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

Abstract

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.

Keywords: 6-Methylsalicylic acid decarboxylase; 6-Methylsalicylic acid synthase; 6-methylsalicylic acid decarboxylase, PatG; 6-methylsalicylic acid synthase, MSAS; 6-methylsalicylic acid, 6-MSA; Acyl carrier protein, ACP; Acyltransferase, AT; Codon-optimization; Polyketide synthase; Saccharomyces cerevisiae; ketoreductase, KR; ketosynthase, KS; m-Cresol; optical density, OD; phosphopantetheinyl transferase, PPT; polyketide synthase, PKS; thioester hydrolase, TH.

Graphical abstract

Fig. 1

Metabolic pathway for m-cresol production in S. cerevisiae via 6-methylsalicylic acid (6-MSA) synthesis. The 6-methylsalicylic acid synthase (MSAS) consists of multiple domains: the ketoacylsynthase (KS), acyltransferase (AT), thioester hydrolase (TH), ketoreductase (KR), and acyl carrier protein (ACP). MSAS must be activated by phosphopantetheinylation, and catalyzes the synthesis of 6-MSA from one acetyl-CoA and three malonyl-CoA under consumption of one NADPH. 6-MSA decarboxylase can further convert 6-MSA to m-cresol, valuable for the flavor and pharmaceutical industry. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

6-MSA formation with strain CEN.PK2–1C carrying the empty vectors pJHV7 and pRS62H as control (green circles), expressing ^PpvarMSAS and native npgA from multi-copy plasmids pJHV17 and pJHV20 (red squares) and expressing ^PpvarMSAS and codon-optimized ^optnpgA from multi-copy plasmids pJHV17 and pJHV2 (blue triangles). Cultures were inoculated at low OD (0.1) and cultivated for 144 h at 30 °C in 25 mL YPD supplemented with G418 and hygromycin. 6-MSA concentrations were determined in the supernatants. Error bars represent the standard deviation of biological duplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

6-MSA production by different MSASs in high-OD fermentations. Yeast strain CEN.PK2–1C carrying the empty vectors pJHV7 and pRS62H as control (purple), and strains expressing ^optnpgA (pJHV2) and the MSAS variants ^PpvarMSAS (pJHV17; light blue), ^AniMSAS (pJHV5; black), ^PpMSAS (pJHV11; green) or ^PpoptMSAS (pJHV36; orange) from multi-copy plasmids were inoculated at an OD of 9, and cultivated for 144 h at 30 °C in 25 mL YPD supplemented with G418 and hygromycin. 6-MSA concentrations were determined in the supernatants. Error bars represent the standard deviation of biological duplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Toxic effects of m-cresol on growth of CEN.PK2–1C in YPD supplemented with different m-cresol concentrations. Cell densities (starting OD = 0.1) were followed over 144 h with the Cell Growth Quantifier (Aquila Biolabs GmbH) and are depicted as arbitrary units (a.u.). Growth curves represent average of two biological replicates including standard deviations (light grey bars). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

6-MSA uptake and conversion. A) 6-MSA consumption and B) m-cresol production of CEN.PK2–1C expressing 6-MSA decarboxylase ^optpatG from multi-copy plasmid pJHV13 or carrying empty vector pJHV7 as reference. Strains were cultivated for 72 h in YPD plus G418 with and without supplementation of 1 mM 6-MSA with an initial OD of 0.2.6-MSA and m-cresol concentrations were determined in the supernatants. Error bars represent standard deviation of biological duplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Production of the intermediate 6-MSA (orange), final product m-cresol (blue) and growth (black) of CEN.PK2–1C expressing ^PpoptMSAS,^optnpgA and ^optpatG from multi-copy plasmid pJHV53. Fermentations (starting OD = 5) were performed in biological duplicates at 30 °C in YPD supplemented with G418.6-MSA and m-cresol concentrations were determined in the supernatants. Error bars represent standard deviation of biological duplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Increase in m-cresol production by genomic integration of the pathway genes. Growth (A) and production of 6-MSA (B) and m-cresol (C) of CEN.PK2–1C expressing ^PpoptMSAS,^optnpgA and ^optpatG from multi-copy plasmid pJHV53 (blue) or from genome (strain JHY162; red). As control, the empty vector pRS42K was transformed into CEN.PK2–1C (black). High-OD fermentations (starting OD = 6) were performed in biological duplicates at 30 °C in YPD supplemented with G418 for plasmid maintenance (error bars represent standard deviations). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Determination of the limiting factors for 6-MSA and m-cresol production. A) 6-MSA titers produced by strain JHY163 (ura3::^PpoptMSAS-^optnpgA) expressing additionally ^PpoptMSAS and ^optnpgA from multi-copy plasmid pJHV49 (pl. M; black) or as control the empty plasmid pRS42K (pl. Empty; light blue). B) m-cresol titers produced by strain JHY162 (ura3::^PpoptMSAS-^optnpgA-^optpatG) carrying additionally as a control the empty plasmid pRS42K (pl. Empty; red), or expressing ^PpoptMSAS,^optnpgA,^optpatG from pJHV53 (pl. M/patG; orange) or ^optpatG from pJHV13 (pl. patG; blue) and strain JHY163 (ura3::^PpoptMSAS-^optnpgA) (g. M) expressing additionally ^PpoptMSAS,^optnpgA,^optpatG from pJHV53 (pl. M/patG; purple) or ^optpatG from pJHV13 (pl. patG; grey). High-OD fermentations (starting OD = 5) were performed in biological duplicates at 30 °C in YPD supplemented with G418 (error bars represent standard deviations). g. M. indicates genomic expression of MSAS/npgA, g. M/patG indicates genomic expression of MSAS/npgA and patG, + pl. indicates additional overexpression from multi-copy plasmids. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)