title: Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.
abstract: The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Barbara U
authors.initials: BU
authors.lastname: Kozak
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Harmen M
authors.initials: HM
authors.lastname: van Rossum
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Marijke A H
authors.initials: MA
authors.lastname: Luttik
authors.affiliation: DSM Biotechnology Center, Delft, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: Akeroyd
authors.affiliation: Amyris Inc., Emeryville, California, USA.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Benjamin
authors.affiliation: DSM Biotechnology Center, Delft, The Netherlands.
authors.forename: Liang
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands A.J.A.vanMaris@TUDelft.nl.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000332:Aerobiosis; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D003470:Culture Media / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D020869:Gene Expression Profiling; D060847:Metabolic Engineering; D064688:Metabolic Flux Analysis; D008969:Molecular Sequence Data; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A