Ferulic acid decarboxylase (FDC) catalyzes the conversion of trans-cinnamic acid to styrene. We codon-optimized S. cerevisiae’s FDC gene for expression in E. coli. This construct includes a T7 inducible promoter, a ribosome binding site, and a FLAG-tag peptide sequence for easy and efficient protein purification. We have sequenced our construct and verified that all these components are indeed present. We were able to successfully clone our construct into E. coli and induce expression with isopropyl β-D-1-thiogalactopyranoside (IPTG). A sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that our FLAG-tag extraction selectively purified the FDC enzyme.
Styrene synthesis pathway The enzymes of interest are phenylalanine ammonia lyase (PAL), ferulic acid decarboxylase (FDC), and a flavin prenyltransferase involved in ubiquinone biosynthesis called UbiX. PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid, while FDC catalyzes the conversion of trans-cinnamic acid to styrene. Recently, it has been discovered that a cofactor is required to activate FDC. This cofactor is a product of the reaction between dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN), which is catalyzed by the enzyme UbiX.
Background
This is a SDS PAGE gel with purified PAL, FDC and UbiX protein. We ran a Mark 12 protein ladder to verify that our proteins were the correct molecular weight.
Styrene AbsorbanceThe absorbance spectrum of pure styrene (1 mM) measured on a Spectramax Pro spectrophotometer
trans-Cinnamic Acid AbsorbanceThe absorbance spectrum of pure trans-Cinnamic Acid (1 mM) measured on a Spectramax Pro spectrophotometer
Flavin Mononucleotide AbsorbanceThe absorbance spectrum of pure Flavin Mononucleotide (1 mM) measured on a Spectramax Pro spectrophotometer
