(27bb) Enzymatic Optimization of the Biosynthetic Production of Paraxanthine By Site-Saturated Mutagenesis with Enhanced Formaldehyde-Sensitive Selection

Paraxanthine (1,7-dimethylxanthine) is a purine alkaloid derivative of caffeine (1,3,7-trimethylxanthine). It is a high-value biochemical with several applications in the pharmaceutical, cosmetic, and nutritional industries. Paraxanthine is a very rare compound and chemical synthesis requires harsh conditions that frequently result in low yield mixtures of non-specifically methylated compounds. We have recently demonstrated the ability to produce paraxanthine biosynthetically using a mutant of the wild-type N-demethylase genes isolated and characterized from the soil bacterium Pseudomonas putida CBB5, ndmABCDE, specifically ndmA4. While attempts at genetic and reaction condition optimization improved the overall conversion of caffeine to paraxanthine by ndmA4, the reaction rate and overall conversion efficiency remained lower than the rates and conversions observed when using the wild-type genes. Our analysis of our data collected thus far led us to the hypothesis that the enzyme itself remains the bottleneck of the process. It is our goal to create a new ndmA mutant capable of producing paraxanthine from caffeine at a rate and efficiency comparable to the wild-type genes.

In this project, ndmA4 will be mutated by targeted, site-saturated mutagenesis at nine amino acid residues in and around the active site. The generated mutant library will be transformed into an “indicator strain” of E. coli designed to fluoresce in accordance with demethylation activity, thereby facilitating identification of functional mutants. We have partially constructed this indicator strain by inserting an sfGFP gene under the control of an optimized, constitutive promoter into the genome of E. coli JM109(DE3), disrupting a formaldehyde-degrading gene, frmA. Our next step is the genomic integration of a formaldehyde-sensitive repressor, frmR, under the control of a strong, constitutive promoter. The FrmR repressor will bind to the optimized repressor controlling sfGFP, preventing expression of the sfGFP gene until formaldehyde is present. Formaldehyde is a byproduct of N-demethylation so this will effectively couple the expression of sfGFP to the demethylation reaction. The more efficient the demethylation of caffeine to paraxanthine, the more formaldehyde that is generated, and subsequently the more sfGFP that will be expressed. This method will facilitate an efficient reduction in size of the generated mutant library.

Following the elimination of inactive or poorly active mutant strains, the remaining mutants will be tested by HPLC analysis to confirm conversion of caffeine to paraxanthine, and the most efficient strain will be selected as the new paraxanthine-producing mutant, ndmA5.