Engineering the Phenylpropanoid Pathway in Rhodosporidium toruloides for Naringenin Production from Tyrosine by Leveraging on its Native PAL Gene

Abstract

Flavonoids are valued for their beneficial properties. This was a proof-of-concept study to show that the PAL gene in Rhodosporidium toruloides (R. toruloides) could be harnessed and only two genes 4CL and CHS needed to be inserted to create the naringenin producing strain, CBS-N. The growth of CBS-N reached a higher OD600 86.1 compared to 31.8 by wildtype. High-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS) confirmed CBS-N successfully produced 0.038 mg/L naringenin and 16.9 mg/L p-coumaric acid via its PAL/TAL activity when supplemented with tyrosine, although the conversion efficiency of 4CL and CHS genes was the limiting factor. Metabolic profiling by gas chromatography–mass spectrometry (GC–MS) showed that, in the CBS-N strain, metabolic flux was toward glycolysis and the TCA cycle to produce malonyl CoA, the precursor for naringenin synthesis, and away from fatty acids synthesis pathway. Our study showed that microorganisms containing a native PAL gene, such as R. toruloides, could be harnessed for flavonoids production via precision fermentation.