IGEM:MIT/2006/System brainstorming/Smell-o-Rama

Description

simultaneously produce scented bacteria

Requirements

Testing

• transfer known coding region/enzyme for up to 4 scent producing proteins into e. coli bacteria first

• try to develop pathway control for intracellular creation of salicylic acid, benzoic acid, jasmonic acid, cinamic acid
• -test different acid concentrations in media for optimal concentration. Want minimum cell death and maximum desired scent

 -could use heat, light, etc. to monitor enzyme activity

• transfer scent coding region into yeast and other cellular organisms
• -experiment with bread, beer brewing

Feasibility & Timeline

The beginning phases of the project include the insertion of the enzyme into E. coli, which has already been done. This first step will take at most 2-3 weeks, giving lots of room to expand into the other possiblities for the remainder of the project timeline.

Parallelizability

The team can work together at first to put the enzyme in E. coli. With that first step done, it will be easy to run multiple parallel subprojects to tackle different applications in yeast, plants, sawdust, etc.

Intermediate goals

The first goal is to acheive results in E. coli. Following that, S. cerevisiae is a good candidate for a solid extended appplication (which will run concurrently with other subprojects).

Safety & ethics

The extended applications in humans (mouthwash etc.) pose potential ethical issues; however, the scope of the project will not encompass testing in humans.

Summary (pros/cons)

There are various advantages to the project, including:

1. Appealing demo possibilities
2. Strong case for extended commercial applications, including everyday uses
3. Solid previous work that shows proof of concept
4. Room for additional controls (light, heat, sense)

However, the difficulty is that there has been a great deal of previous work. To distinguish the project, it would be necessary to fine tune controls, which may make the project ultimately as complex.

Bacterial scents

Precursor	Enzyme	Compound	Scent	References
benzoic acid & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT)	methyl benzoate	pleasant smell	[1]
trans-cinnamic acid & S-adenosyl-L-methionine (SAM)	?	methyl cinnamate	cinnamon?
jasmonic acid & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT)	methyl jasmonate	jasmine
salicylic acid (SA) & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT)	methyl salicylate	wintergreen	[2, 3, 4]

Expert advice

From Natalia Dudareva, Purdue University:

• thinks that you can smell wintergreen from E. coli cultures expressing SAMT with salicylic acid in the media

From Eran Pichersky, University of Michigan:

• E. coli cultures expressing SAMT with salicylic acid in the media will have a detectable wintergreen smell
• eliminate indole pathway (responsible for bad E. coli smell) to strengthen the scent.
• have shown production of several scent compounds in E. coli

SAMT

• C. breweri
  • DNA and protein sequence known
  • Expressed in E. coli
  • Methyl salicylate has been extracted from spent medium of E. coli cells when medium was supplemented with salicylic acid
  • Genbank AF133053
  • also can use benzoic acid as a substrate but with lower efficiency
  • crystal structure available
• A. majus (Snapdragon)
  • DNA and protein sequence known
  • Expressed in E. coli
  • Methyl salicylate has been extracted from spent medium of E. coli cells when medium was supplemented with salicylic acid
  • also can use benzoic acid as a substrate but with lower efficiency
  • Methyl benzoate has been extracted from spent medium of E. coli cells when medium was supplemented with benzoic acid
• S. floribunda
  • Genbank AJ308570
• A belladonna
  • Genbank AB049752

JMT

• A. thaliana AY008434

BAMT

• Snapdragon AF198492

References

1. Pott MB, Hippauf F, Saschenbrecker S, Chen F, Ross J, Kiefer I, Slusarenko A, Noel JP, Pichersky E, Effmert U, and Piechulla B. Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens. Plant Physiol. 2004 Aug;135(4):1946-55. DOI:10.1104/pp.104.041806 |
2. Ross JR, Nam KH, D'Auria JC, and Pichersky E. S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch Biochem Biophys. 1999 Jul 1;367(1):9-16. DOI:10.1006/abbi.1999.1255 |
3. 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 |
4. Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, and Noel JP. Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell. 2003 Aug;15(8):1704-16. DOI:10.1105/tpc.014548 |

Terpenes and terpenoids

• Terpenes are hydrocarbons: combinations of several isoprenes. (Sometimes encompasses terpenoids.)
• Terpernoids are modified terpenes with methyl groups added/removed or oxygens added
• From Wikipedia: "Terpenoids contribute to the scent of eucalyptus, the flavors of cinnamon, cloves and ginger and the color of yellow flowers. Well-known terpenoids include citral, menthol, camphor and the cannabinoids found in the Cannabis plant."

E. coli has the Δ³-isopentenyl-pyrophosphate pathway, and the enzymes to produce geranyl-PP. This pathway is less effective than the mevalonate pathway, but this has been cloned into an E. coli strain by Keastling's group. Many scented compounds can be made from isopentenyl-PP and geranyl-PP with one or two enzymes, including lemon, orange, pine, etc. See Ecocyc for the pathways (type IPP as a compound and look at the synthetic and reactant pathways that link to it).

Terpenes are also the precursor to rubber and many of the resins and gums.

Indole elimination

Indole is the precursor to and degradation product of tryptophan. We could knock out the relevant two enzymes and supply tryptophan exogenously. Also, we could supply tryptophan exogenously and see if that is sufficient to inhibit indole formation via feedback inhibition in a "normal" strain. [from TK]

Indole can act as an extracellular signal so indole can probably get in and out of the cell.

Relevant reactions

In Pathway Reactions as a Reactant:

tryptophan biosynthesis : indole + L-serine = L-tryptophan + H2O

In Pathway Reactions as a Product:

tryptophan biosynthesis : indole-3-glycerol-phosphate = indole + D-glyceraldehyde-3-phosphate

tryptophan degradation II (via pyruvate) : L-tryptophan + H2O = indole + pyruvate + ammonia

Relevant enzymes

trpB (biosynthesis) and tnaA (degradation)

References

5. FREUNDLICH M and LICHSTEIN HC. Inhibitory effect of glucose on tryptophanase. J Bacteriol. 1960 Nov;80(5):633-8. DOI:10.1128/jb.80.5.633-638.1960 |
6. Phillips RS and Dua RK. Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine. Arch Biochem Biophys. 1992 Aug 1;296(2):489-96. DOI:10.1016/0003-9861(92)90602-s |