Engineering Bifunctional Galactokinase/Uridyltransferase Chimera for Enhanced UDP-d-Xylose Production

Abstract

The biotechnological production of uridine diphosphate-d-xylose (UDP-d-xylose), the glycosyl donor in enzymatic for d-xylose, is an important precursor for advancing glycoengineering research on biopharmaceuticals such as heparin and glycosaminoglycans. Leveraging a recently discovered UDP-xylose salvage pathway, we have engineered a series of bifunctional chimeric biocatalysts derived from Solitalea canadensis galactokinase/uridyltransferase, facilitating the conversion of d-xylose to UDP-d-xylose. This study elucidates the novel assembly of eight fusion protein constructs, differing in domain orientations and linker peptide lengths, to investigate their functional expression in Escherichia coli, resulting in the synthesis of the first bifunctional enzyme that orchestrates a direct transformation from d-xylose to UDP-d-xylose. Fusion constructs with a NH₂-GSGGGSGHM-COOH peptide linker demonstrated the highest expression and catalytic tenacity. For the highest catalytic conversion from d-xylose to UDP-d-xylose, we established an optimum pH of 7.0 and a temperature optimum of 30 °C, with an optimal fusion enzyme concentration of 3.3 mg/mL for large-scale UDP-d-xylose production. Insights into ATP and ADP inhibition further helped to optimize the reaction conditions. Testing various ratios of unfused galactokinase and uridyltransferase biocatalysts for UDP-xylose synthesis from d-xylose revealed that a 1:1 ratio was optimal. The K _cat/K _m value for the NH₂-GSGGGSGHM-COOH peptide linker showed a 10% improvement compared with the unfused counterparts. The strategic design of these fusion enzymes efficiently routes for the convenient and efficient biocatalytic synthesis of xylosides in biotechnological and pharmaceutical applications.

Figure 1

(a) Salvage pathway for UDP-xylose biosynthesis from xylose by ScGalK/ScGPUT. (b) Examples of bifunctional enzyme fusions in nature. (c) Model structure of fused ScGalK and ScGPUT.

Figure 2

Overview of constructing the recombinant ScGalK/ScGPUT fusion proteins using different linkers.

Figure 3

Expression, purification, and activity assessment of the various constructs. (a) SDS-PAGE analysis of purified recombinant fusion enzymes highlighted variations in linker peptides. (b) Enzymatic conversion of d-xylose to UDP-xylose by ScGalK/ScGPUT fusion enzymes. (c) TLC analysis results of the ScGalK/ScGPUT enzymes reacting with d-xylose. The conversion rate of linker 8 mentioned in the text (89%) is based on standardized conditions to match the enzyme concentration across different linkers. (d) LC-ESI-MS profile confirming the presence of UDP-xylose in reaction mixtures containing ScGalK/ScGPUT fusion enzymes. (e) Mass spectrum at a retention time of 11.6 min, indicative of UDP-xylose production. (f) TLC separation of products from the reaction of ScGalK/ScGPUT enzymes with d-xylose-1-phosphate.

Figure 4

Biocatalytic evaluation of the bifunctional fusion enzymes. (a) 40 h time-course LC-ESI-MS reaction profiling for d-xylose conversion. (b) Optimal pH for the enzyme’s activity, derived from TLC analysis. (c) Determination of the enzyme’s optimum operating temperature. (d) TLC analysis displaying inhibition by ATP and ADP. (e) TLC results were used for identifying the optimal enzyme concentration for UDP-d-xylose enzymatic synthesis. (f) Determination of the enzymes’ optimum operating ratios of ScGalK/ScGPUT.