The smell (of E. coli) that surrounds you

Anyone that has worked with the molecular biology workhorse, Escherichia coli, is well aware that the bacteria can create a foul odor.  The bad smell made by E. coli is caused by the reaction of an enzyme called tryptophanase that converts the amino acid tryptophan into the noxious chemical indole in the reaction shown below.

L-tryptophan + H2O ---> indole + pyruvate + NH3

Indole is actually responsible for the bad smell of human feces and it can be produced by E. coli living in the intestinal tract.  


It's possible to genetically engineer E. coli to remove the gene (tnaA) that codes for tryptophanase and the bad smell.  This has been done as part of genome-wide functional studies like the Keio knockout collection and also IGEM teams have made use of a tryptophanase knockout to change the smell of E. coli.  This year I mentored a group of students in Hershey, Pennsylvania, as part of the Biobuilder Club, they were trying to make E. coli produce a vanilla smell.  The plan was to add genes that could produce vanillin to a tryptophanase negative strain.  

Vanillin is the compound responsible for the smell of vanilla.  It was first extracted from the seeds of the vanilla orchids and is still obtained from this source as a "natural vanilla".  More recently, chemical methods were developed to produce vanillin synthetically from petroleum byproducts.  The petroleum derived vanillin is sold as "artificial vanilla" and is usually less expensive than "natural vanilla".  A third means of production has been via microbes.  Microbes are capable of converting the naturally occurring plant phenolic compound ferulic acid to vanillin via a multi-step metabolic pathway (see below).  The microbes have no need for vanilla flavors -- the vanillin is just an intermediate in the conversion of ferulic acid to energy and carbon.

 This schematic was copied from Di Gioia, Diana, et al. "Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid."  Journal of biotechnology  156.4 (2011): 309-316.

This schematic was copied from Di Gioia, Diana, et al. "Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid." Journal of biotechnology 156.4 (2011): 309-316.

The genes for ferulic acid catabolism in Pseudomonas species are co-located in an operon in the genome.  To produce vanillin the activities of feruloyl-CoA synthetase and feruloyl-CoA hydratase/aldolase are needed and vanillin dehydrogenase should not be present.  If vanillin dehydrogenase is present then the vanillin will be converted to vanillic acid which doesn't have much smell.  

 Ferulic acid operon from  Pseudomonas fluorescens BF13 .  Only the ech and fcs genes are needed for vanillin production.

Ferulic acid operon from Pseudomonas fluorescens BF13.  Only the ech and fcs genes are needed for vanillin production.

To produce vanillin in E. coli two genes from Pseudomonas were codon optimized for expression in E. coli and purchased as gene fragments.  These genes were originally identified in a 2008 publication.  The fragments were assembled in a stepwise fashion using Golden Gate assembly and the pGLB3 vector to form a lactose inducible promoter fused to the ech and fcs genes.  When this was transformed into the E. coli unfortunately no vanilla smell was generated.  The plasmid was sequence verified and confirmed to be correct.  

More careful inspection of the fcs gene open reading frame suggests that the authors who originally identified the gene may have mispredicted the start codon (see alignment below).  It appears that in the DNA sequenced by the original research group, the N-terminal region is truncated as compared to essentially all other similar Pseudomonas fcs genes.  To fix this potential issue, additional DNA will be synthesized that adds the coding sequence for the probably N-terminus of the genes.  Hopefully this will enable the recombinant E. coli to generate vanillin, and more importantly the pleasant smell of vanilla, in large enough quantities to be easily noticeable.  Expect another update on this progress soon.