title: Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.
abstract: BACKGROUND
  L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling.
  
  
  RESULTS
  Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways.
  
  
  CONCLUSIONS
  Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae.
authors.affiliation: Department of Biology and Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC, H4B 1R6, Canada. nicholas.gold@concordia.ca.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Gold
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada. chris.gowen@utoronto.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Gowen
authors.affiliation: Department of Biology and Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC, H4B 1R6, Canada. fx.lussier@concordia.ca.
authors.forename: Francois-Xavier
authors.initials: FX
authors.lastname: Lussier
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada. sarat.cautha@utoronto.ca.
authors.forename: Sarat C
authors.initials: SC
authors.lastname: Cautha
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada. krishna.mahadevan@utoronto.ca.|Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON, M5S 3G9, Canada. krishna.mahadevan@utoronto.ca.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Mahadevan
authors.affiliation: Department of Biology and Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC, H4B 1R6, Canada. vincent.martin@concordia.ca.
authors.forename: Vincent J J
authors.initials: VJ
authors.lastname: Martin
mesh_terms: D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D055432:Metabolomics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D005954:Glucosephosphate Dehydrogenase