ENZYMATIC PREPARATION OF [1,3-C-13]DIHYDROXYACETONE PHOSPHATE FROM [C-13]METHANOL AND HYDROXYPYRUVATE USING THE METHANOL-ASSIMILATING SYSTEM OF METHYLOTROPHIC YEASTS

Dihydroxyacetone synthase (EC 2.2.1.3), which is a key enzyme of the C -1-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO2. When [C-13]formaldehyde was used as a substr ate with dihydroxyacetone synthase from Candida boidinii 2201, C-13 wa s confirmed to be incorporated to the C-1 and C-3 positions of dihydro xyacetone, and the C-13 content of each carbon (atoms/100 atoms) was e stimated to be 50%. [C-13]Methanol was also useful for the enrichment of dihydroxyacetone with C-13: when alcohol oxidase from a methylotrop hic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was requir ed for the accumulation of C-13-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160 mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Dihydroxyacetone kina se (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacetone. The ph osphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyaceton e production. A fed-batch reaction afforded 185 mM [1: 3-C-13]dihydrox yacetone phosphate from [C-13]methanol; the molar yield of the ester r elative to methanol added was 92.5%.