Increased xylanase yield in Streptomyces lividans: dependence on number of ribosome-binding sites.
The Streptomyces lividans xylanase A (XlnA) signal peptide (sp) was replaced with the signal peptides of either mannanase A (ManA) or cellulase A (CelA), two enzymes secreted by S. lividans. Depending on the location of the ribosome binding sites (RBS) with respect to a potential initiation codon, the length of the putative sps of ManA and CelA is either 34 or 43 amino acids and 27 or 46 amino acids, respectively. The sequence encoding these sps were fused to the xylanase A gene (xlnA). Clones harboring the short sps of ManA and CelA produced as much xylanase as the clone with the control wild-type sp sequence of XlnA. In clones containing the long sps of ManA and CelA, the XlnA production was enhanced 1.5- and 2.5-fold, respectively. These XlnA yields are reduced by half and one third respectively when the internal initiation codons of the long sp sequences of ManA and CelA are mutated. Since these clones exhibited the same transcription levels, the results indicate that both RBSs are used concomitantly in S. lividans to increase the enzyme production at the translational level. However, when the short and long sps of ManA were fused to the long CelA sp sequence, giving constructs containing respectively 3 and 4 RBSs, a decrease in xylanase production was observed.
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Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Ville de Laval, Canada.
Kluepfel
D
D
Shareck
F
F
Morosoli
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D004269:DNA, Bacterial; D043364:Endo-1,4-beta Xylanases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D003062:Codon; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases