Enzymatic preparation of [1, 3- 13C]dihydroxyacetone phosphate from [ 13C]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 CO ₂. When [ ¹³C]formaldehyde was used as a substrate with dihydroxyacetone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100?atoms) was estimated to be 50%. [ ¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumulation of ¹³C-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 kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacetone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185?mM [1, 3- ¹³C]dihydroxyacetone phosphate from [ ¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%.