Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation.

Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

De novo production of aromatic m-cresol in Saccharomyces cerevisiae mediated by heterologous polyketide synthases combined with a 6-methylsalicylic acid decarboxylase.

As a flavor and platform chemical, m-cresol (3-methylphenol) is a valuable industrial compound that currently is mainly synthesized by chemical methods from fossil resources. In this study, we present the first biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid (6-MSA) was introduced into a CEN.PK yeast strain. For synthesis of 6-MSA, expression of different variants of 6-MSA synthases (MSASs) were compared. Overexpression of codon-optimized MSAS from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans resulted in up to 367 mg/L 6-MSA production. Additional genomic integration of the genes had a strongly promoting effect and 6-MSA titers reached more than 2 g/L. Simultaneous expression of 6-MSA decarboxylase patG from A. clavatus led to the complete conversion of 6-MSA and production of up to 589 mg/L m-cresol. As addition of 450-750 mg/L m-cresol to yeast cultures nearly completely inhibited growth our data suggest that the toxicity of m-cresol might be the limiting factor for higher production titers.

High Trap Formation and Low Metabolite Production by Disruption of the Polyketide Synthase Gene Involved in the Biosynthesis of Arthrosporols from Nematode-Trapping Fungus Arthrobotrys oligospora.

A group of morphology regulatory arthrosporol metabolites have been recently characterized from carnivorous fungus Arthrobotrys oligospora that can develop trapping networks to capture their prey. A combination of genetic manipulation and chemical analyses was applied to characterize the function of one polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora, which was putatively involved in the production of 6-methylsalicylic acid. High-performance liquid chromatography analysis showed that the disruption of the PKS gene not only led to the total loss of the arthrosporol A but also resulted in significant reduction in the production of secondary metabolites in the cultural broth of the mutant ΔAOL_s00215g283 strain. Interestingly, the mutant strain displayed significant increases in the trap formation and the nematicidal activity by 10 and 2 times, respectively, higher than the wild-type strain. These findings revealed a pathogenicity-related biosynthetic gene of this agriculturally important biological agent and have implications for establishment of efficient fungal biocontrol agents.

The gene PatG involved in the biosynthesis pathway of patulin, a food-borne mycotoxin, encodes a 6-methylsalicylic acid decarboxylase.

Patulin is a mycotoxin produced by fungal genera such as Aspergillus, Penicillium and Byssochlamys. It induces neurological, gastrointestinal and immunological effects, which is why patulin belongs to a short list of mycotoxins whose level in food is regulated in many countries around the world. Recently, a cluster gathering 15 genes involved in the biosynthesis of patulin has been identified in Aspergillus clavatus, but so far, only 4 genes encoding 6-methylsalicylic acid synthase, m-cresol hydroxylase, m-hydroxybenzyl alcohol hydroxylase and isoepoxydon dehydrogenase have been characterized. Previous studies have shown the involvement of a decarboxylase in the transformation of 6-methylsalicylic acid, the first stable patulin precursor, into m-cresol. In this study a putative decarboxylase gene, PatG, was identified in the genome sequence of A. clavatus. This gene is located near two P450 cytochrome genes PatH and PatI responsible respectively for the hydroxylation of m-cresol and m-hydroxybenzyl alcohol. This decarboxylase encoded by PatG (ACLA_093620) consists of 325 amino acids. The search for putative conserved domain revealed that the gene product belongs to the AminoCarboxyMuconate Semialdehyde Decarboxylase (ACMSD) related protein family. This family includes decarboxylases such as the γ-resorcylate decarboxylase or o-pyrocatechuate decarboxylase. The substrates of these enzymes display strong structural similarities with 6-methylsalicylic acid. PatG was strongly expressed during patulin production whereas it was very weakly expressed in non-patulin permissive conditions. The coding sequence was used to enable heterologous expression of functional enzymes in Saccharomyces cerevisiae. The presence of decarboxylase was confirmed by Western blot. The bioconversion assays showed that PATG catalyzed the decarboxylation of 6-methylsalicylic acid into m-cresol. These results confirm for the first time that 6-methylsalicylic acid is the substrate for PATG, the 6-methylsalicylic acid decarboxylase. With this study, the four genes involved in the four first steps of patulin biosynthesis pathway (acetate→gentisyl alcohol) are now identified.