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Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Engineering, Bio-arming Technology
The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of proteins/peptides library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-ac...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Yeast Metabolomics: Sample Preparation for a GC/MS-Based Analysis
Metabolome sample preparation is one of the key factors in metabolomics analyses. The quality of the metabolome data will depend on the suitability of the experimental procedures to the cellular system (e.g., yeast cells) and the analytical performance. Here, we summarize a protocol for metabolome analysis of yeast cells using gas chromatography–mass spectrometry (GC–MS). First, the main phases of a metabolomics analysis are identified: sample preparation, metabolite extraction, and analysis. We also provide an overview on different methods used to quench samples and extract intracellular metabolites from yeast...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Genetic Engineering of Industrial Saccharomyces cerevisiae Strains Using a Selection/Counter-selection Approach
Gene modification of laboratory yeast strains is currently a very straightforward task thanks to the availability of the entire yeast genome sequence and the high frequency with which yeast can incorporate exogenous DNA into its genome. Unfortunately, laboratory strains do not perform well in industrial settings, indicating the need for strategies to modify industrial strains to enable strain development for industrial applications. Here we describe approaches we have used to genetically modify industrial strains used in winemaking. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

13C-Based Metabolic Flux Analysis in Yeast: The Pichia pastoris Case
Metabolic flux analysis based on tracing patterns of stable isotopes, particularly 13C, comprises a set of methodologies to experimentally quantify intracellular biochemical reaction rates, i.e., to measure carbon flux distributions through a metabolic network. This allows quantifying the response of a metabolic network to an environmental or genetic perturbation (i.e., the metabolic phenotype). Here, we describe a protocol based on growing yeast on a 13C-labelled substrate and subsequent NMR detection of 13C-patterns in proteinogenic amino acids. To calculate metabolic fluxes, we describe two complementary mathematical ap...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Pathway Activity Profiling (PAPi): A Tool for Metabolic Pathway Analysis
Pathway Activity Profiling (PAPi) is a method developed to correlate levels of metabolites to the activity of metabolic pathways operating within biological systems. Based solely on a metabolomics data set and the Kyoto Encyclopedia of Genes and Genomes, PAPi predicts and compares the activity of metabolic pathways across experimental conditions, which considerably improves the hypothesis generation process for achieving the biological interpretation of biological studies. In this chapter, we describe how to apply PAPi to a metabolomics data set using the R-software. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Genome-Scale Metabolic Models of Yeast, Methods for Their Reconstruction, and Other Applications
Here, we present the concept of genome-scale metabolic models and some of their applications in metabolic engineering of yeast and in the analysis of gene expression data. The yeast species for which there are available genome-scale metabolic models are reviewed, as well as the methods for the reconstruction of genome-scale metabolic models for new species. Some commonly used algorithms for metabolic engineering and data integration are described. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

QTL Mapping by Pooled-Segregant Whole-Genome Sequencing in Yeast
Quantitative trait locus (QTL) mapping by pooled-segregant whole-genome sequencing in yeast is a robust methodology for the simultaneous identification of superior genes involved in polygenic traits (e.g., high ethanol tolerance). By crossing two haploid strains with opposite phenotypes, being one of interest, the resulting diploid is sporulated, the meiotic segregants phenotyped, and a pool of selected segregants with the phenotype of interest assembled. The genotyping by pooled-segregant sequencing constitutes a fast and reliable methodology to map all QTL defining the trait of interest. The QTLs can be further analyzed ...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Model-Guided Identification of Gene Deletion Targets for Metabolic Engineering in Saccharomyces cerevisiae
Identification of metabolic engineering strategies for rerouting intracellular fluxes towards a desired product is often a challenging task owing to the topological and regulatory complexity of metabolic networks. Genome-scale metabolic models help tackling this complexity through systematic consideration of mass balance and reaction directionality constraints over the entire network. Here, we describe how genome-scale metabolic models can be used for identifying gene deletion targets leading to increased production of the desired product. Vanillin production in Saccharomyces cerevisiae is used as a case study throughout t...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Patents: A Tool to Bring Innovation from the Lab Bench to the Marketplace
Intellectual property (IP) is creations of the mind. Protecting IP through patents is an important venue for a researcher to reap rewards from his scientific endeavors. It is part of a competitive strategy for bringing one’s invention to the marketplace. Using the US and European patent systems as examples, we provide here an overview of how patents protect innovation, with a focus on biotechnology. We explain what a patent is, what a patent owner can do with a patent, and how patents are granted. The article ends with some recent examples of noteworthy patents in the field of yeast research. (Source: Springer protoc...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Synthetic RNA Switches for Yeast Metabolic Engineering: Screening Recombinant Enzyme Libraries
Directed evolution is a powerful technique for increasing the activity of poorly active enzymes, for example when an enzyme is engineered to accept a new substrate or function in a new environment. Since enzyme activity greatly depends on the enzyme environment, screening should be performed under the same conditions as the ultimate application of the enzyme. When an enzyme will be used in live cells, RNA biosensors offer a powerful and flexible method of linking the desired phenotype, production of a small molecule of interest, to an easily measured phenotype, such as fluorescence. Here, we describe methods for screening ...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news

Methods for Efficient High-Throughput Screening of Protein Expression in Recombinant Pichia pastoris Strains
The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a wh...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news

Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris
Within the last two decades, the methylotrophic yeast Pichia pastoris has become an important alternative to E. coli or mammalian cell lines for the production of recombinant proteins. Easy handling, strong promoters, and high cell density cultivations as well as the capability of posttranslational modifications are some of the major benefits of this yeast. The high secretion capacity and low level of endogenously secreted proteins further promoted the rapid development of a versatile Pichia pastoris toolbox. This chapter reviews common and new “Pichia tools” and their specific features. Special focus is given ...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news

Molecular Tools and Protocols for Engineering the Acid-Tolerant Yeast Zygosaccharomyces bailii as a Potential Cell Factory
Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevis...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news

Tools for Genetic Engineering of the Yeast Hansenula polymorpha
Hansenula polymorpha is a methylotrophic yeast species that has favorable properties for heterologous protein production and metabolic engineering. It provides an attractive expression platform with the capability to secrete high levels of commercially important proteins. Over the past few years many efforts have led to advances in the development of this microbial host including the generation of expression vectors containing strong constitutive or inducible promoters and a large array of dominant and auxotrophic markers. Moreover, highly efficient transformation procedures used to generate genetically stable strains are ...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news

Natural and Modified Promoters for Tailored Metabolic Engineering of the Yeast Saccharomyces cerevisiae
The ease of highly sophisticated genetic manipulations in the yeast Saccharomyces cerevisiae has initiated numerous initiatives towards development of metabolically engineered strains for novel applications beyond its traditional use in brewing, baking, and wine making. In fact, baker’s yeast has become a key cell factory for the production of various bulk and fine chemicals. Successful metabolic engineering requires fine-tuned adjustments of metabolic fluxes and coordination of multiple pathways within the cell. This has mostly been achieved by controlling gene expression at the transcriptional level, i.e., by using...
Source: Springer protocols feed by Biotechnology - April 18, 2014 Category: Biotechnology Source Type: news