Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene.
A beta-1,4-endoglucanase gene (eglA) cloned from C. acetobutylicum P262 was selected for use in the development of a reporter system for C. beijerinckii NCIMB 8052. The reporter plasmid, pER1, was constructed by ligating the promoterless eglA gene into the B subtilis/Clostridium shuttle vector, pFNK1, which can replicate and is stably maintained in C. beijerinckii. The expression of the endoglucanase enzyme from its own promoter was not significantly induced in cells grown in glucose, sucrose or galactose, while growth of cells in cellobiose or fructose resulted in lower levels of activity. The enzyme was efficiently secreted into the culture medium and did not remain associated with the cell in any way. A transcriptional fusion between the glutamine synthetase (glnA) promoter region and the promoterless eglA gene resulted in high levels of endoglucanase expression, which reflected an 11-fold increase in expression levels over the eglA promoter.
Quixley
K W
KW
Department of Microbiology, University of Cape Town, South Africa.
Reid
S J
SJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003013:Clostridium / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
D002480:Cellulase