title: Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.
abstract: The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, 900 University Avenue, Riverside, California 92521 College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China.
authors.forename: Shouqiang
authors.initials: S
authors.lastname: Ouyang
authors.affiliation: Department of Chemistry, University of California, Riverside, 900 University Avenue, Riverside, California 92521.
authors.forename: Consuelo N
authors.initials: CN
authors.lastname: Beecher
authors.affiliation: Department of Chemistry, University of California, Riverside, 900 University Avenue, Riverside, California 92521.
authors.forename: Kang
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, Riverside, 900 University Avenue, Riverside, California 92521 katherine.borkovich@ucr.edu clarive@ucr.edu.
authors.forename: Cynthia K
authors.initials: CK
authors.lastname: Larive
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, 900 University Avenue, Riverside, California 92521 katherine.borkovich@ucr.edu clarive@ucr.edu.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Borkovich
mesh_terms: D003300:Copper / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D005800:Genes, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D005973:Glutamine; D055442:Metabolome; D055432:Metabolomics; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D066244:Proton Magnetic Resonance Spectroscopy
chemical_list: D009566:Nitrates; D005973:Glutamine; D003300:Copper; D009584:Nitrogen
keywords: NMR; heterologous gene expression; inducible promoter; metabolic profiling; metabolomics