Abstract
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Here we present an inexpensive production method for 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) by microbial fermentation of sugars. Heterologous expression in the yeast Saccharomyces cerevisiae of phosphopantetheinyltransferase-activated 6-methylsalicylic acid (6-MSA) synthase (MSAS) and 6-MSA decarboxylase converted acetyl-CoA as a priming unit via 6-MSA into 3-methylphenol (3-MP). We exploited the substrate promiscuity of MSAS to utilize propionyl-CoA and butyryl-CoA as alternative priming units and the substrate promiscuity of 6-MSA decarboxylase to produce 3-EP and 3-PP in yeast fermentations. Increasing the formation of propionyl-CoA by expression of a bacterial propionyl-CoA synthetase, feeding of propionate and blocking propionyl-CoA degradation led to the production of up to 12.5 mg/L 3-EP. Introduction of a heterologous ‘reverse ß-oxidation’ pathway provided enough butyryl-CoA for the production of 3-PP, reaching titers of up to 2.6 mg/L. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa.
Highlights
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease
A heterologous phosphopantetheinyltransferase (NpgA)-activated 6-methylsalicylic acid synthase (MSAS) utilizes acetyl-CoA as priming and malonyl-CoA as extender units to synthesize 6-methylsalicylic acid (6-MSA) that is further converted by 6-MSA decarboxylase (PatG) to 3-MP (Fig. 1)
High-OD fermentations in KPi buffered YPD medium supplemented with 10 mM butyrate did not result in any 3-PP formation in any of the strains (Supplementary Fig. S3C). These results indicate that either butyryl-CoA concentrations cannot be enhanced to levels at which it can compete with acetyl-CoA for priming MSAS or that MSAS and/or MSA decarboxylase are not able to convert butyryl-CoA and/or propylsalicylic acid www.nature.com/scientificreports (PSA), respectively, in vivo
Summary
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa. In order to make 3-alkylphenols accessible for the poor rural communities in sub-saharan Africa, microbial fermentation offers an alternative method for the inexpensive and simple production of these compounds on site. We have recently developed an S. cerevisiae strain with a de novo 3-MP (m-cresol) production pathway from sugars[14] In this recombinant yeast, a heterologous phosphopantetheinyltransferase (NpgA)-activated 6-methylsalicylic acid synthase (MSAS) utilizes acetyl-CoA as priming and malonyl-CoA as extender units to synthesize 6-methylsalicylic acid (6-MSA) that is further converted by 6-MSA decarboxylase (PatG) to 3-MP (Fig. 1). PatG was shown to decarboxylate 6-ESA to 3-EP in vitro[17], while no data are available for the decarboxylation of 6-PSA
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