Abstract
BackgroundMethylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monomethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonasputida, ndmA, ndmB, and ndmD, provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3-methylxanthine by a metabolically engineered strain of E. coli.ResultsHere we report the construction of E. coli strains with ndmA and ndmD, capable of producing 3-methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3-methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3-methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-methylxanthine were also produced, attributed to slight activity of NdmA at the N3-position of theophylline. The 1- and 3-methylxanthine products were separated by preparative chromatography with less than 5 % loss during purification and were identical to commercially available standards. Purity of the isolated 3-methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chromatography-mass spectrophotometry or nuclear magnetic resonance.ConclusionsWe were able to biologically produce and separate 100 mg of highly pure 3-methylxanthine from theophylline (1,3-dimethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD. This microbial conversion represents a first step to develop a new biological platform for the production of methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0395-1) contains supplementary material, which is available to authorized users.
Highlights
Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics
Given that 3MX is a metabolite of TP in humans [16], TP itself may be a prodrug; there is an interest in directly testing 3MX as well. 3MX and TP are used to examine conformational heterogeneity in RNA aptamers and riboswitches [17, 18]. 1-methylxanthine (1MX) is an essential human urinary metabolite of caffeine and TP [19,20,21,22] and exhibits similar activities to other naturally occurring methylxanthines
We previously showed that the ndmA and ndmD genes are expressed partly in soluble form in Escherichia coli [43], and that a strain expressing both genes can be used to convert caffeine to theobromine [44]
Summary
Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many methylxanthines is currently performed by chemical synthesis. Algharrawi et al Microb Cell Fact (2015) 14:203 xanthine derivatives have been synthesized chemically for use in the medical industry [5]. These compounds, natural and synthetic, have been shown to have various biomedical effects, with targets including adenosine receptors [6, 7], phosphodiesterases [8, 9], calcium release channels [10,11,12,13], and GABAA receptors [13, 14].
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