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??
   • The scent comes from methyl benzoate, a volatile ester.
   • Regulation issues
   • Transport issues for substrate/product
5. Ported to E. coli
   • 1.1kb fragment cloned into NdeI/BamHI sites of pET-28a (Kan^R)
   • Contains an N-terminal His-tag
   • They cloned the BAMT gene from two different Met codons. The second Met is two codons downstream (position 4 of the protein) [1].
   • Apparently, they had to induce BAMT expression with IPTG (0.4mM final conc.). I am currently unclear whether this was necessary for expression in E. coli or something done just to get a large amount of protein for purification/characterization.
6. What is the regulation in planta
   • As mentioned above, there are two Met codons at the begnning of the BAMT cDNA. The BAMT resulting from amplification from the second Met shows a 2.4 times higher specific activity than from the first Met in protein purified from cell lysate; on the other hand, BAMT purified from E. coli shows no difference in specific activity.
7. References & Links
   • Genbank accession number(s): AF198492

1. Murfitt LM, Kolosova N, Mann CJ, and Dudareva N. Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus. Arch Biochem Biophys. 2000 Oct 1;382(1):145-51. DOI:10.1006/abbi.2000.2008 |
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: from Antirrhinum majus, common snapdragon flower, SAMT (salicylic acid methyltransferase) expression
2. Sequence & Structure: AF515284, 1333 bp, mRNA

link: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=23957317

1. • Codon optimized?
   • Restriction sites
   • Post-translational modification
   • Material availability: YES - is in a pET 28 vector and is kindly already being shipped by Natalia Dutareva
     • cDNA from arabadopsis: no
     • Request from authors: taken care of, no hassles expected
       • Form of DNA? pET 28 plasmid
     • need to synthesize? no
2. Reaction catalyzed
   • Substrate: salicylic acid
     • Synthesized in our chassis or supplied exogenously? supplied exogeneously (at least for now)
   • Product
     • Coolness (Austin): very cool and pain free
   • Reaction rates - K_m,k_cat: E. coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. Relative activity with substrate salicylic acid is 100%, with substrate benzoic acid is 0%, other acids 0%. Numerically, 2.1 pkat/mg protein was detected with salicylic acid as the substrate
3. Does it smell?? floral scent enzyme in snapdragons
   • Regulation issues
   • Transport issues for substrate/product
4. Ported to E. coli or yeast? Yes, extensively studies with E.coli. -- PMID: 12361714

note: Is already on a pET plasmid. This means that the gene is transcribed by T7 polymerase rather than E. coli polymerase. T7 polymerase is much faster than E. coli so we can expect to get very high expression levels.

1. What is the regulation in planta: Not applicable, works in E.coli
2. References & Links
   • Corresponding authors: Natalia Dudareva
   • Genbank accession numbers: AF515284
   • Pubmed: PMID: 12361714
   • Websites: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12361714&dopt=Abstract

1. • Any companies or patents? just shipping charges

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
   • Reactions done in vitro with purified product- GST is tag?
   • Probable that jasmonic acid and methyl jasmonate (volatile) can be transported (see function)
5. Ported to E. coli (see above for method)
6. Regulated in planta: External stimuli such as wounding or methyl jasmonate can cause gene expression
7. References & Links
   • 3. Seo HS, Song JT, Cheong JJ, Lee YH, Lee YW, Hwang I, Lee JS, and Choi YD. Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4788-93. DOI:10.1073/pnas.081557298 |

1. • 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, Hao for SAM:CCMT
       • 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: PAL=X51513
   • Pubmed:

4. 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 |
5. Rasmussen OF and Oerum H. Analysis of the gene for phenylalanine ammonia-lyase from Rhodosporidium toruloides. DNA Seq. 1991;1(3):207-11. DOI:10.3109/10425179109020772 |
6. Faulkner JD, Anson JG, Tuite MF, and Minton NP. High-level expression of the phenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloides in Saccharomyces cerevisiae and Escherichia coli using a bifunctional expression system. Gene. 1994 May 27;143(1):13-20. DOI:10.1016/0378-1119(94)90598-3 |

1. • Websites:

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

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

http://en.wikipedia.org/wiki/Cinnamic_acid

1. • Any companies or patents?: None