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. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monomethylxanthines and paraxanthine. In this work, we developed a novel biocatalytic platform for the production of methylxanthines from economic feedstocks; bench scale production of three different methlxanthines, theobromine, 3 and 7-methylxanthines has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmA/D or ndmB/D genes combinations. These modifications enabled specific N1 and N3- demethylation of caffeine, theophylline and theobromine to theobromine & paraxanthine, 3-methylxanthine and 7-methylxanthine respectively. This common production platform consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure products.
Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmA/D or ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction to produce the higher value methylxanthines. Strain pDdA had the highest activity for the production of 3-methylxanthine from theophylline; strain pAD1dDD had the highest activity for the production of theobromine from caffeine, and strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. Each of these strains were used to find the optimum amount of cells required to achieve complete conversion of substrates to product(s) within two hours. It was found that 15 mg/mL resting cells concentration of pDdA strain was required to completely N-demethylate 1 mM theophylline to 3-methylxanthine (81% conversion) and 1-methylxanthine (13%). Also, 15 mg/mL resting cells concentration of pAD1dDD strain was required to completely convert 1 mM caffeine to theobromine (98.5% conversion) and paraxanthine (1.5%). The optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. Moreover, coffee post -brew waste was used as a source of caffeine, which was completely utilized by 25 mg/mL resting cells pAD1dDD strain to theobromine by a conversion of 97%.
The cell growth of each specific strain was studied using different growth media, including Luria-Bertani Broth, Terrific Broth, and Super Broth. In all cases, super broth was found to be the best medium to produce the highest amount of cell paste. The amount of cell paste produced from 100 mL Super broth medium after 14-16 hour of growth was found to be 0.9, 0.9, and 1.5 g for pDdA, pAD1dDD, and pBD2dDB strains respectively. Subsequently, each reaction was scaled up to produce 100-300 mg pure methylxanthines products, and therefore cell growth was also scaled up (1-4 L) to produce adequate amount of biocatalyst to carry out these larger scale reactions. 1.3 L reaction volume was used to produce 3-methylxanthine (81%conversion) from 1 mM theophylline catalyzed by 15 mg/mL pDdA strain. 2 L reaction volume was used to produce theobromine (98.5% conversion) from 1 mM caffeine catalyzed by 15 mg/mL pAd1dDD strain. 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. All reactions were carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). All methylxanthines products were separated by preparative chromatography with high recovery, and each product solution was collected in bottles. Products were purified by drying at 120-140 C for 4 hours and 100, 255, and 127 mg 3-methylxanthine, theobromine, and 7-methylxanthine were recovered. Also, 178 mg theobromine was produced form post brew coffee waste from 1.16 L reaction catalyzed by 25 mg/mL pAD1dDD strain.
Purity of the isolated methylxanthine products was comparable to authentic commercially standards with no contaminant peaks, as observed by HPLC, LC-MS and NMR.