title: Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae.
abstract: BACKGROUND
  In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.
  
  
  RESULTS
  We analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600up to 41% while FFA levels were decreased by 63% compared with the control strain.
  
  
  CONCLUSIONS
  Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden.
authors.forename: Paulo Gonçalves
authors.initials: PG
authors.lastname: Teixeira
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden.
authors.forename: Raphael
authors.initials: R
authors.lastname: Ferreira
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden.
authors.forename: Yongjin J
authors.initials: YJ
authors.lastname: Zhou
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden.
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Gothenburg, Sweden. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark. nielsenj@chalmers.se.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics; D003066:Coenzyme A Ligases / Q000235:genetics; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D005233:Fatty Alcohols / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D053520:Marinobacter / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D019910:Mycobacterium marinum / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005230:Fatty Acids, Nonesterified; D005233:Fatty Alcohols; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; C019825:carboxylic acid reductase; D003066:Coenzyme A Ligases; C511244:Faa1 protein, S cerevisiae
keywords: Dynamic control; FAA1; Fatty acid activation; Fatty alcohols; Metabolic engineering; Yeast