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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0252-2) contains supplementary material, which is available to authorized users.

Pterin-Dependent Mono-oxidation for the Microbial Synthesis of a Modified Monoterpene Indole Alkaloid

“…First, we show that tyrosine and tryptophan levels limit the system; thus, increasing aromatic amino acid supply via introduction of feedback-resistant enzyme mutants in aromatic amino acid metabolism, such as Aro4, 70 or utilizing a tyrosine overproducing strain, 71 should enable straightforward increases in amino acid mono-oxidation. Second, our system does not currently take advantage of the power of the BH 4 recycling system, as biogenic amine levels are similar in the presence and absence of the recycling system.…”

Expanding the repertoire of aromatic chemicals by microbial production

“…4). 68 Gold et al 69 also realized a high yield of L-Tyr in S. cerevisiae by a strategy of combining localized pathway engineering with global engineering of central metabolism. Olson et al 67 reported that deletion of pheA encoding chorismate mutase/prephenate dehydratase and its leader peptide gene pheL resulted in a significant increase in L-Tyr production.…”

“…After optimizing the fermentation conditions, the titer of L-Tyr increased to 55 g L −1 in a 200 L fermenter. 68 Gold et al 69 also realized a high yield of L-Tyr in S. cerevisiae by a strategy of combining localized pathway engineering with global engineering of central metabolism. L-Tyr accumulated up to 192 mmol L −1 in the cytosol.…”

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

“…Metabolomics therefore may be used to complement current metabolic engineering strategies for optimizing biological production of chemicals within microorganisms (Oldiges et al, 2007;Putri et al, 2013). Through the detection of relevant metabolic perturbations, metabolomics can identify specific targets for strain improvement that may include detection of pathway bottlenecks, metabolite or product toxicity, imbalanced cofactor supply, or draining of metabolites by alternative pathways (Gold et al, 2015;Hasunuma et al, 2011;Korneli et al, 2012;Noguchi et al, 2016;Ohta et al, 2015;Teoh et al, 2015;Xu et al, 2016). Furthermore, metabolomics provides a comprehensive analysis of the metabolome that allows a deeper understanding of cellular metabolism and how gene modifications can be used for metabolic engineering.…”