scholarly journals The efficiency for recombineering is dependent on the source of the phage recombinase function unit

2019 ◽  
Author(s):  
Yizhao Chang ◽  
Qian Wang ◽  
Tianyuan Su ◽  
Qingsheng Qi
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Corynebacterium Glutamicum ◽  
E Coli ◽  
As Species ◽  
Function Unit ◽  
New Ideas ◽  
Species Evolution ◽  
Low Performance ◽  
Low Efficiency

AbstractPhage recombinase function units (PRFUs) such as lambda-Red or Rac RecET have been proven to be powerful genetic tools in the recombineering of Escherichia coli. Studies have focused on developing such systems in other bacteria as it is believed that these PRFUs have limited efficiency in distant species. However, how the species evolution distance relates to the efficiency of recombineering remains unclear. Here, we present a thorough study of PRFUs to find features that might be related to the efficiency of PRFUs for recombineering. We first identified 59 unique sets of PRFUs in the genus Corynebacterium and classified them based on their sequence as well as secondary structure similarities. Then both PRFUs from this genus and other bacteria were chosen for experiment based on sequential and secondary structure similarity as well as species distance. These PRFUs were compared for their ability in mediating recombineering with oligo or double-stranded DNA substrates in Corynebacterium glutamicum. We demonstrate that the source of the PRFU is more critical than species distance for the efficiency of recombineering. Our work will provide new ideas for efficient recombineering using PRFUs.ImportanceRecombineering using phage recombinase function units (PRFUs) such as lambda-Red or Rac RecET has gained success in Escherichia coli, while efforts applying these systems in other bacteria were limited by the efficiency. It is believed that the species distance may be a major reason for the low efficiency. In this study, however, we showed that it is the source of PRFU rather than the species distance that matters for the recombineering in Corynebacterium glutamicum. Besides, we also showed that the lower transformation efficiency in other bacteria compared to that of E. coli could be a major reason for the low performance of heterogeneously expressed RecET. These findings will be helpful for the recombineering using PRFUs.

Co-Expression of Threonine Dehydratase and Acetolactate Synthase in Escherichia Coli for L-Isoleucine Production

2014 ◽  
Vol 989-994 ◽  
pp. 997-1002 ◽  
Author(s):  
Jian Wang ◽  
Jia Kai Sun ◽  
Qing Yang Xu
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Carbon Flux ◽  
Batch Fermentation ◽  
Acetolactate Synthase ◽  
Fed Batch ◽  
Threonine Dehydratase ◽  
E Coli ◽  
Fed Batch Fermentation ◽  
Fermentation Period

Metabolic engineering ofCorynebacterium glutamicumhas sought to divert carbon into L-isoleucine. However, the fermentation period of this strain is long. TheC.glutamicumYILW strain (LeuL, AHVr, SGr, Leu-MEr) was previously derived by repeated compound mutagenesis which could accumulate 20.2 g/L L-isoleucine in a 5-L jar fermentor. Overexpression of the threonine dehydratase gene (ilvA) fromCorynebacterium glutamicumYILW and coexpression of threonine dehydratase and acetolactate synthase (ilvBN) from it were employed to divert carbon flux toward L-isoleucine. The strainE. coliTRFC with the expression ofilvA could accumulate L-isoleucine of 6.8 g/L without accumulation of any L-threonine by fed-batch fermentation in a 5-L jar fermentor. However, the production of L-isoleucine by the strainE.coliTRFC with the co-expression ofilvA andilvBN was decreased by 19.1%, and the production of L-valine was increased by 40% compared with that ofE. coliTRFC with the expression ofilvA.

Overexpression of the ftsZ gene from Corynebacterium glutamicum (Brevibacterium lactofermentum) in Escherichia coli

2005 ◽  
Vol 51 (1) ◽  
pp. 85-89 ◽  
Author(s):  
María Pilar Honrubia-Marcos ◽  
Angelina Ramos ◽  
José A Gil
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyclonal Antibodies ◽  
T7 Promoter ◽  
Brevibacterium Lactofermentum ◽  
E Coli ◽  
Ftsz Protein ◽  
Pure Protein ◽  
Ftsz Gene ◽  
Dna Deletions

Our goal in this work was to overexpress the essential cell division FtsZ protein from Corynebacterium glutamicum (Brevibacterium lactofermentum) (FtsZCG) in Escherichia coli to produce anti-FtsZCG polyclonal antibodies. Previous results from our laboratory showed that ftsZCG was not expressed in E. coli in a sufficient amount to purify FtsZCG. However, when ftsZCG (without upstream sequences) was transcriptionally fused to the T7 promoter, different truncated FtsZCG proteins (28–32 kDa) were overexpressed in E. coli, and in all cases, stop codons were created because of DNA deletions or rearrangements. Nevertheless, we were able to overexpress and purify an N-terminally hexa-His-tagged FtsZCG from uninduced E. coli cells carrying a pET-28a(+) derivative, yielding about 5 mg of 98% pure protein per 100-mL culture.Key words: Brevibacterium lactofermentum, Corynebacterium glutamicum, FtsZ overexpresssion, hexa-His-tagged FtsZ.

scholarly journals Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

2004 ◽  
Vol 70 (7) ◽  
pp. 3845-3854 ◽  
Author(s):  
Leandro Padilla ◽  
Susanne Morbach ◽  
Reinhard Krämer ◽  
Eduardo Agosin
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Corynebacterium Glutamicum ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
E Coli ◽  
Wide Range ◽  
The Impact ◽  
Synthetic Operon ◽  
Trehalose Synthesis

ABSTRACT Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

scholarly journals Performa Tryptone Bile X-Glucuronide (TBX) yang disuplementasikan dengan Cefotaxime sebagai Medium Selektif Untuk Skrining ESBL-E.coli dari Sampel Lingkungan

2021 ◽  
Vol 49 (1) ◽  
pp. 1-8
Author(s):  
Tati Febrianti ◽  
Sundari Nursofiah ◽  
Novi Amalia ◽  
Dwi Febriyana ◽  
Ratih Dian Saraswati ◽  
...  
Keyword(s):  
Waste Water ◽  
Escherichia Coli ◽  
Selective Medium ◽  
Environmental Sampling ◽  
E Coli ◽  
Sampling Locations ◽  
As Species ◽  
Laboratory Examinations ◽  
Filtration Stage ◽  
Disk Test

Identification of ESBL-E.coli from environment without selective medium will be challenging to do considering that E.coli mixes with various other microorganisms in the environment. This study aimed to determine the performance of TBX Agar supplemented with Cefotaxime as a selective medium for ESBL-E. coli screening from 138 water samples of environmental sampling obtained from rivers, open sewers in the market, poultry slaughterhouses and hospital waste water inlets and outlets around Jakarta. Laboratory examinations were carried out through the filtration stage, inoculation on the TBX Agar supplemented with Cefotaxime medium as well as species confirmation and ESBL with the indol test and double-disk test. The results showed that 87.08% (40-100%) of suspect colonies growing on TBX Agar supplemented with Cefotaxime medium were confirmed as E.coli and 82.51% (12-100%) were confirmed as ESBL-E.coli. However, there was no correlation between TBX Agar supplemented with Cefotaxime performance and sampling locations. Based on the results of the study, it can be concluded that the TBX supplemented with Cefotaxime medium can be used for ESBL-E.coli screening in the environment, but further confirmation is needed using the indole and double-disk tests. Keywords: Escherichia coli, ESBL, TBX Agar suplemented with Cefotaxime Abstrak Identifikasi ESBL-E.coli tanpa medium selektif akan sangat sulit dilakukan pada sampel lingkungan mengingat ESBL-E.coli bercampur dengan berbagai mikroorganisme lainnya. Tujuan penelitian ini yaitu untuk mengetahui performa TBX Agar yang disuplementasi Cefotaxime sebagai medium selektif untuk skrining ESBL-E.coli. Sampel penelitian sebanyak 138 sampel air lingkungan yang diambil dari sungai, saluran pembuangan terbuka di pasar, rumah pemotongan hewan unggas (RPHU) serta inlet dan outlet limbah rumah sakit di sekitar Jakarta. Pemeriksaan laboratorium melalui tahapan filtrasi, inokulasi pada medium TBX Agar dengan suplementasi Cefotaxime serta konfirmasi spesies dan ESBL dengan uji indol dan double-disk test. Hasil menunjukkan bahwa koloni tersangka yang tumbuh pada medium TBX Agar dengan suplementasi Cefotaxime sebanyak 87,08% (40-100%) terkonfirmasi sebagai E.coli dan 82,51% (12-100%) terkonfirmasi sebagai ESBL-E.coli. Namun, tidak ada hubungan antara performa TBX Agar dengan suplementasi Cefotaxime dengan lokasi pengambilan sampel. Berdasarkan hasil penelitian dapat disimpulkan bahwa medium TBX Agar dengan suplementasi Cefotaxime cocok digunakan untuk skrining ESBL-E.coli di lingkungan namun tetap diperlukan konfirmasi lanjut menggunakan uji indol dan double disk test. Kata kunci: Escherichia coli, ESBL, TBX Agar suplemented with Cefotaxime

scholarly journals Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

2003 ◽  
Vol 69 (1) ◽  
pp. 170-176 ◽  
Author(s):  
Katsunori Mizoguchi ◽  
Masatomo Morita ◽  
Curt R. Fischer ◽  
Masatoshi Yoichi ◽  
Yasunori Tanji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Continuous Culture ◽  
Dilution Rate ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Escape Mutant ◽  
E Coli ◽  
Escape Mutants ◽  
Low Efficiency

ABSTRACT The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h−1 and by 3 orders of magnitude at a lower dilution rate (0.327 h−1). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h−1 and persisted until the end of the experiment (∼200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

scholarly journals Engineering Escherichia coli coculture systems for the production of biochemical products

2015 ◽  
Vol 112 (27) ◽  
pp. 8266-8271 ◽  
Author(s):  
Haoran Zhang ◽  
Brian Pereira ◽  
Zhengjun Li ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
Scale Up ◽  
Microbial Consortia ◽  
Biosynthetic Pathways ◽  
Muconic Acid ◽  
E Coli ◽  
Sugar Mixture ◽  
Low Efficiency ◽  
Valuable Compounds ◽  
High Level

Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.

Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum

1999 ◽  
Vol 45 (10) ◽  
pp. 885-890 ◽  
Author(s):  
Min-Ah Han ◽  
Heung-Shick Lee ◽  
Choong-Ill Cheon ◽  
Kyung-Hee Min ◽  
Myeong-Sok Lee
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Auxotrophic Mutant ◽  
Gene Products ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Dehydroquinate Synthase

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.Key words: Corynebacterium glutamicum, aromatic amino acid biosynthetic gene, dehydroquinate synthase, aroB gene.

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

scholarly journals Lac+plasmids are responsible for the strong lactose-positive phenotype found in many strains ofKlebsiellaspecies

1973 ◽  
Vol 22 (3) ◽  
pp. 329-333 ◽  
Author(s):  
E. C. R. Reeve ◽  
J. A. Braithwaite
Keyword(s):  
Escherichia Coli ◽  
Bacterial Strain ◽  
Common Ancestor ◽  
Galactosidase Activity ◽  
Macconkey Agar ◽  
New Host ◽  
E Coli ◽  
Compatibility Group ◽  
Diverse Origin ◽  
Low Efficiency

SUMMARYA variety ofKlebsiellastrains examined all show either a strong (ML+) or a weak (ML−/+) lactose-positive phenotype on MacConkey agar. ML+Klebsiellae have about 10 times the β-galactosidase activity of ML−/+strains in cultures both induced and not induced for this enzyme. Of 14 ML+strains of diverse origin tested, at least 13 carry alacoperon on a plasmid which can be transferred toEscherichia coli. The seven plasmids so far studied in detail all belong to the F compatibility group but are unable to promote their own transfer. To explain these results it is suggested that theKlebsiellagroup derive from a common ancestor with a chromosomallacoperon of low efficiency, which was made good by the acquisition of alacoperon from another bacterial strain, probablyE. coli: the newlacgenes remained as a plasmid, possibly because they could not be integrated in the new host.

scholarly journals A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes

1976 ◽  
Vol 160 (3) ◽  
pp. 505-519 ◽  
Author(s):  
R A Cox ◽  
W Hirst
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Ribonucleic Acid ◽  
Magnesium Ions ◽  
Base Pairs ◽  
E Coli ◽  
Ribosomal Ribonucleic Acid ◽  
The Stability ◽  
Rabbit Reticulocytes

Mg2+ was shown to affect the conformation of rRNA over the range of 0.03-1.2M-KCl. The species studies were Escherichia coli S-rRNA and L-rRNA (the RNA moieties of the smaller and larger subribosomal particles respectively) and rabbits S-rRNA and L-rRNA. 2. The addition of Mg2+ to rRNA in reconstitution buffer (0.35M-KCl0.01M-Tris/HCl, pH7.2) at 20° C let to an increase in bihelical secondary structure through the formation of additional (mainly A-U) base-pairs (e.g. an additional approx. 58 A-U base-pairs per molecule of E. coli S-rRNA as judged by u.v. difference spectrophotometry…

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