scholarly journals Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Erika Yoshida ◽  
Motoki Kojima ◽  
Munenori Suzuki ◽  
Fumio Matsuda ◽  
Kazutaka Shimbo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Proteome Analysis ◽  
Product Formation ◽  
Industrial Biotechnology ◽  
Enzyme Expression ◽  
Biotechnological Production ◽  
Expression Ratio ◽  
Single Strain ◽  
E Coli

Abstract(−)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (−)-carvone is desirable. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (−)-carvone production from 100 mg/L (−)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.

scholarly journals Increased Carvone Production in Escherichia Coli by Balancing Expression of Limonene Conversion Enzymes Through Targeted QconCAT Proteome Analysis

2021 ◽  
Author(s):  
Erika Yoshida ◽  
Motoki Kojima ◽  
Munenori Suzuki ◽  
Fumio Matsuda ◽  
Akiko Onuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Proteome Analysis ◽  
Product Formation ◽  
Industrial Biotechnology ◽  
Expression Ratio ◽  
Environment Friendly ◽  
Single Strain ◽  
E Coli ◽  
Standard Protein

Abstract Background Natural mint flavor is produced by extraction from mint, which is not efficient enough to make it environment friendly process. (−)-Carvone is the monoterpenoid with key flavor of spearmint, and there has been an attempt to produce (−)-carvone by recombinant Escherichia coli. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in E. coli independently, the yield of (−)-carvone was low in the previous study.ResultsWe have found a by-product formation when cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in single cell. These by-products were determined as dihydrocarveol and dihydrocarvone. We hypothesized that the enzymatic kinetics and the expression levels of P450 and CDH are quite different in E. coli. Therefore, two strains independently expressing P450/CPR and CDH were mixed with different mixing ratio, confirming increase in carvone production and decrease in by-product formation when CDH input was reduced. To determine the optimum balance of enzyme expressions, proteome analysis quantification concatemer (QconCAT) method to quantify P450, CPR, and CDH was developed. Using the QconCAT standard protein that was artificially created by concatenating the tryptic peptides, the ratio between P450 and CDH was calculated, and their optimum ratio to maximize (−)-carvone production was shown. Then, a single strain expressing both P450/CPR and CDH was constructed to imitate the superior expression ratio. The upgraded strain showed 15-fold improvement compared to the initial strain, showing 44 ± 6.3 mg/L of (−)-carvone production from 100 mg/L (−)-limonene as a starting substrate.ConclusionsImproved expression balance of P450 and CDH in recombinant E. coli increased the (−)-carvone production using (−)-limonene as the direct substrates by the whole-cell biocatalysis, showing approximately 150 times higher titer than previous report. Our study showed the usefulness of proteome analysis QconCAT method in the strain development for industrial biotechnology field.

scholarly journals A Statistical Model for Assessing Sample Size for Bacterial Colony Selection: A Case Study of Escherichia Coli and Avian Cellulitis

2000 ◽  
Vol 12 (2) ◽  
pp. 118-125 ◽  
Author(s):  
Randall S. Singer ◽  
Wesley O. Johnson ◽  
Joan S. Jeffrey ◽  
Richard P. Chin ◽  
Tim E. Carpenter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Statistical Model ◽  
Clinical Sample ◽  
Agar Plate ◽  
Sample Sizes ◽  
Simple Task ◽  
Single Strain ◽  
Bacterial Colony ◽  
E Coli

A general problem for microbiologists is determining the number of phenotypically similar colonies growing on an agar plate that must be analyzed in order to be confident of identifying all of the different strains present in the sample. If a specified number of colonies is picked from a plate on which the number of unique strains of bacteria is unknown, assigning a probability of correctly identifying all of the strains present on the plate is not a simple task. With Escherichia coli of avian cellulitis origin as a case study, a statistical model was designed that would delineate sample sizes for efficient and consistent identification of all the strains of phenotypically similar bacteria in a clinical sample. This model enables the microbiologist to calculate the probability that all of the strains contained within the sample are correctly identified and to generate probability-based sample sizes for colony identification. The probability of cellulitis lesions containing a single strain of E. coli was 95.4%. If one E. coli strain is observed out of three colonies randomly selected from a future agar plate, the probability is 98.8% that only one strain is on the plate. These results are specific for this cellulitis E. coli scenario. For systems in which the number of bacterial strains per sample is variable, this model provides a quantitative means by which sample sizes can be determined.

scholarly journals Cotrimoxazole-ResistantEscherichia coliBacteremia in Neutropenic Patients at a Regional Oncology Hospital

1992 ◽  
Vol 3 (1) ◽  
pp. 14-18
Author(s):  
Daniel B Gregson ◽  
Anne G Matlow ◽  
Andrew E Simor ◽  
Peter G Tuffnell ◽  
Donald E Low ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Marrow Transplant ◽  
Single Strain ◽  
Gram Negative ◽  
E Coli ◽  
Staff Members ◽  
Cotrimoxazole Prophylaxis ◽  
Neutropenic Patients ◽  
Chemotherapy Induced Neutropenia ◽  
High Level

In a regional oncology hospital using cotrimoxazole (trimethoprim-sulphamethoxazole) prophylaxis during chemotherapy-induced neutropenia, a single strain ofEscherichia coli(indole negative) caused 15 of 27 episodes of Gram-negative rod bacteremia in 1987, and four of 32 such episodes in 1988. This biotype had not been recovered in 1986. Investigations during this ‘outbreak’ of bacteremias revealed enteric colonization with this strain ofE coliin 37% of patients on leukemia or bone marrow transplant wards and in several staff members in July 1987. In 1988, 11 of 32 Gram-negative rod bacteremias were secondary to other strains of indole positiveE coliof several different biotypes and plasmid profiles. Indole negative strains all exhibited low level trimethoprim resistance, whereas indole positive strains which subsequently appeared exhibited high level trimethoprim resistance. Failure of cotrimoxazole prophylaxis was initially due to the clonal dissemination of a single strain ofE coliwithin the institution, with the subsequent appearance of multipleE colistrains with probable differing genetic bases for their resistance.

Survival of Escherichia coli O157:H7 in Ground-Beef Patties during Storage at 2, −2, 15 and then −2°C, and −20°C†

1999 ◽  
Vol 62 (11) ◽  
pp. 1243-1247 ◽  
Author(s):  
SUSAN E. ANSAY ◽  
KIM A. DARLING ◽  
CHARLES W. KASPAR
Keyword(s):  
Escherichia Coli ◽  
Frozen Storage ◽  
Ground Beef ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
Control Strain ◽  
Single Strain ◽  
E Coli ◽  
Beef Patties ◽  
Significant Difference

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2°C for 4 weeks, −2°C for 4 weeks, 15°C for 4 h and then −2°C for 4 weeks, and −20°C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 105 CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log10 CFU/g, and pathogen numbers declined 1.9 log10 CFU/g when patties were stored for 4 weeks at 2°C. When patties were stored at −2°C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log10 CFU/g, respectively. Patties stored at 15°C for 4 h prior to storage at −2°C for 4 weeks resulted in 1.6 and 2.7 log10–CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15°C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (−20°C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log10–CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.

scholarly journals Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1972 ◽  
Vol 126 (5) ◽  
pp. 1081-1090 ◽  
Author(s):  
S. E. Halford ◽  
M. J. Schlesinger ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Steady State ◽  
Analytical Ultracentrifugation ◽  
Kinetic Studies ◽  
Product Formation ◽  
E Coli ◽  
Enzyme Subunits ◽  
Self Association ◽  
The Stability

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

scholarly journals Escherichia coliflagellar serotyping is as reliable as it has always been!

1987 ◽  
Vol 98 (2) ◽  
pp. 221-222 ◽  
Author(s):  
Frits Ørskov ◽  
Ida Ørskov ◽  
K. A. Bettelheim
Keyword(s):  
Escherichia Coli ◽  
Dna Analysis ◽  
Epidemiological Evidence ◽  
Single Strain ◽  
E Coli ◽  
Electrophoretic Patterns ◽  
Cast Doubt ◽  
The Stability ◽  
H Serotypes

In a recent paper in this Journal by Marshallet al.(1985), which described the bacterial endonuclease DNA analysis (BRENDA) of severalEscherichia coli0126 strains, the following statement was found in the summary: ‘The isolates from the outbreak produced indistinguishable DNA electrophoretic patterns in spite of their assignment to seven different H serotypes… These results support the epidemiological evidence that a single-strain outbreak had occurred, and they cast doubt on the value of H typing for this particular investigation.’ The same 0126 strains that were enterotoxigenic (ST) were treated in two earlier papers (Bettelheim & Reeve, 1982; Bettelheim, 1984) and also on these two earlier occasions the authors raised doubt about the stability ofE. coliH typing results.

scholarly journals In Silico Design for Systems-Based Metabolic Engineering In Escherichia Coli for The Bioconversion of Aerobic Glycerol to Succinic Acid

2020 ◽  
Author(s):  
Albert Enrique Tafur Rangel ◽  
Wendy Lorena Rios Guzman ◽  
Carmen Elvira Ojeda Cuella ◽  
Daissy Esther Mejia Perez ◽  
Ross Carlson ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Succinic Acid ◽  
In Silico ◽  
Rational Design ◽  
Industrial Biotechnology ◽  
E Coli ◽  
Cell Factories ◽  
Transcriptomics Data

Abstract BackgroundGlycerol has become an interesting carbon source for industrial processes as consequence of the biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. Selecting the appropriate metabolic targets to build efficient cell factories and maximize the desired chemical production in as little time as possible is a major challenge in industrial biotechnology. The engineering of microbial metabolism following rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, to be proficient is needed known in advance the effects of gene deletions.ResultsAn in silico experiment was performed to model and understand the effects of metabolic engineering over the metabolism by transcriptomics data integration. In this study, systems-based metabolic engineering to predict the metabolic engineering targets was used in order to increase the bioconversion of glycerol to succinic acid by Escherichia coli. Transcriptomics analysis suggest insights of how increase the glycerol utilization of the cell for further design efficient cell factories. Three models were used; an E. coli core model, a model obtained after the integration of transcriptomics data obtained from E. coli growing in an optimized culture media, and a third one obtained after integration of transcriptomics data obtained from E. coli after adaptive laboratory evolution experiments. A total of 2402 strains were obtained from these three models. Fumarase and pyruvate dehydrogenase were frequently predicted in all the models, suggesting that these reactions are essential to increasing succinic acid production from glycerol. Finally, using flux balance analysis results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of importance of each knockout’s (feature’s) contribution.ConclusionsThe combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed versatile molecular mechanisms involved in the utilization of glycerol. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work a guide platform for the selection/engineering of microorganisms for production of interesting chemical compounds.

Proteome Analysis of Virulence Factor Regulated by Autoinducer-2–like Activity in Escherichia coli O157:H7

2007 ◽  
Vol 70 (2) ◽  
pp. 300-307 ◽  
Author(s):  
YOUNGHOON KIM ◽  
SANGNAM OH ◽  
EUN YOUNG AHN ◽  
JEE-YOUNG IMM ◽  
SEJONG OH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Proteome Analysis ◽  
Protein Translation ◽  
Escherichia Coli O157 ◽  
Dependent Manner ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli ◽  
Autoinducer 2

Many pathogenic bacteria, including Escherichia coli O157:H7, can control gene expression in a cell density–dependent manner by producing small signaling molecules (autoinducers) in a process known as quorum sensing. In this study, the effects of the autoinducer-2–like activity on the expression of proteins, including virulence factors, in E. coli O157:H7 were characterized by proteomic analysis. Compared with the control, E. coli O157:H7 strains in the presence of autoinducer-2–like activity exhibited elevated virulence by more rapidly forming cell aggregates on epithelial cells and rapidly killing the nematode Caenorhabditis elegans, the surrogate host. Two-dimensional gel electrophoresis revealed 18 proteins that were upregulated by autoinducer-2–like activity and 4 proteins that were down-regulated. These proteins were further characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and are involved in the metabolic process, adaptation and protection, cell motility, secretion, envelope biogenesis, and protein translation. These results indicate that the newly identified proteins are associated with the control of virulence in E. coli O157:H7 and that these proteins can be potential targets for the development of antibiotics and other antimicrobial agents.

scholarly journals Fermentative production of short-chain fatty acids in Escherichia coli

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1513-1522 ◽  
Author(s):  
Alexandra R. Volker ◽  
David S. Gogerty ◽  
Christian Bartholomay ◽  
Tracie Hennen-Bierwagen ◽  
Huilin Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Short Chain Fatty Acids ◽  
Product Formation ◽  
Efficient Manner ◽  
E Coli ◽  
Odd Chain Fatty Acids ◽  
Coa Reductase ◽  
Dependent Pathway ◽  
Chain Fatty Acids

Escherichia coli was engineered for the production of even- and odd-chain fatty acids (FAs) by fermentation. Co-production of thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase and trans-enoyl-CoA reductase from a synthetic operon allowed the production of butyrate, hexanoate and octanoate. Elimination of native fermentation pathways by genetic deletion (ΔldhA, ΔadhE, ΔackA, Δpta, ΔfrdC) helped eliminate undesired by-products and increase product yields. Initial butyrate production rates were high (0.7 g l−1 h−1) but quickly levelled off and further study suggested this was due to product toxicity and/or acidification of the growth medium. Results also showed that endogenous thioesterases significantly influenced product formation. In particular, deletion of the yciA thioesterase gene substantially increased hexanoate production while decreasing the production of butyrate. E. coli was also engineered to co-produce enzymes for even-chain FA production (described above) together with a coenzyme B12-dependent pathway for the production of propionyl-CoA, which allowed the production of odd-chain FAs (pentanoate and heptanoate). The B12-dependent pathway used here has the potential to allow the production of odd-chain FAs from a single growth substrate (glucose) in a more energy-efficient manner than the prior methods.

scholarly journals Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

2014 ◽  
Vol 80 (21) ◽  
pp. 6828-6836 ◽  
Author(s):  
Yuta Miki ◽  
Yasuhisa Asano
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Culture Conditions ◽  
Culture Broth ◽  
Single Step ◽  
Content Type ◽  
E Coli ◽  
Pharmaceutical Industries ◽  
Aldoxime Dehydratase

ABSTRACTThe biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed inEscherichia coliutilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produceE,Z-phenylacetaldoxime (PAOx) froml-Phe was constructed inE. coliby introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase fromArabidopsis thaliana, yielding theE,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene fromBacillussp. strain OxB-1, yielding the PAN-producing transformant. TheE,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites ofE,Z-PAOx produced byE. colienzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion ofl-Phe toE,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression ofgroE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration ofE,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.

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