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
     • Request from authors: Already requested!!!
       • Form of DNA: pET-28a vector
3. Reaction catalyzed
   • Substrate: Benzoic Acid (BA)
     • Synthesized in our chassis or supplied exogenously: supplied exogenously [1]
   • Product
     • Coolness (Austin)
   • Reaction rates - As described below, 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 (from both Met codons). The K_m and k_cat are the same from both codons in E. coli, also.
4. Does it smell??
   • The scent comes from methyl benzoate, a volatile ester.
   • Regulation issues
   • Transport issues for substrate/product
     • Apparently, they had to induce BAMT expression with IPTG (0.4mM final conc.) in E. coli. 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. **Note: it looks as if IPTG is necessary.
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) [2].

1. What is the regulation in planta
   • Enzyme is active as a dimer.
2. References & Links
   • Genbank accession number(s): AF198492

2. 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 |

Kate's enzyme

1. Species:
   • from Antirrhinum majus or A. majus. This is the common snapdragon flower
2. Sequence & Structure:
   • AF515284, 1333 bp, mRNA
   • link: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=23957317
   • Codon optimized?
     • yes, already been ported
   • Restriction sites
   • Post-translational modification
   • Material availability:
     • YES - it is in a pET 28 vector and is kindly being shipped by Natalia Dutareva
     • cDNA from arabadopsis: no
     • Request from authors: yes, Reshma has secured the vector already
       • Form of DNA? pET 28 plasmid from cDNA
     • need to synthesize? no
3. Reaction catalyzed
   • Substrate:
     • salicylic acid (100%) or benzoic acid (45% relative activity)
     • Synthesized in our chassis or supplied exogenously?
       • supplied exogeneously, genes activated by induction (At least for now)
   • Product
     • Coolness (Austin): very cool, painless
   • 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. (This is pretty small = good). It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM (larger). Relative activity with substrate salicylic acid is 100%, with substrate benzoic acid is 45%, and with all other acids is 0%. Numerically, 2.1 pkat/mg protein was detected with salicylic acid as the substrate.
4. Does it smell??
   • It is not involved in floral scent production in snapdragons because snapdragon flowers do not emit methyl salicylate. Snapdragons seem to use their SAMT gene to create methyl salicylate for defense purposes. Snapdragon flowers do emit their methyl benzoate, which is their most abundant scent compound. When the SAMT gene was put into E.coli, some wintergreen smell was detected by Dutareva, and the gene products ARE DEFINATELY PRESENT, so i don't think we should worry. Specifically, the culture medium of the E. coli cells expressing SAMT contained methyl salicylate (2.1 μg/ml) when the growing medium was supplemented with 5 μg/ml salicylic acid, and methyl benzoate (0.86 μg/ml) when the growing medium was supplemented with 5 μg/ml benzoic acid. Note that E. coli cells that contained a pET-28a plasmid without the SAMT coding region did not have any detectable enzyme activity and did not produce methyl salicylate or methyl benzoate.
   • Regulation issues: no
   • Transport issues for substrate/product: no
5. Ported to E. coli or yeast? Yes, extensively studies with E.coli. -- PMID: 12361714

Also, the SAMT gene Is already on a pET plasmid with ease of access to our team. This also means that the gene is transcribed by T7 polymerase rather than E. coli polymerase, and 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: see description for 'does it smell'
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? no

JMT: 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 or his former advisor Simon for SAM:CCMT Progress: contacted both Simon and Hao about access to Hao's dissertation, contacted Simon for access to cDNA library containing 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

BSMT: Benzoic acid/salicylic acid carboxyl methyltransferases

1. Species: Petunia x hybrida (petunia)
2. Sequence & Structure
   • Codon optimized?
   • Restriction sites?
   • Post-translational modification?
   • Material availability: cDNA from Petunia x hybrida
3. Reaction catalyzed
   • Substrate
     • Synthesized in our chassis or supplied exogenously?
   • Product
     • Coolness (Austin)
   • Reaction rates - K_m,k_cat
4. Can produce both methyl benzoate and methyl salicylate
   • 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) [2].
   • 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. The K_m is the same from both codons, also.
7. References & Links
   • GenBank: 28629494, 28629496