Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate.
In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun's lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze-thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.
Ni
Ye
Y
Chemical Engineering Department, Virginia Commonwealth University, Richmond, VA 23284, USA.
Mao
Zichao
Z
Chen
Rachel R
RR
D000606:Amino Sugars / Q000737:chemistry; D002384:Catalysis; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism; D005615:Freezing; D005822:Genetic Vectors; D007763:Lac Operon; D008070:Lipopolysaccharides / Q000737:chemistry; D008074:Lipoproteins / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D010539:Permeability; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000737:chemistry*
D000606:Amino Sugars; D008070:Lipopolysaccharides; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C000458:N-acetyllactosamine; D014532:Uridine Diphosphate Glucose