Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics

Microbial Cell Factories

Research

Francois-Xavier Lussier¹  Vincent J. J. Martin¹  Nicholas D. Gold¹  Christopher M. Gowen²  Sarat C. Cautha²  Radhakrishnan Mahadevan³ 

[1] Department of Biology and Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, H4B 1R6, Montreal, QC, Canada;Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, M5S 3E5, Toronto, ON, Canada;Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, M5S 3E5, Toronto, ON, Canada;Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, M5S 3G9, Toronto, ON, Canada;

关键词: L-tyrosine;    Saccharomyces cerevisiae;    Metabolic engineering;    Targeted metabolomics;    Glucose-6-phosphate dehydrogenase;    Pyruvate kinase;    Prephenate dehydrogenase;    Cyclohexadienyl dehydrogenase;    Phenylpyruvate decarboxylase;    Aromatic amino acids;   

DOI  :  10.1186/s12934-015-0252-2

 received in 2014-12-09, accepted in 2015-05-06,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundL-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.ResultsAddition 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 ARO4FBR 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.ConclusionsOur 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.

【 授权许可】

Unknown   
© Gold et al. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.