title: Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.
abstract: Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose YSFarcould be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.
authors: Department of Biology and Biological Engineering, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.
authors: Stefan
authors: S
authors: Tippmann
authors: Division of Proteomics and Nanobiotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Science for Life Laboratory , SE171 21 Stockholm, Sweden.
authors: Josefine
authors: J
authors: Anfelt
authors: Department of Biology and Biological Engineering, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.
authors: Florian
authors: F
authors: David
authors: Department of Biology and Biological Engineering, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors: Jacqueline M
authors: JM
authors: Rand
authors: Department of Biology and Biological Engineering, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.
authors: Verena
authors: V
authors: Siewers
authors: Division of Proteomics and Nanobiotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Science for Life Laboratory , SE171 21 Stockholm, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK2970 Hørsholm, Denmark.
authors: Mathias
authors: M
authors: Uhlén
authors: Department of Biology and Biological Engineering, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology , SE412 96 Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , DK2970 Hørsholm, Denmark.
authors: Jens
authors: J
authors: Nielsen
authors: Division of Proteomics and Nanobiotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Science for Life Laboratory , SE171 21 Stockholm, Sweden.
authors: Elton P
authors: EP
authors: Hudson
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D051228:Geranyltranstransferase / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017368:Protein Prenylation; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D051228:Geranyltranstransferase; D011755:Pyrophosphatases; C109459:beta-farnesene synthase
keywords: PHB; affibodies; biofuels; isoprenoids; metabolic engineering; yeast