Engineering Saccharomyces cerevisiae for production of non-natural and glycosylated flavonoids
Flavonoids are plant phenolic compounds that have many interesting medicinal properties. Therefore, there is interest in the synthesis of non-natural and glycosylated flavonoids as they may possess new or enhanced biological activities. In the first study, metabolically engineered Saccharomyces cerevisiae expressing 4-coumaroyl:CoA-ligase and chalcone synthase was explored as a platform for producing non-natural flavanones and dihydrochalcones. By precursor addition of cinnamic acid analogues to the engineered yeast, numerous non-natural flavanones and dihydrochalcones were formed. In a second study, S. cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation flower) was investigated as a whole-cell biocatalyst. GTs are promising biocatalysts for the synthesis of small molecule glycosides. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin 7-O-glucoside was formed and a higher specific GT activity was found using a galactose inducible expression system compared to a constitutive expression system. The constitutive expression system was significantly more productive than the galactose inducible system under in vivo conditions. An unknown naringenin glycoside formed using the Dianthus GT was identified as naringenin 4'-O-glucoside. In a third study, the yeast strain using the constitutive expression system was further characterized using naringenin as a substrate for production of naringenin glycosides. The effect of initial concentration of naringenin and different medium formulations on glycoside yield was examined. A method for controlling overall selectivity of the glycosylation chemistry by the changing the composition of the medium formulation was discovered. There was significant improvement in glycoside yield when orotic acid was added to the medium formulation compared to the control. It was also found that a low-cost nutrient source (yeast extract) could replace yeast nitrogen base in the medium formulation while maintaining high glycoside yield. The highest naringenin glycoside yield achieved using 10 mM orotic acid in the culture medium was 155 mg/L, a 71% conversion of substrate to product. In addition, the yeast strain expressing the Dianthus GT using the constitutive expression system was used to glycosylate other substrates. When phloretin was added as a substrate to the yeast cells, the natural product phlorizin was formed, a compound that has been proposed as a treatment for diabetes and obesity. Also, preliminary data shows the GT accepts several cinnamic acid analogues. This work demonstrates that S. cerevisiae is a promising host for the synthesis of small molecule glycosides using the whole-cell biocatalysis approach.
Morgan, Purdue University.
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