title: Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae.
abstract: Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96, Gothenburg, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tippmann
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96, Gothenburg, Sweden.
authors.forename: Raphael
authors.initials: R
authors.lastname: Ferreira
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96, Gothenburg, Sweden.
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 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, DK2800 Kongens Lyngby, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE412 96, Gothenburg, Sweden. yunc@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE412 96, Gothenburg, Sweden. yunc@chalmers.se.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D008798:Mevalonic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology
chemical_list: D000214:Acyl Coenzyme A; D012717:Sesquiterpenes; C010667:acetoacetyl CoA; C062671:beta-farnesene; D000105:Acetyl Coenzyme A; D000101:Acetyl-CoA C-Acetyltransferase; D003066:Coenzyme A Ligases; D008798:Mevalonic Acid
keywords: Biofuels; Isoprenoids; Metabolic engineering; Mevalonate pathway; Yeast