Averesch, Nils J H; Winter, Gal; Kromer, Jens O

Author(s)

Averesch, Nils J H

Winter, Gal

Kromer, Jens O

Publication Date

2016

Abstract

Background. Biological production of the aromatic compound para-aminobenzoic acid (pABA) is of great interest to the chemical industry. Besides its application in pharmacy and as crosslinking agent for resins and dyes pABA is a potential precursor for the high-volume aromatic feedstocks terephthalic acid and 'para'-phenylenediamine. The yeast 'Saccharomyces cerevisiae' synthesises pABA in the shikimate pathway: Outgoing from the central shikimate pathway intermediate chorismate, pABA is formed in two enzyme-catalysed steps, encoded by the genes 'ABZ1' and 'ABZ2'. In this study 'S. cerevisiae' metabolism was genetically engineered for the overproduction of pABA. Using in silico metabolic modelling an observed impact of carbon-source on product yield was investigated and exploited to optimize production. Results. A strain that incorporated the feedback resistant 'ARO4'K229L and deletions in the 'ARO7' and 'TRP3' genes, in order to channel flux to chorismate, was used to screen different ABZ1 and ABZ2 genes for pABA production. In glucose based shake-flaks fermentations the highest titer (600 µM) was reached when over-expressing the 'ABZ1' and 'ABZ2' genes from the wine yeast strains AWRI1631 and QA23, respectively. 'In silico' metabolic modelling indicated a metabolic advantage for pABA production on glycerol and combined glycerol-ethanol carbon-sources. This was confirmed experimentally, the empirical ideal glycerol to ethanol uptake ratios of 1:2-2:1 correlated with the model. A ¹³C tracer experiment determined that up to 32% of the produced pABA originated from glycerol. Finally, in fed-batch bioreactor experiments pABA titers of 1.57 mM (215 mg/L) and carbon yields of 2.64% could be achieved. Conclusion. In this study a combination of genetic engineering and in silico modelling has proven to be a complete and advantageous approach to increase pABA production. Especially the enzymes that catalyse the last two steps towards product formation appeared to be crucial to direct flux to pABA. A stoichiometric model for carbon-utilization proved useful to design carbon-source composition, leading to increased pABA production. The reported pABA concentrations and yields are, to date, the highest in 'S. cerevisiae' and the second highest in a microbial production system, underlining the great potential of yeast as a cell factory for renewable aromatic feedstocks.

Citation

Microbial Cell Factories, v.15, p. 1-16

ISSN

1475-2859

Link

https://hdl.handle.net/1959.11/20481

Publisher

BioMed Central Ltd

Title

Production of 'para'-aminobenzoic acid from different carbon-sources in engineered 'Saccharomyces cerevisiae'

Type of document

Journal Article

Entity Type

Publication

Author(s)	Averesch, Nils J H Winter, Gal Kromer, Jens O
Publication Date	2016
Abstract	Background. Biological production of the aromatic compound para-aminobenzoic acid (pABA) is of great interest to the chemical industry. Besides its application in pharmacy and as crosslinking agent for resins and dyes pABA is a potential precursor for the high-volume aromatic feedstocks terephthalic acid and 'para'-phenylenediamine. The yeast 'Saccharomyces cerevisiae' synthesises pABA in the shikimate pathway: Outgoing from the central shikimate pathway intermediate chorismate, pABA is formed in two enzyme-catalysed steps, encoded by the genes 'ABZ1' and 'ABZ2'. In this study 'S. cerevisiae' metabolism was genetically engineered for the overproduction of pABA. Using in silico metabolic modelling an observed impact of carbon-source on product yield was investigated and exploited to optimize production. Results. A strain that incorporated the feedback resistant 'ARO4'K229L and deletions in the 'ARO7' and 'TRP3' genes, in order to channel flux to chorismate, was used to screen different ABZ1 and ABZ2 genes for pABA production. In glucose based shake-flaks fermentations the highest titer (600 µM) was reached when over-expressing the 'ABZ1' and 'ABZ2' genes from the wine yeast strains AWRI1631 and QA23, respectively. 'In silico' metabolic modelling indicated a metabolic advantage for pABA production on glycerol and combined glycerol-ethanol carbon-sources. This was confirmed experimentally, the empirical ideal glycerol to ethanol uptake ratios of 1:2-2:1 correlated with the model. A ¹³C tracer experiment determined that up to 32% of the produced pABA originated from glycerol. Finally, in fed-batch bioreactor experiments pABA titers of 1.57 mM (215 mg/L) and carbon yields of 2.64% could be achieved. Conclusion. In this study a combination of genetic engineering and in silico modelling has proven to be a complete and advantageous approach to increase pABA production. Especially the enzymes that catalyse the last two steps towards product formation appeared to be crucial to direct flux to pABA. A stoichiometric model for carbon-utilization proved useful to design carbon-source composition, leading to increased pABA production. The reported pABA concentrations and yields are, to date, the highest in 'S. cerevisiae' and the second highest in a microbial production system, underlining the great potential of yeast as a cell factory for renewable aromatic feedstocks.
Citation	Microbial Cell Factories, v.15, p. 1-16
ISSN	1475-2859
Link	https://hdl.handle.net/1959.11/20481
Publisher	BioMed Central Ltd
Title	Production of 'para'-aminobenzoic acid from different carbon-sources in engineered 'Saccharomyces cerevisiae'
Type of document	Journal Article
Entity Type	Publication

Production of 'para'-aminobenzoic acid from different carbon-sources in engineered 'Saccharomyces cerevisiae'

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