title: Spanning high-dimensional expression space using ribosome-binding site combinatorics.
abstract: Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.
authors.affiliation: Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Lior
authors.initials: L
authors.lastname: Zelcbuch
authors.forename: Niv
authors.initials: N
authors.lastname: Antonovsky
authors.forename: Arren
authors.initials: A
authors.lastname: Bar-Even
authors.forename: Ayelet
authors.initials: A
authors.lastname: Levin-Karp
authors.forename: Uri
authors.initials: U
authors.lastname: Barenholz
authors.forename: Michal
authors.initials: M
authors.lastname: Dayagi
authors.forename: Wolfram
authors.initials: W
authors.lastname: Liebermeister
authors.forename: Avi
authors.initials: A
authors.lastname: Flamholz
authors.forename: Elad
authors.initials: E
authors.lastname: Noor
authors.forename: Shira
authors.initials: S
authors.lastname: Amram
authors.forename: Alexander
authors.initials: A
authors.lastname: Brandis
authors.forename: Tasneem
authors.initials: T
authors.lastname: Bareia
authors.forename: Ido
authors.initials: I
authors.lastname: Yofe
authors.forename: Halim
authors.initials: H
authors.lastname: Jubran
authors.forename: Ron
authors.initials: R
authors.lastname: Milo
mesh_terms: D001665:Binding Sites; D002338:Carotenoids / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009876:Operon; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011506:Proteins; D002338:Carotenoids