Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Abstract

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 wholecell 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 INPbased 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.

Deciphering EGFP production via surface display and self-cleavage intein system in different hosts

“…This revealed that the removal of LPS (components of the cell membrane) did help to reduce the steric hindrances and enhance the outer-membrane protein expression. This result is consistent with that suggested by Bloois et al that the surface display of alditol oxidase might be restricted by LPS in the wild-type E. coli host [31]. Strain JM109 (lon mutation) was observed to give the highest EGFP production.…”

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2Sequestration

“…However, the ratio of cell lysate activity to wholecell activity for the periplasmic CA system was an order of magnitude lower (ϳ6) than that of the cytoplasmic CA system (ϳ102). This observation clearly indicates the effectiveness of periplasmic translocation for overcoming the physical or physiological barriers imposed by the cytoplasmic membrane (22,39). Again, this indirectly demonstrated that ngCA was successfully exported into the periplasm of E. coli.…”

Biotechnological applications of periplasmic expression in E. coli

“…In summary, the choice of export pathway is dictated by the properties of the target protein. This is exemplified by our study on exporting AldO, a flavoprotein oxidase, to the periplasm of E. coli [15]. AldO contains covalently bound FAD, which will only form upon linkage to polypeptide chain.…”

“…The latter is well illustrated by our finding that cells which express AldO in the cytoplasm are unable to convert xylitol. In contrast, xylitol was readily converted by cells that export AldO Tat-dependently to the periplasm [15]. Next, we constructed a xylitol biosensor comprising AldO equipped with a microperoxidase (MP) domain.…”