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Improved Production (improved + production)
Selected AbstractsImproved production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional cry1C gene in its chromosomeBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2005Chaoyin Yue Abstract A cryIC gene, whose product is active against Spodoptera exigua, was introduced into wildtype Bacillus thuringiensis kurstaki strain YBT1520 using an integrative and thermosensitive vector, pBMB-FLCE, which was developed based on B. thuringiensis transposon Tn4430 harboring a tnpI-tnpA gene. With the mediation of TnpI-TnpA, the cry1C gene was integrated into the chromosome of the host strain. To prevent secondary integration, the integrative vector was eliminated by moving recombinant cultures to 46°C for generations. Two integrative recombinant B. thuringiensis strains BMB1520-E and BMB1520-F were obtained. In recombinant BMB1520-F, the cry1C gene was expressed stably at a significant level and did not reduce the expression of endogenous crystal protein genes. Bioassay results indicated that BMB1520-E and BMB1520-F showed a higher level of activity against S. exigua third-instar larvae than did their parent strains, in addition to the high toxicity to Plutella xylostella third-instar later larvae. © 2005 Wiley Periodicals, Inc. [source] Globalization vs. localization: global food challenges and local solutionsINTERNATIONAL JOURNAL OF CONSUMER STUDIES, Issue 3 2010Quaye Wilhelmina Abstract The objective of this study was to examine the effect of global,local interactions on food production and consumption in Ghana, and identify possible local solutions. Primary data were collected using a combination of quantitative-qualitative methods, which included focus group discussions and one-on-one interviews. Approximately 450 household heads were randomly selected and interviewed between August 2007 and August 2008 in Eastern, Central, Upper East and Northern Regions of Ghana. Findings revealed increasing consumption of foreign rice as opposed to decreasing consumption of local rice and other staples like millet, sorghum and yam because of global,local interactions. However, opportunities exist to re-localize production-consumption patterns through the use of ,glocal foods' like improved ,koose and waakye'. Referencing the situation in Ghana, the study recommends improved production and processing practices backed with appropriate technologies that reflect changing consumption dynamics in order to take full advantage of opportunities created as a result of global,local interactions. [source] Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2008Lidia Westers Abstract Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases. [source] Engineering of Pichia pastoris for improved production of antibody fragmentsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Brigitte Gasser Abstract The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor , (MF, leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. © 2006 Wiley Periodicals, Inc. [source] Improving 1,3-Propanediol Production from Glycerol in a Metabolically Engineered Escherichia coliby Reducing Accumulation of sn -Glycerol-3-phosphateBIOTECHNOLOGY PROGRESS, Issue 4 2002Marie M. Zhu High levels of glycerol significantly inhibit cell growth and 1,3-propanediol (1,3-PD) production in anaerobic glycerol fermentation by genetically engineered Escherichia coli( E. coli) strains expressing genes from the Klebsiella pneumoniae dha( K.pneumoniae) regulon. We have previously demonstrated that 1,3-PD production by the engineered E. colican be improved by reducing the accumulation of methylglyoxal. This study focuses on investigation of another lesser-known metabolite in the pathways related to 1,3-PD production-glycerol-3-phosphate (G3P). When grown anaerobically on glycerol in the absence of an exogenous acceptor, the engineered E.colistrains have intracellular G3P levels that are significantly higher than those in K. pneumoniae, a natural 1,3-PD producer. Furthermore, in the engineered E. colistrains, the G3P levels increase with increasing glycerol concentrations, whereas, in K. pneumoniae, the concentrations of G3P remain relatively constant. Addition of fumarate, which can stimulate activity of anaerobic G3P dehydrogenase, into the fermentation medium led to a greater than 30-fold increase in the specific activity of anaerobic G3P dehydrogenase and a significant decrease in concentrations of intracellular G3P and resulted in better cell growth and an improved production of 1,3-PD. This indicates that the low activity of G3P dehydrogenase in the absence of an exogenous electron acceptor is one of the reasons for G3P accumulation. In addition, spent media from E. coliLin61, a glycerol kinase (responsible for conversion of glycerol to G3P) mutant, contains greatly decreased concentrations of G3P and shows improved production of 1,3-PD (by 2.5-fold), when compared to media from its parent strain E. coliK10. This further suggests that G3P accumulation is one of the reasons for the inhibition of 1,3-PD production during anaerobic fermentation. [source] Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterizationBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Marie-Hélène Rodriguez Summary Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII. [source] Altering the Substrate Specificity of RhlI by Directed EvolutionCHEMBIOCHEM, Issue 3 2009Pavan Kumar Reddy Kambam Abstract Reducing virulence: RhlI catalyzes the synthesis of N -butanoyl homoserine lactone (BHL), with a minor product N -hexanoyl homoserine lactone (HHL). By using directed evolution and a genetic screen, RhlI has been engineered for enhanced production of both BHL and HHL at a similar level. Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhlI, an enzyme in the RhlI,RhlR quorum-sensing system of P. aeruginosa, to alter its substrate specificity and gain insight into the molecular mechanisms of quorum sensing. By using a genetic screen, we identified a mutant with improved production of RhlI's two signaling molecules, N -butanoyl- and N -hexanoyl-homoserine lactone (BHL and HHL). In particular, production of BHL has been enhanced by more than two-fold, and the synthesis of HHL has been improved from an undetectable level to a level similar to BHL; this change indicates a significant change in substrate specificity. No significant change in the gene expression level was observed. Sequence alignments suggest that the mutations are most likely to facilitate interactions between the enzyme and the two acylated ACP substrates. This work also demonstrates that the genetic screen/selection should be useful in engineering additional quorum-sensing components. [source] | |||||||||||||