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

Averesch, Nils J. H., Winter, Gal and Kromer, Jens O. (2016) Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae. Microbial Cell Factories, 15 89: 1-16. doi:10.1186/s12934-016-0485-8


Author Averesch, Nils J. H.
Winter, Gal
Kromer, Jens O.
Title Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae
Formatted title
Production of para-aminobenzoic acid from different carbon-sources in engineered Saccharomyces cerevisiae
Journal name Microbial Cell Factories   Check publisher's open access policy
ISSN 1475-2859
Publication date 2016-05-26
Year available 2016
Sub-type Article (original research)
DOI 10.1186/s12934-016-0485-8
Open Access Status DOI
Volume 15
Issue 89
Start page 1
End page 16
Total pages 16
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2017
Language eng
Formatted 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.
Keyword pABA
Phenylethanol
Aromatics
Yeast
Glycerol
Ethanol
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
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