Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol.
The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V (max) value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli.
Zhang
Hongfang
H
School of Chemical & Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459, Singapore.
Lountos
George T
GT
Ching
Chi Bun
CB
Jiang
Rongrong
R
D002072:Butylene Glycols / Q000378:metabolism*; D032621:DNA Shuffling; D004269:DNA, Bacterial / Q000235:genetics; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D006885:Hydroxybutyrates / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D015202:Protein Engineering; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*
D002072:Butylene Glycols; D004269:DNA, Bacterial; D006885:Hydroxybutyrates; D009580:Nitroblue Tetrazolium; D008773:Methylphenazonium Methosulfate; C028491:1,3-butylene glycol; C035142:4-hydroxy-2-butenoic acid; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase