Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them

1999 ◽  
Vol 65 (11) ◽  
pp. 5158-5162 ◽  
Author(s):  
Hiroaki Iwaki ◽  
Yoshie Hasegawa ◽  
Masahiro Teraoka ◽  
Tai Tokuyama ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Expression System ◽  
Transcriptional Activator ◽  
Fold Increase ◽  
Gene Arrangement ◽  
Catabolic Pathway ◽  
Gene Encoding ◽  
Cyclohexanone Monooxygenase ◽  
E Coli ◽  
Molecular Masses

ABSTRACT We identified chnR, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression ofchnB, the structural gene for cyclohexanone monooxygenase (CHMO) in Acinetobacter sp. strain NCIMB 9871. The gene sequence of chnE, which encodes an NADP+-linked 6-oxohexanoate dehydrogenase, the enzyme catalyzing the fifth step of cyclohexanol degradation, was also determined. The gene arrangement ischnB-chnE-chnR. The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system. Inducible expression of cloned chnB inEscherichia coli depended upon the presence ofchnR. A transcriptionalchnB::lacZ fusion experiment revealed that cyclohexanone induces chnB expression in E. coli, in which a 22-fold increase in activity was observed.

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

scholarly journals Synthesis of the novel transporter YdhC, is regulated by the YdhB transcription factor controlling adenosine and adenine uptake

2020 ◽  
Author(s):  
Irina A. Rodionova ◽  
Ye Gao ◽  
Anand Sastry ◽  
Reo Yoo ◽  
Dmitry A. Rodionov ◽  
...  
Keyword(s):  
Nitrogen Sources ◽  
Gene Arrangement ◽  
The Novel ◽  
Rna Seq ◽  
Gene Encoding ◽  
E Coli ◽  
Binding Sequence ◽  
Predicted Binding Site ◽  
Lysr Family ◽  
Mg1655 Strain

AbstractThe YdhB transcriptional factor, re-named here AdnB, homologous to the allantoin regulator, AllS, was shown to regulate ydhC gene expression in Escherichia coli, which is divergently transcribed from adnB, and this gene arrangement is conserved in many Protreobacteria. The predicted consensus DNA binding sequence for YdhB is also conserved in Entrobacterial genomes. RNA-seq data confirmed the activation predicted due to the binding of AdnB as shown by Chip-Exo results. Fluorescent polarization experiments revealed binding of YdhB to the predicted binding site upstream of ydhC in the presence of 0.35 mM adenine, but not in its absence. The E. coli MG1655, strain lacking the ydhB gene, showed a lower level of ydhC mRNA in cells grown in M9-glucose supplemented with 2 mM adenosine. Adenosine and adenine are products of purine metabolism and provide sources of ammonium for many organisms. They are utilized under nitrogen starvation conditions as single nitrogen sources. Deletion of either the ydhC or the ydhB gene leads to a substantially decreased growth rate for E. coli in minimal M9 medium with glycerol as the carbon source and adenosine or adenine as the single nitrogen source. The ydhC mutant showed increased resistance to Paromomycine, Sulfathiazole and Sulfamethohazole using Biolog plates. We provide evidence that YdhB, (a novel LysR family regulator) activates expression of the ydhC gene, encoding a novel adenosine/adenine transporter in E. coli. The YdhB binding consensus for different groups of Enterobacteria was predicted.

scholarly journals Secretory expression of cyclohexanone monooxygenase by methylotrophic yeast for efficient omeprazole sulfide bio-oxidation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ya-Jing Li ◽  
Yu-Cong Zheng ◽  
Qiang Geng ◽  
Feng Liu ◽  
Zhi-Jun Zhang ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
Enantiomeric Excess ◽  
Catalytic Efficiency ◽  
Methylotrophic Yeast ◽  
Fold Increase ◽  
Dry Weight ◽  
Cyclohexanone Monooxygenase ◽  
E Coli ◽  
A Cell ◽  
Omeprazole Sulfide

AbstractProchiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with  >  99% enantiomeric excess.

scholarly journals Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

2004 ◽  
Vol 70 (6) ◽  
pp. 3292-3297 ◽  
Author(s):  
Gerard M. Gibbs ◽  
Barrie E. Davidson ◽  
Alan J. Hillier
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
Scale Production ◽  
Gene Encoding ◽  
Strong Activity ◽  
E Coli ◽  
Large Scale Production ◽  
Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

scholarly journals Genes from Pseudomonas sp. Strain BS Involved in the Conversion of l-2-Amino-Δ2-Thiazolin-4-Carbonic Acid to l-Cysteine

2002 ◽  
Vol 68 (5) ◽  
pp. 2179-2187 ◽  
Author(s):  
Toshikazu Shiba ◽  
Kohji Takeda ◽  
Misako Yajima ◽  
Makoto Tadano
Keyword(s):  
Carbonic Acid ◽  
Open Reading Frames ◽  
Synthetic Activity ◽  
Gene Products ◽  
Gene Encoding ◽  
E Coli ◽  
Cysteine Synthesis ◽  
Molecular Masses ◽  
Dna Fragment ◽  
Derivatives Of

ABSTRACT dl-2-amino-Δ2-thiazolin-4-carbonic acid (dl-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of l-ATC to l-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert l-ATC to cysteine via N-carbamyl-l-cysteine (l-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has l-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the l-cysteine synthesis from dl-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of l-ATC to l-NCC and the conversion of l-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with l-N-carbamoylases. Since both enzymes can only catalyze the l-specific conversion from l-ATC to l-NCC or l-NCC to l-cysteine, it is thought that atcB and atcC encode l-ATC hydrolase and N-carbamyl-l-cysteine amidohydrolase, respectively.

scholarly journals Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Trang Thi Phuong Phan ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fusion Protein ◽  
Expression System ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

Secretory Expression of Cyclohexanone Monooxygenase by Methylotrophic Yeast for Efficient Omeprazole Sulfide Bio-oxidation

2021 ◽  
Author(s):  
Ya-Jing Li ◽  
Yu-Cong Zheng ◽  
Qiang Geng ◽  
Feng Liu ◽  
Zhi-Jun Zhang ◽  
...  
Keyword(s):  
Fermentation Broth ◽  
Enantiomeric Excess ◽  
Catalytic Efficiency ◽  
Methylotrophic Yeast ◽  
Fold Increase ◽  
Dry Weight ◽  
Cyclohexanone Monooxygenase ◽  
E Coli ◽  
A Cell ◽  
Omeprazole Sulfide

Abstract Prochiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with >99% enantiomeric excess.

scholarly journals Improved production of the non-native cofactor F420 in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mihir V. Shah ◽  
Hadi Nazem-Bokaee ◽  
James Antoney ◽  
Suk Woo Kang ◽  
Colin J. Jackson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Carbon Sources ◽  
Fold Increase ◽  
High Yield ◽  
Antibiotic Biosynthesis ◽  
Bacterial Strains ◽  
E Coli ◽  
A Genome

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

scholarly journals Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ida Lauritsen ◽  
Pernille Ott Frendorf ◽  
Silvia Capucci ◽  
Sophia A. H. Heyde ◽  
Sarah D. Blomquist ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Metabolic Flux ◽  
Global Gene Expression ◽  
Catabolic Pathway ◽  
Gene Encoding ◽  
Adaptive Mutations ◽  
E Coli ◽  
Dna And Rna ◽  
Pathway Expression ◽  
Sequential Generation

AbstractThe evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

scholarly journals Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

2012 ◽  
Vol 78 (16) ◽  
pp. 5831-5838 ◽  
Author(s):  
Yun Zhang ◽  
Xiuling Shang ◽  
Shujuan Lai ◽  
Guoqiang Zhang ◽  
Yong Liang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Expression System ◽  
Carbon Sources ◽  
Inducible Expression ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Inducible Expression System ◽  
Green Fluorescent

ABSTRACTCorynebacterium glutamicumis currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species uselac-derived promoters fromEscherichia colithat exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains thel-arabinose regulator AraC, thePBADpromoter from thearaBADoperon, and thel-arabinose transporter AraE, all of which are derived fromE. coli. The level of induciblePBAD-based expression could be modulated over a wide concentration range from 0.001 to 0.4%l-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control ofPBADpromoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case inE. coli,PBADinduction was not significantly affected in the presence of different carbon sources inC. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of theodhIgene fromC. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering ofC. glutamicum.

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