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A Quick Look-Around of Microbial Enzymes in Modern Food Industries and Dietary Research
Published in Pankaj Bhatt, Industrial Applications of Microbial Enzymes, 2023
Vineet Singh, Anjali Pande, Jae-Ho Shin
Enzymes are valuable bioactive compounds available in all living organisms, but for industrial purposes, enzymes are usually obtained from microbial sources, such as bacteria, yeast, actinomycetes, and fungi. Though enzymes can be obtained from plants and animals, but microbes can be manipulated very easily to get the desired product with a better yield. For example, initially, rennin was obtained from calves, but nowadays, most of the rennin is obtained from the microbe Rhizomucor miehei [3]. Additionally, microbial productivity can be enhanced further by inserting multiple copies of a specific gene and by adding a strong promoter. Moreover, a suitable foreign gene can be inserted into the microbial genome to create a heterologous expression system that also enhances enzyme production [4]. Other than that, screening of selected microbes of interest can be simplified by adding any selectable marker, such as antibiotic resistance genes. After successful manipulation, a high-yielding microbial strain is obtained that can be used in downstream processing for the commercial production of enzymes. Presently, most of the enzymes utilized in the food and feed industry are obtained from such recombinant microorganisms.
Agrobacterium and Plant Genetic Engineering
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Masami Sekine, Atsuhiko Shinmyo
Regardless of transformation strategy, genetic markers are required for the selection of transformed cells that have acquired the DNA of interest. Genetic markers include dominant selectable markers for the direct selection of transformed cells, assayable markers that allow the detailed analysis of gene expression, and negative selection markers that prevent the development of normal cells. In this section we will describe dominant selectable markers and negative selection markers, and the assayble markers will be outlined in the following section.
Cell Line Development
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Typically, the vector is propagated in E. coli to generate sufficient quantities for transfection into the host cell. The vector typically carries a selectable marker gene so that the cells that have acquired the vector can be enriched after transfection using a selection agent (Figure 6.1). In vitro screening of post-selection cells is then performed to screen for clones in this population that produce the product at high levels.
Enhancement of anti-TNFα monoclonal antibody production in CHO cells through the use of UCOE and DHFR elements in vector construction and the optimization of cell culture media
Published in Preparative Biochemistry & Biotechnology, 2021
Chinh Chung Doan, Nguyen Quynh Chi Ho, Thi Thuy Nguyen, Thi Phuong Thao Nguyen, Dang Giap Do, Nghia Son Hoang, Thanh Long Le
In this study, pN vectors containing both UCOE and DHFR sequences were kindly provided by Dr. Bruce May, The University of British Columbia, Vancouver, Canada. The construct of pN vectors had a selection marker (G418 resistance gene) and an amplifiable selectable marker (DHFR gene) isolated from pSV2-DHFR (Thermo Fisher Scientific, Waltham, MA, USA). The CMV promoter was used to regulate the expression of the target gene while the SV40 promoter was used to regulate the expression of the G418 resistance gene. The UCOE fragment (1.5 Kb) from the human HNRPA2B1 gene, as an enhancer was inserted into pN vector upstream of the LC or HC expression units. The IRES was inserted in front of the DHFR gene to ensure regulated co-expression of the DHFR gene and the target gene. The expression vectors also carry the bacterial ColE1-like origin of replication and the bacterial beta-lactamase gene from transposon Tn3 (AmpR), conferring ampicillin resistance. In addition, the terminator region of pN consists of an SV40 enhancer positioned downstream of the bGH polyadenylation signal. The codon-optimized genes encoding the heavy chain (HC; 1441 bp) and light chain (LC; 733 bp) of adalimumab (Drugbank, ID: DB00051) expressed in CHO cells were synthesized by GenScript Biotech (Piscataway, NJ, USA). To construct the expression vectors, LC or HC genes were cloned into the pN vectors, and named pN-ADA-LC and pN-ADA-HC, respectively. The constructs of the plasmid vectors used in the present study are presented in Figure 1A. The nucleotide sequence for each plasmid was primarily examined using restriction digestion analysis (Figure 1B) and then confirmed by DNA sequencing.
Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase
Published in International Journal of Phytoremediation, 2020
René Piña-Olavide, Luz M. T. Paz-Maldonado, M. Catalina Alfaro-De La Torre, Mariano J. García-Soto, Angélica E. Ramírez-Rodríguez, Sergio Rosales-Mendoza, Bernardo Bañuelos-Hernández, Ramón Fernando García De la-Cruz
The vector used for subcloning was pGEM®-T Easy, of 3015 bp and including the gene Ampr (Promega Co., Madison, WI). The expression vector for transforming the chloroplast was p463, of 8500 bp and carrying the gene aadA for spectinomycin resistance as a selectable marker, flanked by the regulatory regions 5’UTR and 3’UTR, and under the control of the ribulose bisphosphate carboxylase large-chain (rbcL) gene promoter. The strain used for gene propagation was E. coli DH5α.
Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa
Published in International Journal of Phytoremediation, 2019
V. Kumar, S. AlMomin, A. Al-Shatti, H. Al-Aqeel, F. Al-Salameen, A. B. Shajan, S. M. Nair
Genetic transformation of recalcitrant plant species such as Alfalfa requires optimization of several critical factors. Although there are several reports on the somatic embryogenesis of alfalfa, most reported methods are highly genotype-specific (Walker and Sato 1981; Novak and Konečná 1982; Dijak et al. 1986; Strickland et al. 1987; Hernandez-Fernandez and Christie 1989; Song et al. 1990; Kielly and Bowley 1992; Parrott and Bailey 1993; Shetty and McKersie 1993; Ninković et al. 1995; Shao et al. 2000; Pasternak et al. 2002; Tian et al. 2002; Liu et al. 2013; Fu et al. 2015). Therefore, optimization of the embryogenesis method is a very important factor, prior to any transformation attempt. Here, we report the development and optimization of a protocol to successfully generate transgenic alfalfa plants of the Regen SY genotype, using the somatic embryo-based regeneration method (Fu et al. 2014). The interaction between Agrobacterium and explants during the co-cultivation step is a critical factor affecting genetic transformation. Previous alfalfa transformation protocols involved ultra-sonication of leaf explants to induce micro wounds (Fu et al. 2014). However, this caused bleaching of the explants and permit over-growth of Agrobacteria. Consequently, manual wounding was performed to facilitate Agrobacterium infection in a more controlled manner. Since only a few cells in a plant tissue or organ explant can be genetically transformed using currently available methods, the selective growth of the transformed cells is crucial for the recovery of transgenic plants. The common strategy is that of constitutive expression of a selectable marker gene (Tian et al. 2002); however, we found that kanamycin selection of putative transgenic embryos was very efficient in generating transgenic alfalfa plants.