scholarly journals Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli

2021 ◽  
Vol 9 (7) ◽  
pp. 1520
Author(s):  
Minjae Han ◽  
Pyung Cheon Lee
Keyword(s):  
Escherichia Coli ◽  
Retinoic Acid ◽  
Acid Production ◽  
Early Stage ◽  
Fold Increase ◽  
Coli Strain ◽  
Microbial Production ◽  
Bacterial Strains ◽  
Culture Optimization ◽  
Β Carotene

Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15’-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5’-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid–producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli.

scholarly journals YdgG (TqsA) Controls Biofilm Formation in Escherichia coli K-12 through Autoinducer 2 Transport

2006 ◽  
Vol 188 (2) ◽  
pp. 587-598 ◽  
Author(s):  
Moshe Herzberg ◽  
Ian K. Kaye ◽  
Wolfgang Peti ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Acid Production ◽  
Polysialic Acid ◽  
Fold Increase ◽  
Uncharacterized Protein ◽  
Biofilm Thickness ◽  
Autoinducer 2 ◽  
Membrane Spanning ◽  
K 12

ABSTRACT YdgG is an uncharacterized protein that is induced in Escherichia coli biofilms. Here it is shown that deletion of ydgG decreased extracellular and increased intracellular concentrations of autoinducer 2 (AI-2); hence, YdgG enhances transport of AI-2. Consistent with this hypothesis, deletion of ydgG resulted in a 7,000-fold increase in biofilm thickness and 574-fold increase in biomass in flow cells. Also consistent with the hypothesis, deletion of ydgG increased cell motility by increasing transcription of flagellar genes (genes induced by AI-2). By expressing ydgG in trans, the wild-type phenotypes for extracellular AI-2 activity, motility, and biofilm formation were restored. YdgG is also predicted to be a membrane-spanning protein that is conserved in many bacteria, and it influences resistance to several antimicrobials, including crystal violet and streptomycin (this phenotype could also be complemented). Deletion of ydgG also caused 31% of the bacterial chromosome to be differentially expressed in biofilms, as expected, since AI-2 controls hundreds of genes. YdgG was found to negatively modulate expression of flagellum- and motility-related genes, as well as other known products essential for biofilm formation, including operons for type 1 fimbriae, autotransporter protein Ag43, curli production, colanic acid production, and production of polysaccharide adhesin. Eighty genes not previously related to biofilm formation were also identified, including those that encode transport proteins (yihN and yihP), polysialic acid production (gutM and gutQ), CP4-57 prophage functions (yfjR and alpA), methionine biosynthesis (metR), biotin and thiamine biosynthesis (bioF and thiDFH), anaerobic metabolism (focB, hyfACDR, ttdA, and fumB), and proteins with unknown function (ybfG, yceO, yjhQ, and yjbE); 10 of these genes were verified through mutation to decrease biofilm formation by 40% or more (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR). Hence, it appears YdgG controls the transport of the quorum-sensing signal AI-2, and so we suggest the gene name tqsA.

A Conductance Method for the Identification of Escherichia coli O157:H7 Using Bacteriophage AR1

2002 ◽  
Vol 65 (1) ◽  
pp. 12-17 ◽  
Author(s):  
TSUNG C. CHANG ◽  
HWIA C. DING ◽  
SHIOWWEN CHEN
Keyword(s):  
Escherichia Coli ◽  
Positive Reaction ◽  
Present Method ◽  
Culture Broth ◽  
Coli Strain ◽  
Bacterial Suspension ◽  
Escherichia Coli O157 ◽  
Bacterial Strains ◽  
E Coli ◽  
Conductance Method

The feasibility of using a specific phage (AR1) in conjunction with a conductance method for the identification of Escherichia coli O157:H7 was evaluated. The multiplication of strains of E. coli O157:H7 was inhibited by AR1; therefore, a time point (detection time, DT) at which an accelerating change in conductance in the culture broth was not obtained. Bacterial strains were subcultured on sorbitol-MacConkey agar and incubated at 35°C for 24 h, and the ability of the bacteria to ferment sorbitol was recorded. An aliquot of 0.5 ml of the bacterial suspension (107 CFU/ml) and 0.5 ml of the phage suspension (108 PFU/ml) were added to the conductance tube of a Malthus analyzer containing 5 ml of culture broth. The tubes were incubated at 35°C, and conductance changes in the tubes were continuously monitored at 6-min intervals for 24 h by the instrument. A positive reaction was defined as an E. coli strain that could not utilize sorbitol and caused no conductance change (i.e., no DT) within an incubation period of 24 h. Of the 41 strains of E. coli O157:H7 tested, all produced positive reactions. When a total of 155 strains of non-O157:H7 E. coli were tested, 14 did not have a DT within 24 h. However, among these 14 strains, 13 were sorbitol fermenters, and the remaining one was a nonfermenter. Therefore, by definition, only one strain produced a false-positive reaction. The sensitivity and specificity of the present method were 100% (41 of 41) and 99.4% (154 of 155), respectively. The present method incorporating conductimetric measurement and phage AR1 for the identification of E. coli O157:H7 was simple and capable of automation.

Mitomycin C-Activity Effected by Vitamins B1, C, E and β-Carotene under Irradiation with γ-Rays

2003 ◽  
Vol 58 (3-4) ◽  
pp. 244-248 ◽  
Author(s):  
Edith Heinrich ◽  
Nikola Getoff
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Vitamin B1 ◽  
Fold Increase ◽  
Oh Radicals ◽  
Strong Decrease ◽  
Γ Rays ◽  
Escherichia Coli Bacteria ◽  
Β Carotene

Vitamin B1 (thiamine) can essentially effect the activity of mitomycin C (MMC), added individually or in combination with antioxidant vitamins (C, E-acetate, β-carotene) as found in experiments in vitro (Escherichia coli bacteria, AB 1157) under irradiation with γ-rays. The environment plays a crucial role. In airfree media vitamin B1 leads to a 2-fold increase of the MMC-efficiency, but adding vitamin C it decreases. In the presence of all vitamins (B1, C, E-ac., and β-carotene) the MMC-action increases about 1.8-fold. In aerated media vitamin B1 causes an about 4-times increase of the MMC-efficiency, but by adding vitamin B1 and C the MMC-activity decreases by a factor of two, whereas in the presence of B1, C, E-ac., and β-carotene it rises again to 2.6-fold. In environment saturated with N2O (conversion of e-aq into OH radicals) a different picture is observed. The presence of vitamin B1 or vitamin B1 + C causes a strong decrease of the MMC-efficiency, but the addition of all vitamins (B1, C, E-ac., and β-car.) leads to a small increase of the cytostatic action. The results demonstrate the influence of vitamin B1 used individually or in combination with other antioxidants on the MMC-efficiency and the strong effect of the environment. The results are of interest for the application of MMC in radiotherapy.

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.

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