Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions

• Published in: Applied microbiology and biotechnology Vol. 99; no. 8; pp. 3469 - 3477
• Main Authors: Wang, Tianwen David, Ma, Fei, Ma, Xingyuan, Wang, Ping
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.04.2015; Springer; Springer Nature B.V
• Subjects: Acids; Analysis; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Biocatalysts; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Biotransformation; China; Coenzymes - metabolism; Dehydrogenase; Dehydrogenases; Deoxyribonucleic acid; DNA; DNA, Single-Stranded - metabolism; E coli; Efficiency; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Glutamate Dehydrogenase - genetics; Glutamate Dehydrogenase - metabolism; Glycerol; Glycerol - metabolism; Influence; Ketoglutaric Acids - metabolism; Kinetics; Laboratories; Life Sciences; Liquid chromatography; Metabolism; Metabolites; Microbial Genetics and Genomics; Microbiology; Oxidoreductases; Physiological aspects; Plasmids; Protein Conformation; Protein Multimerization; Proteins; Reagents; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recycling; Recycling (Waste, etc.); Studies; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohol Dehydrogenases - metabolism; China; Proximity effect; Spatially assembled enzymes; Cofactor recycling; Single-stranded DNA; Substrate channeling; Coupled oxidoreductive reaction
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-014-6172-y

Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.