Team:Rice University/STRATEGY

(Difference between revisions)

Revision as of 23:40, 29 October 2008

[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=169745 Tyrosine Ammonia Lyase] (aka TAL, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 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 [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=36575&Template=fungiYeast Rhodotorula glutinis] TAL because its ratio of TAL to PAL activity is high compared with 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.

[http://www.rcsb.org/pdb/explore.do?structureId=1Z1F Peanut STS] monomer bound to resveratrol.

[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_001084228.1 4-coumarate CoA ligase] :: [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=nuccore&term=&query_key=18&dopt=gb&dispmax=20&page=1&qty=1&WebEnv=1BAyWdNufvUWxpNM-oKD-9JoRgEPTXzU_kF02A2hfcePWB3nxyPvHO3gqlDJksRdZq9GmTDHNDmKEFuabzP4VKB%40263F5D1B8F754EA0_0062SID&WebEnvRq=1 Stilbene Synthase] Fusion Protein (aka 4CL:STS, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 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).

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010].

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 express the resveratrol pathway primarily under anaerobic conditions.

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-=== Circuit Design for Expression During Fermentation in Yeast===
+=== Circuit Design for Recombination in Yeast===
 [[Image:circuit.png|right|350px|thumb|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010].]]
-*To engineer our yeast in a less [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=PGK1 3-phosphoglycerate kinase 1] (aka PGK1) - Locus chosen for genomic integration of expression circuit.
+*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 express the resveratrol pathway primarily under anaerobic conditions.