth of streptomyces and other filamentous microorganisms by methylene blue adsorption-desorption. Appl Environ Microbiol 79:4499–4502
Kensy F, Zang E, Faulhammer C, Tan R-K, Büchs J (2009) Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates. Microb Cell Factories 8:31
Josefsen KD, Nordborg A, Sletta H (2017) Bioconversion of Phytosterols into androstenedione by mycobacterium. Methods Mol Biol Clifton NJ 1645:177–197
Calibration data BioLector https://www.m2p-labs.com/services/calibration-data/
Acknowledgments
This work was supported by a grant of the European Union program ERA CoBioTech Syntheroids. Funding was received from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. [722361] and financed through the Research Council of Norway (project number 285849). The authors thank Andrea Draget Hoel, Marit Dyrendahl, and Susan Maleki for their technical support and Curia Global for supplying AD. They are also grateful to have had the Syntheroids consortium members as valuable discussion partners during the work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Le, S.B., Nordborg, A., Josefsen, K.D., Olsen, S.M., Sletta, H. (2023). Cultivation of Mycolicibacterium spp. Mutants in Miniaturized and High-Throughput Format to Characterize Their Growth, Phytosterol Conversion Ability, and Resistance to the Steroid Products. In: Barreiro, C., Barredo, JL. (eds) Microbial Steroids. Methods in Molecular Biology, vol 2704. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3385-4_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3385-4_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3384-7
Online ISBN: 978-1-0716-3385-4
eBook Packages: Springer Protocols