7-Methylxanthine is a rare compound that is not readily found in nature, except as an intermediate of caffeine biosynthesis in plants [29]. Caffeine (1,3,7-trimethylxanthine) derivatives like 7-methylxanthine are often noted for their ability to cross the blood-brain barrier and act as adenosine receptor antagonists [15], making them attractive as scaffolds for the synthesis of more complex compounds with more finely tuned medical applications [16]. For example, N-heterocyclic carbenes have been constructed from a variety of methylxanthines and have been reported to demonstrate selective toxicity towards certain cancer cell lines and not towards healthy cells [17–20]. Additionally, two specific 7-methylxanthine derivatives clearly demonstrate the diversity and tunability achievable through the use of methylxanthines as scaffolds. KF17837 ((E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) was designed as a potent adenosine receptor antagonist highly specific for the A2 adenosine receptor [21], which could have applications in areas such as Parkinson’s disease treatment [22]. In contrast, 1,3-dipropyl-7-methylxanthine was designed to sensitize lung carcinoma cells to radiation treatments by inducing apoptotic responses and modifying checkpoints within the cell cycle [23]. Investigations into the medical applications of 7-methylxanthine strongly suggest that the compound can be used to treat and slow the progression of myopia, or nearsightedness, and even prevent its formation. Studies supporting these findings have been conducted in rabbits [24], guinea pigs [25], rhesus monkeys [15], and human children [26]. Additional studies have concluded that 7-methylxanthine is safe for long-term oral administration [27, 28].
Purely chemical methods of 7-methylxanthine synthesis that have attempted to overcome the lack of natural availability are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, such as tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) [30–33]. Attempts at direct N-substitution of xanthine are further complicated by a lack of selectivity resulting from a high similarity in acidity of N3-H and N7-H, closely followed by N1-H. Substitution of the N-H groups therefore follows the expected pattern of N3 ≥ N7 > N1 as indicated by the acidity [34]. Traube purine synthesis, a method considered to be the classical technique for chemically synthesizing substituted purines, is a lengthy process constrained by harsh conditions and poor specificity, even with recent updates and modifications [34–36]. With so many obstacles to the chemical synthesis of 7-methylxanthine, a biosynthetic route offers an alternative method that is simple, safe, reliable, and cost-effective. The retail price of pure 7-methylxanthine can exceed 10,000 times the cost of caffeine (Fig. 1), thus providing an economic incentive to produce 7-methylxanthine from caffeine.
We have characterized a family of five N-demethylase enzymes, NdmABCDE, which are able to metabolize caffeine to xanthine in Pseudomonas putida CBB5 [1–4]. Initial enzyme characterization conducted in vitro revealed that NdmA is responsible for N1-demethylation of caffeine to theobromine, NdmB carries out the N3-demethylation of theobromine to 7-methylxanthine, and NdmCDE form a complex for N7-demethylation of 7-methylxanthine to xanthine [1]. Within the NdmCDE complex, NdmC was specifically identified as responsible for N7-demethylation. NdmE plays a role as a structural support and is non-catalytic [2]. NdmD is a reductase that is highly specific to the Ndm enzymes and is absolutely required for the biocatalysis of the N-demethylation reactions by transferring electrons to NdmA, NdmB, and NdmC.
In the past few years, several metabolically engineered Escherichia coli strains have been constructed harboring various combinations of the ndmABD genes for the purpose of selective methylxanthine production. For example, 3-methylxanthine has been produced from theophylline (1,3-dimethylxanthine) using E. coli strain pDdA [5], theobromine (3,7-dimethylxanthine) has been produced from caffeine using E. coli strain pAD1dDD [6], and 7-methylxanthine has been produced from theobromine using E. coli strain pBD2dDB [7]. We recently generated a mutant of NdmA, known as NdmA4, that is capable of carrying out N3-demethylation of caffeine to generate paraxanthine (1,7-dimethylxanthine) as the primary metabolite [8, 9] while also retaining N1-demethylation activity toward paraxanthine (Fig. 1) [10]. Genetic strain optimization resulted in the creation of E. coli strain MBM019 utilizing simultaneous expression of ndmA4 and ndmDP1, an N-terminally truncated version of the NdmD reductase (Fig. S1). Using strain MBM019, we have established optimized processes for the biocatalytic production and purification of paraxanthine and 7-methylxanthine from caffeine [9, 10]. However, these processes are limited by the low reaction rate of the NdmA4 mutant enzyme and result in low yields over longer time periods when compared to processes using wild-type enzymes [5–7].
Here, we demonstrate an optimized mixed-culture microbial platform for the production of 7-methylxanthine from caffeine that is more efficient than previously described methods and generates minimal quantities of side products. This platform uses a mixed culture of E. coli strains expressing either ndmA or ndmB in conjunction with ndmDP1, hereafter referred to as pADP1 cells and pBDP1 cells, respectively, thus harnessing the abilities of NdmA and NdmB to jointly convert caffeine to 7-methylxanthine using theobromine as an intermediate. The mixed-culture process constitutes a marketable improvement in conversion efficiency of caffeine to 7-methylxanthine from our previously described method using four rounds of reaction with cells expressing ndmA4 [9].