title: Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae.
abstract: Saccharomyces cerevisiae is an important platform organism for synthesis of chemicals and fuels. However, the promoters used in most pathway engineering studies in S. cerevisiae have not been characterized and compared in parallel under multiple conditions that are routinely operated in laboratory and the number of known promoters is rather limited for the construction of large biochemical pathways. Here a total of 14 constitutive promoters from S. cerevisiae were cloned and characterized using a green fluorescent protein (GFP) as a reporter in a 2 µ vector pRS426, under varying glucose and oxygen concentrations. The strengths of these promoters varied no more than sixfold in the mean fluorescence intensity of GFP, with promoter TEF1p being the strongest and promoter PGI1p the weakest. As an example of application for these promoters in metabolic engineering, the genes involved in xylan degradation and zeaxanthin biosynthesis were subsequently cloned under the control of promoters with medium to high strength and assembled into a single pathway. The corresponding construct was transformed to a S. cerevisiae strain integrated with a D-xylose utilizing pathway. The resulting strain produced zeaxanthin with a titer of 0.74 ± 0.02 mg/L directly from birchwood xylan.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Zengyi
authors.initials: Z
authors.lastname: Shao
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.forename: Nikhil
authors.initials: N
authors.lastname: Nair
authors.forename: Fei
authors.initials: F
authors.lastname: Wen
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D019677:Artificial Gene Fusion; D001709:Biotechnology / Q000379:methods; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D024341:Xanthophylls / Q000378:metabolism; D065146:Zeaxanthins
chemical_list: D024341:Xanthophylls; D065146:Zeaxanthins; D049452:Green Fluorescent Proteins