Part:BBa_K1692002

codon optimized FDC with T7 promoter and Flag Tag

Overview

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 [1]. 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 [2].

Experiments and Results

After obtaining our synthesized gene, we needed to insert it into the standard pSB1C3 backbone so we could transform it and submit as a biobrick. To do this we digested our linear gene and standard iGEM RFP plasmid (BBa_J04450) with a combination of EcoRI and SpeI or PstI restriction enzymes. We then ligated with T4 ligase and transformed into NEB 5-alpha competent E. coli cells. Now that we had our gene in a plasmid with a promoter and RBS we transformed it into T7 expressing NEB E. coli. We grew up large cultures, which we initiated T7 polymerase gene expression by adding IPTG to our cultures. Because all of our synthesized genes had a FLAG tag at the end of their sequence, we were able to purify our proteins from the cell lysate. To do this we used the Anti-FLAG Tag protein purification method. We then used a BCA protein assay to determine the concentrations of our purified proteins. Finally we ran all three of our purified enzymes on SDS PAGE with a Mark 12 protein ladder to verify that our proteins were the correct molecular weight, which they were.

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.

Given the success of the spectrophotometric approach in our PAL assays, we attempted to proceed in a similar manner with FDC and UbiX. Unfortunately, there were several complicating factors. In the case of UbiX, the main problem lay in the impossibility of distinguishing UbiX’s reactant from its product. UbiX catalyzes the prenylation of flavin mononucleotide. Unfortunately, this chemical transformation does not result in a change in the overall absorbance of the reaction solution that we are able to detect. The reactant and the product are simply to chemically similar. We believe that this is one reason why no isozyme of UbiX has ever been kinetically characterized. In the case of FDC, we initially set out to use tCA’s unique absorbance spectrum to our advantage: whereas we measured an increase in tCA to track the activity of PAL (which produces tCA), we endeavored to measure a decrease in tCA to probe the activity of FDC (which consumes tCA). Unfortunately, FDC cannot function in the absence of its prenylated FMN cofactor, which is supplied by UbiX. This presents a challenge, since FMN has a strong absorbance peak in precisely the same region as tCA’s peak. Consequently, we could not apply the same spectrophotometric method to quantify FDC activity. As an alternative, we adapted a purely computational approach to obtain at least some of the results that we would have gained from the same genre of experimental analysis that we performed in our PAL assay. Although we could not perform a multiplexed time course experiment on FDC or UbiX, we carried out a sensitivity analysis to determine FDC’s role in the overall synthetic pathway which is detailed in the modeling section of our wiki.

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

Reference

[1] Mckenna, Rebekah, Luis Moya, Matthew Mcdaniel, and David R. Nielsen. "Comparing in Situ Removal Strategies for Improving Styrene Bioproduction." Bioprocess Biosyst Eng Bioprocess and Biosystems Engineering (2014): 165-74. Print.

[2] White, Mark D., Karl A. P. Payne, Karl Fisher, Stephen A. Marshall, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "UbiX Is a Flavin Prenyltransferase Required for Bacterial Ubiquinone Biosynthesis." Nature (2015): 502-06. Print.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 233
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]