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Structure of the gene encoding the exoglucanase of Cellulomonas fimi and its expression and secretion in Escherichia coli O’Neill, Gary Paul


In the Gram-positive bacterium Cellulomonas fimi, the pex gene encodes a secreted exoglucanase (Exg) involved in the degradation of cellulose. In order to study the relationship between the structure and function of this cellulase, the nucleotide sequence of the pex gene was determined, and its overproduction in Escherichia coli was obtained to facilitate purification of large amounts of this enzyme. The pex gene was localized to a 2.58-kb BamHI-Sall fragment contained within a 6.6-kb BamHI fragment of C. fimi DNA, and its nucleotide sequence was determined. The pex coding region of 1452-bp (484 codons) was identified by comparison of the DNA sequence to the amino terminal amino acid sequence of the Exg produced by C. fimi. In the gene, the region encoding the amino terminus of the mature Exg is preceded by a sequence encoding a putative signal peptide of 41 amino acids, a translational initiation codon and a ribosome binding site. The nucleotide sequence immediately following the translational stop codon contained four inverted repeats, two of which overlap. A dramatic (98.5%) bias occurs for guanosineor cytosine in the third position of the 35 codons utilized in the pex, The ability of the proposed 41 amino acid signal peptide to direct secretion from E. coli was shown by gene fusion experiments. A hybrid leader sequence containing the 6 amino-terminal amino acids of β-galactosidase (βGal) followed by the 37 amino acids of the carboxyl end of the leader peptide directed export of Exg into the periplasm of Lpoli In contrast, hybrid βGal-Exg proteins in which the leader sequence was not present were located in the cytoplasm. In an attempt to optimize the secretion of the Exg from E. poll, the Exg signal peptide W8S replaced with the signal peptide from the OmpA protein to yield an OmpA-Exg hybrid protein. The OmpA-Exg hybrid protein retained biological activity and was secreted into the periplasmic space of E. poll. A series of operon and gene fusions between various E. coli operons and the Exg coding sequence was carried out in order to optimize the expression of Exg in E. coli. The highest level of Exg production exceeded 20% of the total cellular protein. To obtain this level of overproduction, the Exg coding sequence was fused to a synthetic ribosome binding site, with an initiating ATG, and was placed under the control of the leftward promoter of phage lambda contained on a runaway replication plasmid vector.

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