Improving the growth rate of Escherichia coli DH5alpha at low temperature through engineering of GroEL/S chaperone system.

GroEL/S is a molecular chaperone system in Escherichia coli which not only assists the folding of intracellular proteins but also affects the cellular activity against the change of environmental condition. Here we show that the growth rate of E. coli DH5alpha can be improved at low temperature by expressing a GroEL/S variant achieved through irrational protein engineering approach. The GroELS variant (GroELS(var)) accelerating the growth of E. coli DH5alpha was screened through enrichment culture of the mutant libraries obtained by random mutagenesis. E. coli DH5alpha harboring the groELS(var) gene exhibited approximately 1.5-2 times higher growth rate compared to the strain with wild-type GroELS at 15-30 degrees C. At 10 degrees C, a temperature that the growth of E. coli DH5alpha almost stops, the GroELS(var) triggered the growth of E. coli DH5alpha. We identified that seven nucleotides of groELS gene and six amino acids of the GroELS were changed through the mutagenesis and screening. Site directed mutagenic analysis revealed that H360 in GroEL(var) is the most crucial residue determining the activity of GroELS(var) and more than one of the other residues in GroEL(var) may be additionally involved in the activity of GroELS(var). The improvement of growth rate induced by the GroELS(var) was observed only in the strain DH5alpha and not detected in other E. coli strains, such as BL21, BW25113, codon+, JM110, Top10, and XL1-blue. (c) 2007 Wiley Periodicals, Inc.