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

van Bloois E, Winter RT, Janssen DB, Fraaije MW - Appl. Microbiol. Biotechnol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

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 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.

Show MeSH

Related in: MedlinePlus

Subcellular localization of AldO of wild-type AldO (a), Sec-AldO (b), SRP-AldO (c), and Tat-AldO (d). E. coli cells expressing the indicated constructs were fractionated into total cells (T), cytoplasm (C), and periplasm (P). Samples were normalized on the basis of OD660 and analyzed by immunoblotting with the indicated antisera. Black lines indicate that intervening lanes have been spliced out. Oxidase activity in the cytoplasmic and periplasmic fraction was determined using xylitol as substrate. Plus sign, oxidase activity; minus sign, no oxidase activity
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2690846&req=5

Fig2: Subcellular localization of AldO of wild-type AldO (a), Sec-AldO (b), SRP-AldO (c), and Tat-AldO (d). E. coli cells expressing the indicated constructs were fractionated into total cells (T), cytoplasm (C), and periplasm (P). Samples were normalized on the basis of OD660 and analyzed by immunoblotting with the indicated antisera. Black lines indicate that intervening lanes have been spliced out. Oxidase activity in the cytoplasmic and periplasmic fraction was determined using xylitol as substrate. Plus sign, oxidase activity; minus sign, no oxidase activity

Mentions: Previous work indicated that AldO expressed in E. coli is a soluble protein in agreement with a cytoplasmic localization as predicted by PSORT (Nakai and Horton 1999; Heuts et al. 2007). To unambiguously assess its cellular localization, we first studied the localization of wild-type AldO in E. coli. Therefore, cells expressing wild-type AldO were grown to saturation at 17°C, harvested, converted to spheroplasts by EDTA/lysozyme treatment, after which the periplasmic fraction was obtained by osmotic shock. Subsequently, the spheroplasts were disrupted by sonication and subjected to ultracentrifugation to obtain the cytoplasmic fraction. The different subcellular fractions were analyzed by immunoblotting using antisera directed against AldO or riboflavin to study (1) the AldO content of the samples and (2) whether AldO contained covalently bound FAD. Figure 2a shows that wild-type AldO is cytoplasmically localized and contains covalently bound FAD as previously reported (Heuts et al. 2007). This indicates that AldO is properly folded, as evidenced by the presence of covalent FAD, and functional, as confirmed by the detection of oxidase activity in the cytoplasmic fraction.Fig. 2


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

van Bloois E, Winter RT, Janssen DB, Fraaije MW - Appl. Microbiol. Biotechnol. (2009)

Subcellular localization of AldO of wild-type AldO (a), Sec-AldO (b), SRP-AldO (c), and Tat-AldO (d). E. coli cells expressing the indicated constructs were fractionated into total cells (T), cytoplasm (C), and periplasm (P). Samples were normalized on the basis of OD660 and analyzed by immunoblotting with the indicated antisera. Black lines indicate that intervening lanes have been spliced out. Oxidase activity in the cytoplasmic and periplasmic fraction was determined using xylitol as substrate. Plus sign, oxidase activity; minus sign, no oxidase activity
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690846&req=5

Fig2: Subcellular localization of AldO of wild-type AldO (a), Sec-AldO (b), SRP-AldO (c), and Tat-AldO (d). E. coli cells expressing the indicated constructs were fractionated into total cells (T), cytoplasm (C), and periplasm (P). Samples were normalized on the basis of OD660 and analyzed by immunoblotting with the indicated antisera. Black lines indicate that intervening lanes have been spliced out. Oxidase activity in the cytoplasmic and periplasmic fraction was determined using xylitol as substrate. Plus sign, oxidase activity; minus sign, no oxidase activity
Mentions: Previous work indicated that AldO expressed in E. coli is a soluble protein in agreement with a cytoplasmic localization as predicted by PSORT (Nakai and Horton 1999; Heuts et al. 2007). To unambiguously assess its cellular localization, we first studied the localization of wild-type AldO in E. coli. Therefore, cells expressing wild-type AldO were grown to saturation at 17°C, harvested, converted to spheroplasts by EDTA/lysozyme treatment, after which the periplasmic fraction was obtained by osmotic shock. Subsequently, the spheroplasts were disrupted by sonication and subjected to ultracentrifugation to obtain the cytoplasmic fraction. The different subcellular fractions were analyzed by immunoblotting using antisera directed against AldO or riboflavin to study (1) the AldO content of the samples and (2) whether AldO contained covalently bound FAD. Figure 2a shows that wild-type AldO is cytoplasmically localized and contains covalently bound FAD as previously reported (Heuts et al. 2007). This indicates that AldO is properly folded, as evidenced by the presence of covalent FAD, and functional, as confirmed by the detection of oxidase activity in the cytoplasmic fraction.Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

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 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.

Show MeSH
Related in: MedlinePlus