IGEM:MIT/2006/Notebook/2006-6-6

A very brief description of the mechanism for making cinnamon smells:

http://docs.lib.purdue.edu/dissertations/AAI9939353/

• Brainstorming page for scents

We'll dump all this information on today's page for now but you guys should think about what page structure you want to use to store all your information!

BAMT: SAM-Benzoic Acid Carboxyl Methyltransferase

1. Species
   • Antirrhinum majus (snapdragon); Just to note, this is the species that Dudareva is apparently sending us.
2. Sequence & Structure
   • Codon optimized?
   • Restriction sites
   • Post-translational modification
   • Material availability
     • cDNA from arabadopsis
     • Request from authors
       • Form of DNA?
     • need to synthesize?
3. Reaction catalyzed
   • Substrate
     • Synthesized in our chassis or supplied exogenously?
   • Product
     • Coolness (Austin)
   • Reaction rates - K_m,k_cat
4. Does it smell??
   • Regulation issues
   • Transport issues for substrate/product
5. Ported to E. coli or yeast?
   • 1.1kb fragment cloned into NdeI/BamHI sites of pET-28a (Kan^R)
   • Contains an N-terminal His-tag
6. What is the regulation in planta
7. References & Links
   • Corresponding authors
   • Genbank accession numbers
   • Pubmed
   • Websites
   • Any companies or patents?

1. Negre F, Kolosova N, Knoll J, Kish CM, and Dudareva N. Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers. Arch Biochem Biophys. 2002 Oct 15;406(2):261-70. DOI:10.1016/s0003-9861(02)00458-7 |
2. Dudareva N, Murfitt LM, Mann CJ, Gorenstein N, Kolosova N, Kish CM, Bonham C, and Wood K. Developmental regulation of methyl benzoate biosynthesis and emission in snapdragon flowers. Plant Cell. 2000 Jun;12(6):949-61. DOI:10.1105/tpc.12.6.949 |

Kate's enzyme

1. Species
2. Sequence & Structure
   • Codon optimized?
   • Restriction sites
   • Post-translational modification
   • Material availability
     • cDNA from arabadopsis
     • Request from authors
       • Form of DNA?
     • need to synthesize?
3. Reaction catalyzed
   • Substrate
     • Synthesized in our chassis or supplied exogenously?
   • Product
     • Coolness (Austin)
   • Reaction rates - K_m,k_cat
4. Does it smell??
   • Regulation issues
   • Transport issues for substrate/product
5. Ported to E. coli or yeast?
6. What is the regulation in planta
7. References & Links
   • Corresponding authors
   • Genbank accession numbers
   • Pubmed
   • Websites
   • Any companies or patents?

Jasmonic acid carboxyl methyltransferase

1. Species: Arabidopsis thaliana (thale cress)
2. Sequence & Structure
   • Use cDNA from A. thaliana (multistep- screened with Brassica NTR1)
   • JMT cDNA inserted into the pGEX-2T vector (Amersham Pharmacia-GE) at the EcoRI site and fused with glutathione-S-transferase gene (GST).
   • The recombinant was transformed into E. coli BL21. Fusion protein GST-JMT was expressed
     • Purified to get JMT- issue?
3. Reaction catalyzed
   • Substrate: Jasmonic acid and SAM added exogenously
   • Product: Methyl jasmonate
     • Given off by plants as defense, also in jasmin oil
   • Reaction rates - K_m: 38.5 µM; K_cat:25 S⁻¹
4. Methyl jasmonate has jasmine smell
   • Introduced into E. coli with fused GST gene, resulting in fused product
   • Reactions done in vitro with purified product
5. Ported to E. coli
6. Regulated in planta: External stimuli such as wounding or methyl jasmonate can cause gene expression
7. References & Links
   • Corresponding authors: Hak Soo Seo, Jong Tae Song, Jong-Joo Cheong, Yong-Hwan Lee, Yin-Won Lee, Ingyu Hwang, Jong Seob Lee, and Yang Do Choi
   • PubMed Link: http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=31912s
   • Genbank accession numbers: BD441046, BD441047
   • Patents for synthesizing inferior jasmine substitutes are commercial

PAL & SAM:CCMT

1. Species: PAL (Phenylalanine Ammonia-lyase) is found in several organisms, but for our interests, it seems the best option is the gene from Rhodosporidium toruloides. SAM:CCMT (S-adenosyl-L-methionine:cinnamate carboxyl methyltransferase) can only be found in Ocimum basilicum L., Lamieaceae (a basil plant) as far as I can tell.
2. Sequence & Structure
   • Codon optimized?: The good news is that PAL from Rhodosporidium toruloides has been successfully transported and expressed in E. coli and S. cerevisiae via plasmids by Faulkner, et al in 1994. The bad news is that SAM:CCMT seems to be only present in the cDNA library created by Dr. Hao in 1998 and in the raw Ocimum basilicum L., Lamieaceae form.
   • Restriction sites
   • Post-translational modification
   • Material availability
     • cDNA from arabadopsis: No
     • Request from authors: Yes Faulkner for PAL
       • Form of DNA? PAL- E. coli & S. cerevisiae vector form; SAM:CCMT- Ocimum bailicum cDNA library
     • need to synthesize? An option for SAM:CCMT
3. Reaction catalyzed: (1) Phenylalanine-->Trans-cinnamic Acid + NH4+ with PAL as a catalyst (2) Trans-cinnamic Acid-->Methylcinnamate with SAM:CCMT as a catalyst
   • Substrate: Phenylalanine *Advantage-Phenylalanine already needed for other cell processes in E. coli. Thus, no extraneous compound would need to be added to the media.
     • Synthesized in our chassis or supplied exogenously?: Phenylalanine must be supplied in the media.
   • Product: Methylcinnamate
     • Coolness (Austin): Very cool.
   • Reaction rates - K_m,k_cat
4. Does it smell?: Smells like cinnamon
   • Regulation issues
   • Transport issues for substrate/product: None.
5. Ported to E. coli or yeast?: Ported to both for PAL; not ported to either for SAM:CCMT
6. What is the regulation in planta
7. References & Links
   • Corresponding authors
   • Genbank accession numbers: X51513
   • Pubmed
   • Websites:

http://www.ncbi.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=X51513&doptcmdl=GenBank

3. Orum H and Rasmussen OF. Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides. Appl Microbiol Biotechnol. 1992 Mar;36(6):745-7. DOI:10.1007/BF00172186 |

1. • Any companies or patents?: None