title: ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae.
abstract: BACKGROUND
  With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.
  
  
  RESULTS
  We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate.
  
  
  CONCLUSIONS
  By combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. srodriguez@lbl.gov.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. srodriguez@lbl.gov.
authors.forename: Sarah
authors.initials: S
authors.lastname: Rodriguez
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. cmdenby@lbl.gov.|California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. cmdenby@lbl.gov.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Denby
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. vvu@lbl.gov.|Department of Bioengineering, University of California, Berkeley, CA, USA. vvu@lbl.gov.
authors.forename: T
authors.initials: T
authors.lastname: Van Vu
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. eebaidoo@lbl.gov.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. eebaidoo@lbl.gov.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. georgewang@lbl.gov.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. georgewang@lbl.gov.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA, USA. JDKeasling@lbl.gov.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. JDKeasling@lbl.gov.|California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.|Department of Bioengineering, University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA. JDKeasling@lbl.gov.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000096:biosynthesis*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008798:Mevalonic Acid / Q000378:metabolism*; D009584:Nitrogen / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000105:Acetyl Coenzyme A; D001275:ATP Citrate (pro-S)-Lyase; D009584:Nitrogen; D008798:Mevalonic Acid