scholarly journals Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production

2021 ◽  
Vol 9 ◽  
Author(s):  
Gen Zou ◽  
Juanbao Ren ◽  
Di Wu ◽  
Henan Zhang ◽  
Ming Gong ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genetic Manipulation ◽  
Hericium Erinaceus ◽  
E Coli ◽  
Macrophage Activity ◽  
Medicinal Fungus ◽  
Polysaccharide Composition ◽  
High Production ◽  
The Many

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Molecular characterization, heterologous expression and resistance analysis of OsLEA3-1 from Oryza sativa

Biologia ◽  
2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Tingzhang Hu ◽  
Junnian Yang ◽  
Yongwei Yang ◽  
Yingmei Wu
Keyword(s):  
Fusion Protein ◽  
Heterologous Expression ◽  
Abiotic Stresses ◽  
Structure Prediction ◽  
Bioinformatics Analysis ◽  
Secondary Structure Prediction ◽  
Mature Embryo ◽  
Lactate Dehydrogenase Activity ◽  
E Coli

AbstractLate embryogenesis abundant (LEA) proteins in organisms are closely associated with resistance to abiotic stresses. Here we characterized a rice LEA protein, OsLEA3-1, by bioinformatics analysis and heterologous expression in Escherichia coli. Bioinformatics analysis showed that OsLEA3-1 contains a 603-bp open reading frame encoding a putative polypeptide of 200 amino acids, which contains a “LEA_4” motif at positions 5–48 and belongs to a typical group 3 LEA. OsLEA3-1 polypeptide is rich in Ala, Lys, and Thr, but depleted in Cys, Pro, and Trp residues; and is strongly hydrophilic. Secondary structure prediction showed that OsLEA3-1 polypeptide contained an α-helical domain in positions 4-195 but not any β-sheet domain. OsLEA3-1 gene can express in shoot and root of germinating seeds, seedling, panicles, mature embryo, seed, and callus; and was also up-regulated by ultraviolet (UV), heat, cold, salt, and emergency drought. OsLEA3-1 gene was introduced into E. coli. A fusion protein of about 28.03 kDa was expressed in recombinant E. coli cells after the induction by isopropylthio-β-D-galactoside. Compared with control E. coli cells harbouring pET30a, the accumulation of the OsLEA3-1 fusion protein increased the tolerance of the E. coli recombinants under diverse abiotic stresses: high salinity, metal ions, hyperosmotic, heat, and UV radiation. The OsLEA3-1 has the ability to protect the lactate dehydrogenase activity under heating, drying, and MnCl2 treatment in vitro. The findings suggested that the OsLEA3-1 gene may contribute to the ability of adapting to stressful environments of plants.

scholarly journals Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

2006 ◽  
Vol 75 (4) ◽  
pp. 1661-1666 ◽  
Author(s):  
Theresa Deland Ho ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Enterohemorrhagic Escherichia Coli ◽  
Deletion Strain ◽  
Growth Defect ◽  
Intestinal Colonization ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system

2019 ◽  
Vol 74 (9) ◽  
pp. 2559-2565 ◽  
Author(s):  
Pengxia Wang ◽  
Dongmei He ◽  
Baiyuan Li ◽  
Yunxue Guo ◽  
Weiquan Wang ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Clinical Isolates ◽  
Plasmid Curing ◽  
Insertion Element ◽  
E Coli ◽  
Guide Rnas ◽  
Gene Copies ◽  
Conserved Genes ◽  
One Step

Abstract Objectives To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. Methods Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. Results In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. Conclusions CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

scholarly journals Functional reconstitution of TatB into thylakoidal Tat translocase

2019 ◽  
Author(s):  
Sarah Zinecker ◽  
Mario Jakob ◽  
Ralf Bernd Klösgen
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Experimental System ◽  
Receptor Complex ◽  
Transport Activity ◽  
E Coli ◽  
Purified Antigen

AbstractWe have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat1) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 - 25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

scholarly journals Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

2020 ◽  
Author(s):  
Joshua G. Klein ◽  
Yang Wu ◽  
Bashkim Kokona ◽  
Louise K. Charkoudian
Keyword(s):  
Heterologous Expression ◽  
Polyketide Synthase ◽  
Analytical Ultracentrifugation ◽  
Acyl Carrier Protein ◽  
Unmet Need ◽  
Type Ii ◽  
E Coli ◽  
Potential Impact

AbstractEnzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable success in the clinic, the biosynthetic prowess of these enzymes has been stymied by: 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. Towards filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KSCLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer. Using E. coli as a heterologous host, we obtained soluble proteins in titers representing significant improvements over previous KSCLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKSCLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. Given the potential impact of obtaining soluble core type II PKS biosynthetic enzymes to enable in vitro characterization studies, these results lay important groundwork for optimizing the interaction between KrKSCLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

scholarly journals Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

2021 ◽  
Author(s):  
Gloria Gamiz-Arco ◽  
Valeria A. Risso ◽  
Eric A. Gaucher ◽  
Jose A. Gavira ◽  
Athi N. Naganathan ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
3D Structure ◽  
Sequence Divergence ◽  
Redox Activity ◽  
Structure Stability ◽  
Heterologous Protein Expression ◽  
Ancestral Reconstruction ◽  
E Coli ◽  
Statistical Mechanical Model

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

scholarly journals 18-Hydroxydolabella-3,7-diene synthase – a diterpene synthase from Chitinophaga pinensis

2017 ◽  
Vol 13 ◽  
pp. 1770-1780 ◽  
Author(s):  
Jeroen S Dickschat ◽  
Jan Rinkel ◽  
Patrick Rabe ◽  
Arman Beyraghdar Kashkooli ◽  
Harro J Bouwmeester
Keyword(s):  
Heterologous Expression ◽  
Nicotiana Benthamiana ◽  
Terpene Synthase ◽  
In Planta ◽  
Isotopic Labelling ◽  
E Coli ◽  
Diterpene Synthase ◽  
Diterpene Alcohol ◽  
Isotopic Labelling Experiments

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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