title: Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.
abstract: A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-744, Republic of Korea.
authors.forename: Sujin
authors.initials: S
authors.lastname: Kim
authors.forename: Kyusung
authors.initials: K
authors.lastname: Lee
authors.forename: Sang-Jeong
authors.initials: SJ
authors.lastname: Bae
authors.forename: Ji-Sook
authors.initials: JS
authors.lastname: Hahn
mesh_terms: D000093:Acetoin / Q000378:metabolism; D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D002072:Butylene Glycols / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D024322:Amino Acids, Aromatic; D002072:Butylene Glycols; D050485:GABA Plasma Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C513599:UGA4 protein, S cerevisiae; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C026978:2,3-butylene glycol; D005680:gamma-Aminobutyric Acid; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D000095:Acetolactate Synthase; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase