Recombinant protein production in the new Millennium

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Applications of cell sorting in biotechnology

Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology

Adaptive laboratory evolution principles and applications for biotechnology

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.(VLID)90682

Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data

BACKGROUND: Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. RESULTS: Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and sporozoite stages. CONCLUSION: Using integrative analysis techniques, we can integrate knowledge from different levels and obtain a wider view of the system under study. The overlap between method-specific and common results is considerable, even if the basic mathematical assumptions are very different. The three-fold validated network of life cycle stage characteristics of Plasmodium falciparum could identify a large amount of the known associations from literature in only one study

Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>High cell density cultures of <it>Pichia pastoris </it>grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of <it>P. pastoris </it>for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes.</p> <p>Results</p> <p>Overexpression of the first gene of the riboflavin biosynthetic pathway (<it>RIB1</it>) is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P_<it>GAP</it>) increases the riboflavin accumulation significantly.</p> <p>Conclusion</p> <p>The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a <it>P. pastoris </it>strain overexpressing all six <it>RIB </it>genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in <it>P. pastoris </it>has thus been established.</p

Pathway design for mixotrophic production of biochemicals from CO2 and methanol in yeasts

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Impact of oxygen availability on organelle-specific redox potentials and stress in recombinant protein producing Pichia pastoris

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Semi-rational engineering of Adh2 for improved methanol utilization in Komagataella phaffii

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Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

Besides bakers' yeast, the methylotrophic yeast Komagataella phaffii (also known as Pichia pastoris) has been developed into the most popular yeast cell factory for the production of heterologous proteins. Strong promoters, stable genetic constructs and a growing collection of freely available strains, tools and protocols have boosted this development equally as thorough genetic and cell biological characterization. This review provides an overview of state-of-the-art tools and techniques for working with P. pastoris, as well as guidelines for the production of recombinant proteins with a focus on small-scale production for biochemical studies and protein characterization. The growing applications of P. pastoris for in vivo biotransformation and metabolic pathway engineering for the production of bulk and specialty chemicals are highlighted as well

Going beyond the limit: impact of increasing global translation activity on the productivity of recombinant secreted proteins in Pichia pastoris

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