China 
Africa 
Explore chapters and articles related to this topic
by Genetically Engineered Filamentous Fungs
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
T. Vichitsoonthonkul, Y. W. Chu, H. S. Sodhi, G. Saunders
Genes can be identified by complementing mutations in recipient strains with wild-type DNA. Genes (trpC, pyr4) of several fungi (Phycomyces blaksleeanus and Neurospora crassa) have been cloned by the approach of transformation and complementation of E. coli mutants [13,14], The argB gene of Aspergillus nidulans has been cloned by expression in yeast [15]. The promoter and intron recognition problems faced during the cloning of fungal genes in bacteria and yeast soon led to the development of fungal transformation systems. However, cloning by this approach in filamentous fungi largely depends on the transformation frequencies. The transforming sequence, along with the bacterial vector sequences (usually antibiotic resistance and plasmid origin of replication), complementing the mutation of interest, integrate into the nuclear DNA, but can be rescued by partial digestion of the total transformant DNA, religation, and transformation of E. coli to antibiotic resistance (marker rescue). The desirable sequences obtained in the rescued plasmid must be tested by retransformation [16]. It is also possible to isolate genes from one organism by heterologous expression in another. For example, a gene encoding pisatin de-methyl ase from a plant pathogen Nectria haematococca has been cloned by expression in A. nidulans [7].
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Obtaining pure protein samples is a critical first step for many experiments. While a small number of highly abundant proteins can be purified directly from natural sources, most interesting proteins must be obtained by heterologous expressions or total chemical synthesis. The purification of a protein from a natural source requires large amounts of tissue containing the desired protein and extensive knowledge of the protein’s physicochemical properties or the use of immunoaffinity methodologies. As such, natural source isolation of the quantity of protein necessary for biochemical or biophysical studies is limited to a small number of proteins and will not be discussed here. Total chemical synthesis and expressed chemical ligation (ECL) technologies provide intriguing methods for the preparation of modified proteins, but these methods require techniques that are beyond the scope of many laboratories. A brief discussion of these techniques and their applications is provided at the end of the chapter. Generally, proteins are prepared by overexpression and purification using heterologous expression systems; as such, this will be the focus of this chapter. Heterologous expression has the advantage that it can be used for the preparation of small or large amounts of purified proteins, including modified and engineered proteins. Moreover, it can be used to express proteins from pathogenic organisms without the extensive biosafety precautions that would be required for handling the source organism and allows for the functional analysis of open reading frames.
Production of VNPs, VLPs, and Chimeras
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Heterologous expression systems are of great interest as they allow the production of VLPs. VLPs are devoid of infectious nucleic acids and thus cannot replicate themselves. VLPs are considered safer from an agricultural point of view and human health perspective. Heterologous expression systems used to generate VLPs include use of bacteria, yeast, insect cells, and mammalian cells. All these systems have advantages and disadvantages in terms of yield, scale, time, costs, assembly efficiency, and biological integrity (reviewed in Schneemann & Young, 2003). In the following sections, we will discuss different systems and highlight their benefits and pitfalls (summarized in Table 3.1).
Standardized production of a homogeneous latex enzyme source overcoming seasonality and microenvironmental variables
Published in Preparative Biochemistry & Biotechnology, 2021
Sandro Rios Silveira, Raphael Alves Coelho, Brandon Ferraz e Sousa, Jefferson Soares de Oliveira, Laura Maria Isabel Lopez, José Vitor Moreira Lima-Filho, Pedro Abílio Vieira Rocha Júnior, Diego Pereira de Souza, Cleverson Diniz Teixeira de Freitas, Márcio Viana Ramos
Bioprospection, for new sources of enzymes, suitable for application in industrial processes is attracting increasing attention. In general, the use of enzymes, instead of chemicals, in industrial technologies is more environmentally friendly.[1] The challenge of prospecting for new enzymes is complex and laborious. The first challenges involve the choice of the biological source to be screened and possible systems for production. Heterologous expression of enzymes, in microorganisms, has been exploited. However, raw materials, obtained of agricultural practices to produce enzymes of market interest are still interesting. Due to the peculiar characteristics of enzymes and the particularities of the process associated to their use, many other aspects should be observed. It involves investigating the desired specificity and catalytic efficiency, pH and temperature requirements, suitability for large-scale use of the enzyme source and adequate storage conditions, among many other requirements.
Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale
Published in Preparative Biochemistry and Biotechnology, 2019
Miguel Angel Marín Muñoz, Juan Jáuregui Rincón, Leobardo Serrano Carreón, Norma Angélica Chávez Vela
Several heterologous expression systems are available for the expression of recombinant proteins; for instance, the use of methylotrophic yeast Pichia pastoris has increased extensively, especially in pharmaceutical applications.[6,7] This yeast presents certain advantages: (i) it is a Generally Recognized As Safe (GRAS) microorganism; (ii) it prefers an aerobic over an anaerobic metabolic pathway, which prevents the production of fermentation metabolites such as acetic acid and ethanol; (iii) as an eukaryote, it has the capacity to carry out post-translational protein modifications; (iv) it possesses an efficient protein excretion system, and (v) it grows at high cell densities in minimal defined media.[8] The capacity of P. pastoris to oxidize methanol through enzymes alcohol oxidase (AOX1 and AOX2) is used in the construction of genetically modified strains.[9] The promoter of the AOX1 gene (pAOX1) is the most frequently used gene to control the expression of recombinant proteins, although other promoters have been developed.[10]
Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Eva Agustriana, Isa Nuryana, Fina Amreta Laksmi, Kartika Sari Dewi, Hans Wijaya, Nanik Rahmani, Danu Risqi Yudiargo, Astadewi Ismadara, Moch Irfan Hadi, Awan Purnawan, Apridah Cameliawati Djohan
In this study, E. coli was employed for the expression of Bst polymerase. E. coli has been routinely used as a heterologous expression system because it offers a short production time, is genetically easy to manipulate, cheap growth medium, has high cell density, and is capable of producing a wide variety of proteins. However, codon usage bias between E. coli host and native gene sequence has a notable impact on the recombinant protein expression level. Codon optimization by using E. coli preferred codons is an efficient measure to improve the yield of heterologous expression products. Generally, this is completed by replacing rarely used codons with the frequently used ones, adjusting G + C content, and eliminating AT-rich stretch.