Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics. Issue 1 (December 2015)

• Record Type: Journal Article
• Authors: Gold, Nicholas; Gowen, Christopher; Lussier, Francois-Xavier; Cautha, Sarat; Mahadevan, Radhakrishnan; Martin, Vincent
• Is Part Of: Microbial cell factories. Volume 14:Issue 1(2015)
• Page Start: 1
• Page End: 16
• Publication Date: 2015-12
• Subjects: L-tyrosine -- Saccharomyces cerevisiae -- Metabolic engineering -- Targeted metabolomics -- Glucose-6-phosphate dehydrogenase -- Pyruvate kinase -- Prephenate dehydrogenase -- Cyclohexadienyl dehydrogenase -- Phenylpyruvate decarboxylase -- Aromatic amino acids; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-015-0252-2
• Languages: English
• ISSNs: 1475-2859

Abstract

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 yeastSaccharomyces 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 fromS. 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 fromZymomonas mobilis . Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. TheTAL gene fromRhodobacter 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. TheZWF1 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 expressingTYRC 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 inS. cerevisiae .