Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis.
Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.
van Bloois
Edwin
E
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Winter
Remko T
RT
Janssen
Dick B
DB
Fraaije
Marco W
MW
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology*; D021382:Protein Sorting Signals; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014993:Xylitol / Q000378:metabolism*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C515743:alditol oxidase (FAD), Streptomyces coelicolor; D014993:Xylitol