scholarly journals Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation ofcry3AaPromoter and Single Cysteine Mutations in the Protoxin Region

2013 ◽  
Vol 79 (22) ◽  
pp. 6969-6973 ◽  
Author(s):  
Blanca I. García-Gómez ◽  
Jorge Sánchez ◽  
Diana L. Martínez de Castro ◽  
Jorge E. Ibarra ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Expression System ◽  
Efficient Production ◽  
Content Type ◽  
E Coli ◽  
Amino Terminal ◽  
Expression Activity ◽  
Efficient Expression ◽  
Carboxyl Terminal ◽  
Domain I

ABSTRACTBacillus thuringiensisCry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic toEscherichia colicells, since thecry1Apromoter that drives its expression inB. thuringiensishas readthrough expression activity inE. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of thecry3Apromoter region to drive its expression inB. thuringiensiswithout expression inE. colicells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals inB. thuringiensiscells that were not soluble at pH 10.5 and showed no toxicity toPlutella xylostellalarvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity toP. xylostella. These results show that combining thecry3Apromoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins inB. thuringiensis.

scholarly journals One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

2015 ◽  
Vol 81 (11) ◽  
pp. 3648-3654 ◽  
Author(s):  
Ryotaro Hara ◽  
Masashi Nakano ◽  
Kuniki Kino
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Maximum Yield ◽  
Test Tube ◽  
Efficient Production ◽  
Scale Production ◽  
Deficient Mutant ◽  
One Pot ◽  
Content Type ◽  
E Coli

ABSTRACTWe developed a novel process for efficient synthesis ofl-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation ofl-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesizel-THA. By using these recombinant enzymes,l-THA was obtained byl-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube.l-THA was obtained in a small amount with a molar yield of 0.076% by using intactEscherichia coliexpressing theasnOgene, and thus, two asparaginase-deficient mutants ofE. coliwere investigated. A remarkably increasedl-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of theasnOgene was enhanced by using the T7 promoter inE. coliinstead of thelacpromoter, thel-THA yield was significantly increased to 92%. By using a combination of theE. coliasparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently,l-THA was successfully obtained froml-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production ofl-THA.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

2014 ◽  
Vol 80 (12) ◽  
pp. 3576-3584 ◽  
Author(s):  
Gaoyan Wang ◽  
David C. Manns ◽  
John J. Churey ◽  
Randy W. Worobo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Gene Cluster ◽  
Expression Vector ◽  
Expression System ◽  
Structural Genes ◽  
Cry Protein ◽  
Content Type ◽  
Thurincin H

ABSTRACTThurincin H is an antimicrobial peptide produced byBacillus thuringiensisSF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1,thnA2, andthnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designatedB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter,thnA1, and a Cry protein terminator into theEscherichia coli-B. thuringiensisshuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in thethnA1gene, were generated and separately transformed intoB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities ofB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3carrying different pGW133 variants against three different indicator strains were subsequently compared.

scholarly journals Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

2016 ◽  
Vol 82 (21) ◽  
pp. 6414-6422 ◽  
Author(s):  
Ryuki Miyauchi ◽  
Chiho Ono ◽  
Takashi Ohnuki ◽  
Yoichiro Shiba
Keyword(s):  
Genetic Engineering ◽  
Heterologous Expression ◽  
Production System ◽  
Therapeutic Potential ◽  
Biosynthetic Gene Cluster ◽  
Efficient Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
E Coli ◽  
Glycosidase Inhibitor

ABSTRACTThe fungusThelonectria discophoraSANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified fromT. discophoracultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster inT. discophorawas identified from the contiguous genome sequence around thenecCgene. Gene disruption and complementation studies and heterologous expression of the gene showed thatnecA,necB, andnecCcould be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinantEscherichia colicoexpressing thenecA,necB, andnecCgenes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering.IMPORTANCEIminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine inThelonectria discophoraSANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinantE. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

scholarly journals Efficient Expression of Soluble Recombinant Protein Fused with Core-Streptavidin in Bacterial Strain with T7 Expression System

2020 ◽  
Vol 3 (4) ◽  
pp. 82
Author(s):  
Ammar Tarar ◽  
Esmael M. Alyami ◽  
Ching-An Peng
Keyword(s):  
Fusion Protein ◽  
Thymidine Phosphorylase ◽  
Expression System ◽  
Model System ◽  
Carcinoma Cells ◽  
T7 Promoter ◽  
E Coli ◽  
Efficient Expression ◽  
T7 Expression System ◽  
Soluble Recombinant Protein

The limited amount of fusion protein transported into cytosol milieu has made it challenging to obtain a sufficient amount for further applications. To avoid the laborious and expensive task, T7 promoter-driving pET-30a(+) coding for chimeric gene of thymidine phosphorylase and core streptavidin as a model system was constructed and transformed into a variety of E. coli strains with T7 expression system. Our results demonstrated that the pET-30a(+)-TP-coreSA/Lemo21(DE3) system is able to provide efficient expression of soluble TP-coreSA fusion protein for purification. Moreover, the eluted TP-coreSA fusion protein tethered on biotinylated A549 carcinoma cells could effectively eliminate these malignant cells after administrating prodrug 5′-DFUR.

scholarly journals Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

2013 ◽  
Vol 79 (19) ◽  
pp. 6033-6039 ◽  
Author(s):  
Toshiki Furuya ◽  
Mika Hayashi ◽  
Kuniki Kino
Keyword(s):  
Escherichia Coli ◽  
Soluble Fraction ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
Functional Expression ◽  
Content Type ◽  
E Coli ◽  
Efficient Expression ◽  
Coupling Protein ◽  
Binuclear Iron

ABSTRACTBacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly inEscherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD inEscherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of themimABCDgene expression inE. colirevealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction ofE. colicells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformedE. colicells inactive. We found that the following factors are important for functional expression of MimABCD inE. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble inE. colicells, and the optimization of themimDnucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed inE. coli. The strategy described here should be generally applicable to theE. coliexpression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

scholarly journals Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

2021 ◽  
Author(s):  
Neeraj Kapoor ◽  
Esther Ndungo ◽  
Lucy Pill ◽  
Girmay Desalegn ◽  
Aym Berges ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Expression System ◽  
Efficient Production ◽  
Biophysical Characterization ◽  
Antimicrobial Drug Resistance ◽  
E Coli ◽  
Key Points ◽  
Sds Page ◽  
A Cell ◽  
Conventional Cell

Abstract Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

scholarly journals Production of Ophthalmic Acid Using EngineeredEscherichia coli

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Tomokazu Ito ◽  
Maiko Tokoro ◽  
Ran Hori ◽  
Hisashi Hemmi ◽  
Tohru Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Synthetic Medium ◽  
Product Inhibition ◽  
Efficient Production ◽  
Biosynthetic Enzymes ◽  
Phosphate Binding ◽  
Content Type ◽  
Food Industries ◽  
E Coli ◽  
Pharmaceutical Application

ABSTRACTOphthalmic acid (OA;l-γ-glutamyl-l-2-aminobutyryl-glycine) is an analog of glutathione (GSH;l-γ-glutamyl-l-cysteinyl-glycine) in which the cysteine moiety is replaced byl-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineeredEscherichia colicells.yggS-deficientE. coli, which lacks the highly conserved pyridoxal 5′-phosphate-binding protein YggS and naturally accumulates OA, was selected as the starting strain. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletion ofmiaAandridAwith the aim of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 μmol/liter of OA in 24 h without the formation of by-products in a minimal synthetic medium containing 1 mM each glutamate, 2-aminobutyrate, and glycine.IMPORTANCEOphthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would allow us to expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA usingEscherichia colicells in which OA biosynthetic enzymes and degradation enymes were engineered. We also showed that unique approaches, including the use of a ΔyggSmutant as a starting strain, the establishment of an S495F mutation in GshA, and the deletion ofridAormiaA, facilitated the efficient production of OA inE. coli.

scholarly journals Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

2003 ◽  
Vol 185 (7) ◽  
pp. 2178-2186 ◽  
Author(s):  
Anindya S. Ghosh ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Globular Domain ◽  
Morphological Effect ◽  
E Coli ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Domain I

ABSTRACT Penicillin binding protein (PBP) 5, a dd-carboxypeptidase that removes the terminal d-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp218 and Lys219 residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

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