1887

Abstract

The facultatively anaerobic, thermophilic bacterium is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in . Initially, a minimal three-part sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 origin of replication and multiple cloning site (MCS) of pUC18, the sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (), respectively. could be transformed with pUCG3.8 at an increased efficiency [2.8×10 c.f.u. (µg DNA)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from as a secretion target. Expression of was placed under the control of a cellobiose-inducible promoter (P) together with a signal peptide encoding sequence from a C56-YS93 endo-β-1,4-xylanase. The interchange of parts was demonstrated by exchanging the gene with the 3′ region of a gene with homology to from and substituting P for the synthetic, constitutive promoter P, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

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2013-07-01
2022-05-27
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