Team:Rice University/STRATEGY

{| 
 * align="center" style="background:#C0C0C0"| 

OUR TEAM ::: SUMMARY ::: BACKGROUND ::: STRATEGY ::: CONSTRUCTS :::  RESULTS :::  ONGOING WORK

{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}

{|align="justify" style="background-color:#FFFFFF;15pt;text-align:justify" cellpadding="30" width="90%"

Pathway Design for Resveratrol Biosynthesis

 * Tyrosine Ammonia-Lyase (TAL, BBa_K122010) - TAL catalyzes the conversion of L-tyrosine to p-coumaric acid and ammonia. TAL also exhibits Phenylalanine Ammonia-Lyase (PAL) activity, converting L-phenylalanine to trans-cinnamic acid and ammonia.  Our work has focused on using Rhodotorula glutinis TAL because its ratio of TAL to PAL activity is high compared to other TAL homologs. In addition, previous studies have shown that this enzyme can be expressed as a functional protein in Saccharomyces cerevisiae and Escherichia coli.  While the p-coumaric acid produced by TAL will serve as a substrate for resveratrol biosynthesis, the trans-cinnamic acid is expected to add a "floral" and "honey-like" bouquet to the beer.

<BR><BR><BR>

.
 * 4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein (4CL:STS, BBa_K122005) - This enzyme fusion is comprised of Arabidopsis thaliana 4-coumarate-CoA ligase (4CL), which catalyzes the conversion of p-coumaric acid to 4-coumaroyl-CoA, and Vitis vinifera Stilbene Synthase, which catalyzes the condensation of resveratrol from 4-coumaroyl-CoA and three malonyl-CoA molecules. This 4CL:STS fusion protein was selected for our project because it has been shown to more efficiently produce resveratrol than coexpression of the proteins separately (possibly due to substrate channeling), see Zhang Y, et al. (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. J Am Chem Soc. 128(40):13030-1.

<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>

Circuit Design for Recombination in Yeast
<BR>
 * Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and highly express the resveratrol pathway under anaerobic conditions.

<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>

Selection of Brewing Strain


<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR>

OUR TEAM ::: SUMMARY ::: INTRODUCTION :::  STRATEGY :::   RESULTS :::  ONGOING WORK
 * }
 * }