(481e) Understanding the Lpp Deletion Effects in Membrane Permeability and Consequences for Whole-Cell Biocatalysis | AIChE

(481e) Understanding the Lpp Deletion Effects in Membrane Permeability and Consequences for Whole-Cell Biocatalysis

Authors 

Ni, Y. - Presenter, Virginia Commonwealth University
Chen, R. R. - Presenter, Georgia Institute of Technology


Previous studies in our lab showed that the outer membrane permeability for a number of substrates could be dramatically increased with cells carrying a transposon mutation in the Braun's lipoprotein. As a result, whole-cell catalyzed reactions were dramatically accelerated (up to 14-fold increase in reaction rate). These studies were exclusively carried out with the E609L strain. In order to understand the mechanism behind the dramatic rate acceleration and establish this as a generally applicable methodology for accelerating whole-cell biocatalysis, we sequenced the lpp region of the lipoprotein mutant E609L. The sequencing revealed multiple stop codons present in the mutated lpp gene, indicating that the mutant does not synthesize Braun's lipoprotein at all. This result suggests that lpp deletion will generate the same phenotype as the insertion mutation. More importantly, it suggests that the same useful phenotype could be easily transferred to other E. coli strain or other gram-negative bacteria strain with a simple genetic manipulation. To demonstrate this, several lpp deletion mutants were generated with strains from different genetic background. The deletion was found to have the same effect on substrate permeability as the transposon mutagenesis, consistent with our earlier results. While exhibiting the same phenotype, the deletion does not alter cell growth, carbon metabolism, and lipid compositions. Using L-carnitine as a model product, we found that both rate and yield of its synthesis could be increased significantly in cells with the lpp deletions, owing to enhanced permeability through the outer membrane, allowing its escape from degradation inside the cells. These results demonstrate that a useful phenotype can now be transferred to any E. coli strain with a simple genetic manipulation as a method to effectively enhance the rate of a whole-cell biocatalysis reaction. This method is expected to be applicable to biocatalysis processes involving non-E.coli gram-negative bacteria strains.