title: A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzymes.
abstract: Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼ 90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba 263-8522, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Furubayashi
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba 263-8522, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Ikezumi
authors.affiliation: Department of Biology, Nippon Medical School, Musashino, Tokyo 180-0023, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Takaichi
authors.affiliation: Research Institute for Production Development, Kyoto 606-0805, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Maoka
authors.affiliation: Department of Applied Molecular Biosciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
authors.affiliation: Department of Applied Molecular Biosciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Applied Chemistry and Biotechnology, Chiba University, Chiba 263-8522, Japan.
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Saito
authors.affiliation: DuPont Industrial Biosciences, Experimental Station, Wilmington, Delaware 19803, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Tobias
authors.affiliation: 1] Department of Applied Chemistry and Biotechnology, Chiba University, Chiba 263-8522, Japan [2] Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Umeno
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002338:Carotenoids / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000378:metabolism*; D051228:Geranyltranstransferase / Q000378:metabolism*; D024341:Xanthophylls / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D024341:Xanthophylls; D002338:Carotenoids; C005948:astaxanthine; C091056:CrtM protein, Staphylococcus aureus; D051228:Geranyltranstransferase; D013186:Farnesyl-Diphosphate Farnesyltransferase