title: Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro.
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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA.
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Cacho
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA. yitang@ucla.edu.|Department of Chemistry and Biochemistry, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA. yitang@ucla.edu.|Department of Bioengineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA. yitang@ucla.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics*; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D005656:Fungal Proteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Fungal secondary metabolite; Nonribosomal peptide synthetase; Transformation-assisted recombination; Yeast protein expression