scholarly journals Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2779
Author(s):  
Anuwatchakij Klamrak ◽  
Jaran Nabnueangsap ◽  
Natsajee Nualkaew
Keyword(s):  
Web Service ◽  
Sodium Benzoate ◽  
Culture Medium ◽  
Rhodopseudomonas Palustris ◽  
Garcinia Mangostana ◽  
E Coli ◽  
Catalytic Function ◽  
Coa Ligase ◽  
Malonyl Coa ◽  
Artificial Pathway

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.

scholarly journals The crystal structure of benzophenone synthase from Garcinia mangostana L. pericarps reveals the basis for substrate specificity and catalysis

2020 ◽  
Vol 76 (12) ◽  
pp. 597-603
Author(s):  
Chomphunuch Songsiriritthigul ◽  
Natsajee Nualkaew ◽  
James Ketudat-Cairns ◽  
Chun-Jung Chen
Keyword(s):  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Docking Studies ◽  
Structural Basis ◽  
Garcinia Mangostana ◽  
Type Iii Polyketide Synthase ◽  
Catalytic Function ◽  
Malonyl Coa ◽  
Horizontal Cavity ◽  
Polyketide Chain

Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αβαβα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.

scholarly journals Production of 7-O-Methyl Aromadendrin, a Medicinally Valuable Flavonoid, in Escherichia coli

2011 ◽  
Vol 78 (3) ◽  
pp. 684-694 ◽  
Author(s):  
Sailesh Malla ◽  
Mattheos A. G. Koffas ◽  
Romas J. Kazlauskas ◽  
Byung-Gee Kim
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Avermitilis ◽  
Flavonoid Glycosides ◽  
Petroselinum Crispum ◽  
Cell Factory ◽  
Coumaric Acid ◽  
Content Type ◽  
E Coli ◽  
Coa Ligase ◽  
Malonyl Coa

ABSTRACT7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such asPopulus albaandEucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, anEscherichia colicell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor,p-coumaric acid, inE. colifor the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feedingp-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate–coenzyme A (CoA) ligase fromPetroselinum crispum, chalcone synthase fromPetunia hybrida, and chalcone isomerase fromMedicago sativa.In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890andnfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) fromNocardia farcinicawere also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase fromArabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase fromStreptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction inE. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μMp-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.

scholarly journals Efficient Synthesis of Eriodictyol from l-Tyrosine in Escherichia coli

2014 ◽  
Vol 80 (10) ◽  
pp. 3072-3080 ◽  
Author(s):  
Saijie Zhu ◽  
Junjun Wu ◽  
Guocheng Du ◽  
Jingwen Zhou ◽  
Jian Chen
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Antioxidant Activities ◽  
Efficient Synthesis ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
Coa Ligase ◽  
Malonyl Coa ◽  
Two Factors

ABSTRACTThe health benefits of flavonoids for humans are increasingly attracting attention. Because the extraction of high-purity flavonoids from plants presents a major obstacle, interest has emerged in biosynthesizing them using microbial hosts. Eriodictyol is a flavonoid with anti-inflammatory and antioxidant activities. Its efficient synthesis has been hampered by two factors: the poor expression of cytochrome P450 and the low intracellular malonyl coenzyme A (malonyl-CoA) concentration inEscherichia coli. To address these issues, a truncated plant P450 flavonoid, flavonoid 3′-hydroxylase (tF3′H), was functionally expressed as a fusion protein with a truncated P450 reductase (tCPR) inE. coli. This allowed the engineeredE. colito produce eriodictyol froml-tyrosine by simultaneously coexpressing the fusion protein with tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). In addition, metabolic engineering was employed to enhance the availability of malonyl-CoA so as to achieve a new metabolic balance and rebalance the relative expression of genes to enhance eriodictyol accumulation. This approach made the production of eriodictyol 203% higher than that in the control strain. By using these strategies, the production of eriodictyol froml-tyrosine reached 107 mg/liter. The present work offers an approach to the efficient synthesis of other hydroxylated flavonoids froml-tyrosine or even glucose inE. coli.

scholarly journals Biosynthesis of resveratrol derivatives and evaluation of their anti-inflammatory activity

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Yoojin Chong ◽  
Hye Lim Lee ◽  
Jihyeon Song ◽  
Youngshim Lee ◽  
Bong-Gyu Kim ◽  
...  
Keyword(s):  
Biological Activities ◽  
Stilbene Synthase ◽  
Inflammatory Activity ◽  
E Coli ◽  
Prephenate Dehydrogenase ◽  
Coa Ligase ◽  
Final Yield ◽  
Resveratrol Derivatives ◽  
Anti Inflammatory ◽  
Tyrosine Biosynthesis

AbstractResveratrol is a typical plant phenolic compound whose derivatives are synthesized through hydroxylation, O-methylation, prenylation, and oligomerization. Resveratrol and its derivatives exhibit anti-neurodegenerative, anti-rheumatoid, and anti-inflammatory effects. Owing to the diverse biological activities of these compounds and their importance in human health, this study attempted to synthesize five resveratrol derivatives (isorhapontigenin, pterostilbene, 4-methoxyresveratrol, piceatannol, and rhapontigenin) using Escherichia coli. Two-culture system was used to improve the final yield of resveratrol derivatives. Resveratrol was synthesized in the first E. coli cell that harbored genes for resveratrol biosynthesis including TAL (tyrosine ammonia lyase), 4CL (4-coumaroyl CoA ligase), STS (stilbene synthase) and genes for tyrosine biosynthesis such as aroG (deoxyphosphoheptonate aldolase) and tyrA (prephenate dehydrogenase). Thereafter, culture filtrate from the first cell was used for the modification reaction carried out using the second E. coli harboring hydroxylase and/or O-methyltransferase. Approximately, 89.8 mg/L of resveratrol was synthesized and using the same, five derivatives were prepared with a conversion rate of 88.2% to 22.9%. Using these synthesized resveratrol derivatives, we evaluated their anti-inflammatory activity. 4-Methoxyresveratrol, pterostilbene and isorhapontigenin showed the anti-inflammatory effects without any toxicity. In addition, pterostilbene exhibited the enhanced anti-inflammatory effects for macrophages compared to resveratrol.

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

scholarly journals EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

2015 ◽  
Vol 2 (1) ◽  
pp. 25 ◽  
Author(s):  
Sri Sumarsih
Keyword(s):  
Culture Medium ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Agar Medium ◽  
Nitrilotriacetic Acid ◽  
Protoplast Transformation ◽  
E Coli ◽  
Recombinant Cells ◽  
Relative Molecular Weight ◽  
Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

scholarly journals Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community

2019 ◽  
Author(s):  
Ryan K Fritts ◽  
Jordan T Bird ◽  
Megan G Behringer ◽  
Anna Lipzen ◽  
Joel Martin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nutrient Uptake ◽  
Rhodopseudomonas Palustris ◽  
Biogeochemical Cycles ◽  
Wild Type ◽  
Fermentation Products ◽  
E Coli ◽  
Constitutive Activation ◽  
Host Health ◽  
Nitrogen Scavenging

ABSTRACTInteractive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered mutualistic cross-feeding between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excreted essential nitrogen as NH4+ to E. coli while E. coli excreted essential carbon as fermentation products to R. palustris. Here, we enriched for nascent cross-feeding in cocultures with wild-type R. palustris, not known to excrete NH4+. Emergent NH4+ cross-feeding was driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of organic acids from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

scholarly journals Antibiofilm Activity of an Experimental Ricinus Communis Dentifrice on Soft Denture Liners

2019 ◽  
Vol 30 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Maurício Malheiros Badaró ◽  
Vanessa Maria Fagundes Leite-Fernandes ◽  
Luciano Trevisan Martin ◽  
Viviane de Cássia Oliveira ◽  
Evandro Watanabe ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Culture Medium ◽  
Solid Medium ◽  
Ricinus Communis ◽  
Positive Control ◽  
Antibiofilm Activity ◽  
Candida Spp ◽  
E Coli ◽  
Liquid Culture Medium ◽  
Denture Liners

Abstract The disadvantage of liners materials is the difficulty of biofilm control. It was compared an experimental dentifrice contained Ricinus communis, with commercials dentifrices as antibiofilm activity against microorganisms on denture liner. Six hundred specimens were distributed in 5 groups (n=18/ microorganism): water; experimental dentifrice; specific dentifrice for denture and two conventional dentifrices against C. albicans; C. glabrata; S. mutans; S. aureus; E. coli. Each group had a negative (n=5; without contamination) and positive control (n=15/ microorganism; without cleaning). The antibiofilm activity was evaluated by the method of biofilm formation in triplicate. The specimens were contaminated in a standard way and incubated. After that, manual brushing was performed (60 s), washed with PBS, immersed in liquid culture medium for resuspension and sowing in solid medium. The results (mean of triplicates) were expressed in CFU/mL. The data was submitted to Shapiro-Wilk, ANOVA and Tukey test (p<0.05). The specific dentifrice (1.27±1.20) was the most effective against S. mutans, followed by conventional (Trihydral, 3.13±0.88; Colgate, 2.16±2.02) and experimental (3.81±1.37) dentifrices, which were similar to each other (p=0.008). All of them were different from water (4.79±1.42). The specific (0.21±0.21) and experimental (0.36±0.25) dentifrices were similar against S. aureus, with a higher mean of CFU when compared to conventional (Colgate, 0.06±0.13), which was more efficient (p=0.000). For C. albicans, C. glabrata and E. coli, all dentifrices were similar to water (p=0.186). It was concluded, that the experimental dentifrice was effective against S. aureus and had not efficacy against Candida spp.; S. mutans; E. coli, as occurred with the commercials dentifrices.

scholarly journals Adhesion ofE. coliBacteria Cells to Prosthodontic Alloys Surfaces Modified by TiO2Sol-Gel Coatings

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Katarzyna Banaszek ◽  
Witold Szymanski ◽  
Bożena Pietrzyk ◽  
Leszek Klimek
Keyword(s):  
Titanium Dioxide ◽  
Bacterial Adhesion ◽  
Culture Medium ◽  
Bacterial Culture ◽  
Bare Metal ◽  
Bacterial Cells ◽  
E Coli ◽  
Modified Surfaces ◽  
Bare Substrate ◽  
Scanning Electron

The evaluation of the degree of bacteriaE. coliadhesion to modified surfaces of the chosen prosthodontic alloys was presented. The study was carried out on Co-Cr (Wironit), Ni-Cr (Fantocer), and Fe-Cr-Ni (Magnum AN) alloys. Bare substrate as a control and titanium dioxide coated samples were used. The samples were placed for 24 hours in bacterial culture medium. After incubation period, a number of bacterial cells were evaluated by scanning electron microscope. The study revealed that modification of the alloy surfaces by titanium dioxide coating significantly decreases the amount of bacteria adhering to the surfaces and that additionally bare metal alloy substrates have a different degree of susceptibility to bacterial adhesion.

scholarly journals Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria

2018 ◽  
Vol 115 (40) ◽  
pp. 9835-9844 ◽  
Author(s):  
Dongsoo Yang ◽  
Won Jun Kim ◽  
Seung Min Yoo ◽  
Jong Hyun Choi ◽  
Shin Hee Ha ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput Screening ◽  
Polyketide Synthase ◽  
Rapid Development ◽  
Type Iii Polyketide Synthase ◽  
E Coli ◽  
Type Iii ◽  
Small Regulatory Rna ◽  
Enhanced Production ◽  
Malonyl Coa

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only inEscherichia coliandSaccharomyces cerevisiaeand require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria:E. coli,Pseudomonas putida, andCorynebacterium glutamicum. RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor inE. coli. Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level inE. coli. These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple differentE. colistrains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA–derived chemicals.

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