Quandt et al. said that derivatives of their so-called “decaffeination operon” could be used to: (1) Clean wastewater contaminated with caffeine, (2) Recapture valuable byproducts of coffee processing, and (3) Bio-produce specific methylated xanthines (methylxanthines) from caffeine to produce new pharmaceuticals.
Introducing a research letter published in the American Chemical Society journal, ACS Synthetic Biology (stemming from a 2012 iGem synthetic biology competition entry) the student team from the universities of Texas and Iowa noted that sodas, energy drinks, tea and coffee contain caffeine and other methylxanthines, which are also used as cardiac, respiratory stimulants in pharmaceuticals.
Yet due to widespread use, the scientists said the compounds were now common pollutants in wastewater and surface waters around population centers, where caffeine levels often served as measures of human impact.
Although high caffeine levels are toxic to some bacteria, soil bacterium Pseudomonas putida CBB5 (P.putida) can live on caffeine, as its sole nitrogen source, Quandt et al. said.
Genes in that bacterium contained proteins that converted caffeine into xanthine and formaldehyde, the scientists added, which E.Coli bacteria could then use, as nitrogen and carbon sources respectively, to grow.
Coca-Cola, Monster, Red Bull, Starbucks Espresso…
The scientists built a ‘genetic selection’ using P.putida genes, refactored to port degradation functionality into an E.coli strain with its guaB gene knocked out, and thus unable to synthesize guanine, the DNA base necessary for its cell growth, otherwise than in the presence of caffeine.
Such E.coli cells with this synthetic operon (an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a singular regulatory signal or promotor) degraded caffeine to produce guanine precursor xanthine, then guanine itself, causing the engineered bacteria to grow in the presence of caffeine.
When Quandt et al. added the bacteria to cultures supplemented with caffeinated sodas, espresso or energy drinks – Coca-Cola (pictured) Diet Coke, Monster, Red Bull, Starbucks Espresso – they said that E.coli thrived, but not in cultures using decaffeinated drinks (Caffeine-Free Coke).
Final bacterial cell densities provided the team with the data they needed to measure caffeine density in μM or micro meters. For instance, 1740μM for Monster Energy, 508μM for Coca-Cola and 0μM for Caffeine-Free Coca-Cola.
“The combination of the guaB knockout and the decaffeination operon results in E.coli that can be considered ‘addicted’ to caffeine. We were able to show that these E.coli could grow in minimal media supplemented with various sodas and energy drinks containing caffeine, but not a caffeine-free soda,” Quandt et al. wrote.
Transform waste into valuable resource
Given a strong correlation between the saturating cell optical density and the amount of added caffeine, the scientists said they, “reasoned that this strain could be used as a quantitative biosensor for measuring the total concentration of guanine, xanthine and methylxanthines in an unknown sample”.
However, Quandt et al. said that typical caffeine concentrations in coffee berry processing byproducts could be as high as ∼15 mM (micro molars) which would inhibit growth of the E.coli strain they used.
But they added that it was “potentially possible” to evolve an E.coli host strain with a higher methylxanthine tolerance for some of these applications.
Valuable coffee bean processing byproducts include carbohydrates, proteins and other nutrients, but may be unsuitable as agricultural or biofuel feedstocks due to toxic caffeine levels.
“The ability to decaffeinate this waste could alleviate this problem and transform what is currently a cost of production into a valuable resource,” Quandt et al. wrote.
Title: ‘Decaffeination and Measurement of Caffeine Content by Addicted Escherichia Coli with a Refactored N-Demethylation Operon from Pseudomonas Putida CBB5.’
Authors: Quandt, E.M, Hammerling, M.J, Summers, R.M, Otoupal, P.B, Slater, B., Alnahhas, R.N, Dasgupta, A., Bachman, J.L, Subramanian, M.V, Barrick, J.E.
Sources: ACS Synthetic Biology, Published Online, March 8 2013 dx.doi.org/10.1021/sb4000146
Team UT Austin, IGem 2012 Competition Page, 'Caffeinated Cola' http://2012.igem.org/Team:Austin_Texas/Caffeinated_coli