title: Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis.
abstract: Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3' of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.
authors: UCSF-UCB Joint Graduate Group in Bioengineering, Berkeley, CA 94720, USA.
authors: Jeffrey A
authors: JA
authors: Dietrich
authors: David L
authors: DL
authors: Shis
authors: Azadeh
authors: A
authors: Alikhani
authors: Jay D
authors: JD
authors: Keasling
mesh_terms: D020001:1-Butanol / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D003998:Dicarboxylic Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods; D011401:Promoter Regions, Genetic; D054852:Small Molecule Libraries / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003998:Dicarboxylic Acids; D029968:Escherichia coli Proteins; D054852:Small Molecule Libraries; D014157:Transcription Factors; D020001:1-Butanol
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