Abstract
BackgroundAnthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce.ResultsWe subjected Saccharomyces cerevisiae, a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l−1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula. When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l−1.ConclusionsIn this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.
Highlights
Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds
Here, we first knocked out the TRP4 gene from the genome of S. cerevisiae and further engineered its metabolism for improved ANTH production
By carrying out batch cultivations with a strain optimized for ANTH production, we could obtain a titer of 199.7 mg l−1 in a minimal medium and a titer of 567.9 mg l−1 in SCD medium
Summary
Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Aromatic compounds varying from platform chemicals, such as polymer precursors, to high-value fine chemicals including flavour, fragrance and pharmaceutical substances are widely used in different industries. These compounds are typically derived from benzene, toluene and xylene obtained from petroleum or natural gas, or Kuivanen et al Microb Cell Fact (2021) 20:34 plant-derived compound [7], constitutes a food flavour and cosmetic ingredient of significant demand. S. cerevisiae has been engineered to convert ANTH to cinnamoyl, dihydrocinnamoyl and benzoyl anthranilates, potential pharmaceuticals [13] These compounds were not produced de novo, but ANTH feeding was required. Development and investigation of a S. cerevisiae platform for de novo ANTH production could provide a platform for ANTH and its many derivatives, and give useful insight of the less frequently engineered Trp pathway in S. cerevisiae
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