scholarly journals Biosynthesis of morin-3-O-rhamnopyranoside in a genetically engineered Escherichia coli

2018 ◽  
Vol 15 (1) ◽  
pp. 161-168
Author(s):  
Nguyễn Huy Thuần ◽  
Nguyễn Thành Trung ◽  
Đồng Văn Quyền ◽  
Vũ Thị Thu Hằng ◽  
Jae Kyung Sohng
Keyword(s):  
Escherichia Coli ◽  
Liquid Chromatography ◽  
High Performance ◽  
Extraction Methods ◽  
Biosynthetic Gene Cluster ◽  
Culture Broth ◽  
Genetically Engineered ◽  
E Coli ◽  
Biotransformation Process ◽  
Tandem Mass Spectrometric

Flavonoids are significant secondary metabolites of vascular plants. They have been proven to possess numerous types of anti-bacterial, anti-tumor, anti-oxidant and anti-inflammatory bioactivities etc. Thereby, there are many flavonoids extracted and purified from plants and have been used for biological tests. Nevertheless, the traditional extraction methods require a large amount of initial sample, tedious and professional techniques and low yield of target compound. Until present, the advanced development of genetic engineerings and recombinant protein as platform allow synthesis of high amount of different types of flavoid in short time. Escherichia coli is one of the most popular host for whole-cell biotransformation of flavonoids and their derivatives due to its simple genetical, physiological system and fast growth. In this paper, we described the biosynthetic method of morin-3-O-rhamnopyranoside from a genetically engineered E. coli using morin as substrate. In particular, the E. coli harboring biosynthetic gene cluster of activated TDP-L-rhamnose and gene encoding for glycosyltransferase was used as host for biotransformation. Morin was fed into the culture broth of recombinant E. coli for rhamnosylation. The formation of morin-3-O-rhamnopyranoside was then quantitied and qualified using Thin Layer Chromatography (TLC), Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) assays. The highest yield of product (56.5 µM) was achieved after 48h, 32 oC and pH = 7.2-8.0. This result showed that morin-3-O-rhamnopyranoside was succesfully synthesized in the genetically engineered E. coli. Furthermore, metabolic engineering of intra-cellular system for improving absorption of substrate as well as excretion of product or enhancement of glycosyltransferase activity may increase the final yield of biotransformation process.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

scholarly journals Production of Sepiapterin in Escherichia coli by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase

2002 ◽  
Vol 68 (6) ◽  
pp. 3138-3140 ◽  
Author(s):  
Hyun Joo Woo ◽  
Jee Yun Kang ◽  
Yong Kee Choi ◽  
Young Shik Park
Keyword(s):  
Escherichia Coli ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Aldose Reductase ◽  
High Performance ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
E Coli ◽  
Sepiapterin Reductase ◽  
Pcc 6803

ABSTRACT Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.

scholarly journals MhpA Is a Hydroxylase Catalyzing the Initial Reaction of 3-(3-Hydroxyphenyl)Propionate Catabolism in Escherichia coli K-12

2019 ◽  
Vol 86 (4) ◽  
Author(s):  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Liquid Chromatography ◽  
High Performance ◽  
Initial Reaction ◽  
Functional Identification ◽  
Bacterial Strains ◽  
Content Type ◽  
E Coli ◽  
C Terminus ◽  
K 12

ABSTRACT Escherichia coli K-12 and some other strains have been reported to be capable of utilizing 3-(3-hydroxyphenyl)propionate (3HPP), one of the phenylpropanoids from lignin. Although other enzymes involved in 3HPP catabolism and their corresponding genes from its degraders have been identified, 3HPP 2-hydroxylase, catalyzing the first step of its catabolism, has yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characteristics of MhpA from E. coli strain K-12 (MhpAK-12). Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA deletion strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpAK-12. MhpAK-12 was overexpressed and purified, and it was likely a polymer and tightly bound with an approximately equal number of moles of FAD. Using NADH or NADPH as a cofactor, purified MhpAK-12 catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpAK-12 was confirmed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpAK-12 and its putative homologues belonged to taxa that were phylogenetically distant from functionally identified FAD-containing monooxygenases (hydroxylases). Interestingly, MhpAK-12 has approximately an extra 150 residues at its C terminus in comparison to its close homologues, but its truncated versions MhpAK-12400 and MhpAK-12480 (with 154 and 74 residues deleted from the C terminus, respectively) both lost their activities. Thus, MhpAK-12 has been confirmed to be a 3HPP 2-hydroxylase catalyzing the conversion of 3HPP to DHPP, the initial reaction of 3HPP degradation. IMPORTANCE Phenylpropionate and its hydroxylated derivatives resulted from lignin degradation ubiquitously exist on the Earth. A number of bacterial strains have the ability to grow on 3HPP, one of the above derivatives. The hydroxylation was thought to be the initial and vital step for its aerobic catabolism via the meta pathway. The significance of our research is the functional identification and characterization of the purified 3HPP 2-hydroxylase MhpA from Escherichia coli K-12 at biochemical and genetic levels, since this enzyme has not previously been expressed from its encoding gene, purified, and characterized in any bacteria. It will not only fill a gap in our understanding of 3HPP 2-hydroxylase and its corresponding gene for the critical step in microbial 3HPP catabolism but also provide another example of the diversity of microbial degradation of plant-derived phenylpropionate and its hydroxylated derivatives.

scholarly journals Understanding the compositional changes in LB Lennox medium during growth of Escherichia coli DH5α in a 1L bioreactor

2020 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Liquid Chromatography ◽  
High Performance ◽  
Model Building ◽  
Reversed Phase ◽  
Culture Broth ◽  
Weight Changes ◽  
Rp Hplc ◽  
Compositional Changes

AbstractCompositional changes in growth medium represents dynamic interplay between cell growth, biomass formation, and energy maintenance, with concomitant decrease in nutrients and increase in secreted metabolites and metabolic byproducts. Such information is important for quantifying microbial physiological response both at the population and cellular level, with respect to understanding subtle differences in microbial growth response, as well as supporting model building efforts in metabolic engineering. With the desire to understand molecular weight changes in components of LB Lennox medium as well as broth fractions present during cultivation of Escherichia coli DH5α (ATCC 53868) at 37 °C, 400 rpm stirring and 1 VVM aeration in a 1 L bioreactor, this study used a combination of gel filtration chromatography (GFC) and reversed phase high performance liquid chromatography (RP-HPLC) for determining changes to molecular weight of different fractions of the growth medium. Experiment results revealed the difficulty of fractionating the culture broth with RP-HPLC, where no distinct peaks of narrow retention time width were obtained. More importantly, the column used for GFC was unable to differentiate small molecular weight changes on the order of a few tens to few hundred Da through a refractive index detector. Together, GFC and RP-HPLC highlighted the difficulty of fractionating LB Lennox culture broth into different distinct fractions. Finally, the study validated the use of 194 nm as detection wavelength for visualizing the chromatogram of LB Lennox medium eluted from a C-18 reversed phase column during liquid chromatography. Collectively, GFC and RP-HPLC could not fractionate LB Lennox broth of E. coli DH5α into distinct fractions for further analysis by identification techniques such as mass spectrometry. Given the inherent complexity of complex medium such as LB Lennox, clean separation of the medium into every component with high purity may be impossible.Graphical abstractShort descriptionBroad elution profile and lack of distinct peaks in chromatogram of LB Lennox broth at 6 hours post inoculation with Escherichia coli DH5α (ATCC 53868), after attempted fractionation by C-18 reversed phase high performance liquid chromatography (RP-HPLC), revealed that separation could not be achieved with a complex mixture such as a microbiology broth. Specifically, while the objective of understanding compositional changes to culture medium during growth presents tremendous opportunities for determining the dynamic conversion of nutrients into metabolites and byproducts, difficulty of profiling all chemical constituents in a complex broth mixture meant that current model building efforts for metabolic engineering remains primitive with respect to the consortia of metabolic reactions occurring in situ at the cellular level.Subject areasbiotechnology, biochemistry, cell biology, microbiology, analytical chemistry

scholarly journals Escherichia coli as a genetic tool

1985 ◽  
Vol 95 (3) ◽  
pp. 611-618
Author(s):  
Naomi Datta
Keyword(s):  
Escherichia Coli ◽  
Academic Research ◽  
Genetically Engineered ◽  
Cloning And Expression ◽  
Biological Research ◽  
New Techniques ◽  
E Coli ◽  
Genetic Tool ◽  
The Past ◽  
Made In

SUMMARYThe study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli

2019 ◽  
Vol 366 (24) ◽  
Author(s):  
Yan Ge ◽  
Senlin Guo ◽  
Tao Liu ◽  
Chen Zhao ◽  
Duanhua Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Yersinia Enterocolitica ◽  
Inclusion Bodies ◽  
Genetically Engineered ◽  
Biological Research ◽  
Dilution Method ◽  
E Coli ◽  
Protein Renaturation ◽  
Engineered Escherichia Coli ◽  
Pharmaceutical Production

ABSTRACT A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

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