Abstract
The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. Here we describe the use of yeast recombination strategies in S. cerevisiae for cloning of the NRPS coding sequence in 2μ-based expression vector; the use of affinity chromatography for purification of NRPS from the total S. cerevisiae soluble protein fraction; and strategies for reconstitution of NRPSs activities in vitro.
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Acknowledgements
We think Dr. Wei Xu for the construction of pXW55 plasmid. Work from our group is supported by the NIH grants 1R01GM085128 and 1DP1GM106413. R.A.C. is supported by the NRSA grant GM008496 and the UCLA Graduate Division.
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Cacho, R.A., Tang, Y. (2016). Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_7
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DOI: https://doi.org/10.1007/978-1-4939-3375-4_7
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