title: Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.
abstract: BACKGROUND
  In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs.
  
  
  RESULTS
  Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed.
  
  
  CONCLUSIONS
  In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Kanchana R
authors.identifier: http://orcid.org/0000-0001-9885-380X
authors.initials: KR
authors.lastname: Kildegaard
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.|Evolva Biotech A/S, Lersø Park Allé 42-44, 2100, Copenhagen, Denmark.
authors.forename: Niels B
authors.initials: NB
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Schneider
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University, Worringer Weg 1, 52056, Aachen, Germany.
authors.forename: Eik
authors.initials: E
authors.lastname: Czarnotta
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Emre
authors.initials: E
authors.lastname: Özdemir
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Tobias
authors.initials: T
authors.lastname: Klein
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Maury
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University, Worringer Weg 1, 52056, Aachen, Germany.
authors.forename: Birgitta E
authors.initials: BE
authors.lastname: Ebert
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Christensen
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.|The Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.|The Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.|Bio R&D Center, Paikkwang Industrial Co. Ltd, 57 Oehang-4 gil, Gunsan-si, Jellabukdo, Korea.
authors.forename: Il-Kwon
authors.initials: IK
authors.lastname: Kim
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Herrgård
authors.affiliation: Institute of Applied Microbiology, RWTH Aachen University, Worringer Weg 1, 52056, Aachen, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.
authors.forename: Jochen
authors.initials: J
authors.lastname: Forster
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark.|Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. irbo@biosustain.dtu.dk.
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
mesh_terms: D041861:Chloroflexus / Q000201:enzymology / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D030781:Organisms, Genetically Modified; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000201:enzymology / Q000235:genetics
chemical_list: D019344:Lactic Acid; C031601:hydracrylic acid; D010088:Oxidoreductases; C454539:malonyl-Coa reductase
keywords: 3-Hydroxypropionic acid; Metabolic engineering; Redox metabolism; Saccharomyces cerevisiae