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Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis. Claudia Donnini, 2004.The secreted production of heterologous proteins in Kluyveromyces lactis was studied . A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K . lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae . Among the laboratory strains tested, strain JA6 was the best producer of GAA . Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain . One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1ß compared to the parent strain . dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K . lactis (a homologue of S . cerevisiae HXK2) . The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain . Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation . Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided . Insufficient Expression of the ilv-leu Operon Encoding Enzymes of Branched-Chain Amino Acid Biosynthesis Limits Growth of a Bacillus subtilis ccpA Mutant. Holger Ludwig, 2002.Bacillus subtilis ccpA mutant strains exhibit two distinct phenotypes: they are defective in catabolite repression, and their growth on minimal media is strongly impaired . This growth defect is largely due to a lack of expression of the gltAB operon . However, growth is impaired even in the presence of glutamate . Here, we demonstrate that the ccpA mutant strain needs methionine and the branched-chain amino acids for optimal growth . The control of expression of the ilv-leu operon by CcpA provides a novel regulatory link between carbon and amino acid metabolism . Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans. Yoshiko Okamoto-Hosoya, 2003.Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp . These rpsL mutations are known to be located in the two conserved regions within the S12 protein . To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans . Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant . Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin . Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement .
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