title: Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay.
abstract: Engineered metabolic pathways often suffer from flux imbalances that can overburden the cell and accumulate intermediate metabolites, resulting in reduced product titers. One way to alleviate such imbalances is to adjust the expression levels of the constituent enzymes using a combinatorial expression library. Typically, this approach requires high-throughput assays, which are unfortunately unavailable for the vast majority of desirable target compounds. To address this, we applied regression modeling to enable expression optimization using only a small number of measurements. We characterized a set of constitutive promoters in Saccharomyces cerevisiae that spanned a wide range of expression and maintained their relative strengths irrespective of the coding sequence. We used a standardized assembly strategy to construct a combinatorial library and express for the first time in yeast the five-enzyme violacein biosynthetic pathway. We trained a regression model on a random sample comprising 3% of the total library, and then used that model to predict genotypes that would preferentially produce each of the products in this highly branched pathway. This generalizable method should prove useful in engineering new pathways for the sustainable production of small molecules.
authors.affiliation: The UC Berkeley & UCSF Graduate Program in Bioengineering, Berkeley, CA 94720, USA, Department of Bioengineering, University of California, Berkeley, CA 94720, USA, Energy Biosciences Institute, Berkeley, CA 94720, USA, Department of Industrial Engineering and Operations Research, University of California, Berkeley, CA 94720, USA and Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA 94720, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lee
authors.forename: Anil
authors.initials: A
authors.lastname: Aswani
authors.forename: Audrey S
authors.initials: AS
authors.lastname: Han
authors.forename: Claire J
authors.initials: CJ
authors.lastname: Tomlin
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics*; D005786:Gene Expression Regulation; D015723:Gene Library; D060005:Genotyping Techniques; D007211:Indoles / Q000378:metabolism; D016014:Linear Models; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D007211:Indoles; C063155:violacein
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