scholarly journals Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

2003 ◽  
Vol 69 (11) ◽  
pp. 6569-6576 ◽  
Author(s):  
Jörg Overhage ◽  
Alexander Steinbüchel ◽  
Horst Priefert
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Alcohol Oxidase ◽  
Coniferyl Alcohol ◽  
Vanillyl Alcohol ◽  
Resting Cells ◽  
Fermentation Time ◽  
Molar Yield ◽  
E Coli ◽  
Maximum Production Rate

ABSTRACT The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter−1. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter−1 after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter−1, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter−1. The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.

Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives1

1982 ◽  
Vol 45 (3) ◽  
pp. 241-243 ◽  
Author(s):  
M. KRUK ◽  
J. S. LEE
Keyword(s):  
Escherichia Coli ◽  
Benzoic Acid ◽  
Sorbic Acid ◽  
Reductase Activity ◽  
Resting Cells ◽  
E Coli

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

scholarly journals PSV-41 Antibacterial activity of ferulic acid and sodium chlorate against Escherichia coli F18 and K88 during incubations with porcine fecal bacteria

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 296-297 ◽  
Author(s):  
Claudio Arzola ◽  
Elizabeth Latham ◽  
Robin Anderson ◽  
Jaime Salinas-Chavira ◽  
Yamicela Castillo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Sodium Chlorate ◽  
Fecal Bacteria ◽  
Aerobic Incubation ◽  
E Coli ◽  
Mueller Hinton ◽  
Fecal Suspension ◽  
Porcine Feces ◽  
Mueller Hinton Broth

Abstract The influence of ferulic acid (FA) and sodium chlorate (SC) was evaluated in two trials on the growth of Escherichia coli F18 and K88 (F18 and K88) incubated with porcine fecal bacteria. Treatments were 2 levels of FA (0 and 5 mg/mL) and 2 levels of SC (0 and 10 mM/mL). In trial one, ½-strength Mueller Hinton broth mixed with porcine feces (0.5% w/v) was inoculated with a novobiocin and naladixic acid resistant F18-strain. This fecal suspension was transferred to tubes (3/treatment) and anaerobically incubated at 39 oC for enumeration at 0, 6 and 24 h using MacConkey agar supplemented with novobiocin and naladixic acid with aerobic incubation at 37 oC. An interaction (FA x SC) at 6 and 24 h was observed (P < 0.01). At 6 h of incubation, SC alone or combined with FA had the lowest counts (P < 0.05); FA alone was lower than control but higher than SC or SC+FA (P < 0.05). At 24 h, FA alone or combined with SC had the lowest counts (P < 0.05); SC was lower than control but higher than FA or SC+FA (P < 0.05). In trial 2 were used the same procedures of trial 1, except that K88 was used. There was an interaction at 6 h (P < 0.01) where the lowest counts were in FA+SC (P < 0.05). SC alone or FA alone were lower than control but higher than SC+FA (P < 0.05). There was no interaction at 24 h (P = 0.16), where FA reduced the K88 counts (P < 0.01), however it was not affected by SC (P = 0.12). In conclusion, SC reduced E. coli counts; however, at 24 h of incubation greater reductions were observed when FA alone or combined with SC was added into the incubation fluid with porcine feces.

The Role of the ydiB Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS- Strain of Escherichia coli

2016 ◽  
Vol 27 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Sofía García ◽  
Noemí Flores ◽  
Ramón De Anda ◽  
Georgina Hernández ◽  
Guillermo Gosset ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shikimic Acid ◽  
Sharp Decrease ◽  
Quinic Acid ◽  
Shikimate Dehydrogenase ◽  
Molar Yield ◽  
E Coli ◽  
Downstream Processes ◽  
Precise Role

The culture of engineered <i>Escherichia coli</i> for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, <i>ydiB</i>) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of <i>E. coli</i> for SA production remains unclear. We report the effect of the inactivation or the overexpression of <i>ydiB</i> in <i>E. coli</i> strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of <i>ydiB </i>resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of <i>ydiB</i> caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative <i>ydiB </i>strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

scholarly journals Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Kyung Taek Heo ◽  
Byeongsan Lee ◽  
Jae-Hyuk Jang ◽  
Jung-Oh Ahn ◽  
Young-Soo Hong
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Chain Elongation ◽  
Piper Nigrum ◽  
Glucose Medium ◽  
E Coli ◽  
Acid Catalyzed ◽  
Simple Sugar

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

scholarly journals OPTIMASI PROSES UNTUK EKSPRESI GEN ENDOGLUKANASE DARI Bacillus sp. RP1 OLEH Escherichia coli BL21 (DE3)/ egc

2018 ◽  
Vol 5 (1) ◽  
pp. 44
Author(s):  
Hans Victor ◽  
Maelita Ramdani Moeis
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Process Optimization ◽  
Rice Hull ◽  
Bacillus Sp ◽  
Fermentation Time ◽  
E Coli ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Dry Cell

Process Optimization for Endoglucanase Gene Expression Derived from Bacillus sp. RP1 by Escherichia coli BL21 (DE3)/egcABSTRACTCellulases are one of the most used enzymes in industrial processes. In an effort to increase production, industries have developed strategies such as isolating new cellulase producing strains, genetic engineering and process optimization since the last 50 years. One endoglucanase producing strain, Bacillus sp. RP1 was isolated from hot springs. The ribosome binding site and coding sequence of the endoglucanase gene (egc) from Bacillus sp. RP1 was cloned into pGEM-T Easy. The recombinant plasmid was used to transform E. coli BL21 (DE3). Cloning was followed by process optimization. Medium composition was selected using Plackett-Burman design. The medium components tested were rice hull, molasses, ammonium chloride, urea and fishmeal. Rice hull and molasses were found to be the factors most influencing enzyme activity and dry cell weight, respectively. The next step involved Box-Behnken method and response surface methodology to optimize the responses against molasses concentration, rice hull concentration and fermentation time. The concentration intervals used to test were 1%, 5.5% and 10% while the fermentation time used were 24, 36 and 48 hours. The conditions which optimized both enzyme activity and dry cell weight were 7.45% molasses, 6.45% rice hull and 39.52 hours of fermentation.Keywords: Bacillus sp. RP1, E. coli BL21 (DE3), egc, Endoglucanase, optimization ABSTRAKSelulase adalah salah satu enzim yang banyak dimanfaatkan dalam berbagai industri. Sebagai upaya untuk memenuhi kebutuhan, 50 tahun terakhir dikembangkan beberapa strategi untuk meningkatkan produksi selulase yang mencakup rekayasa genetika dan optimasi proses. Karena itu, dilakukan kloning gen egc dan RBS yang berasal dari Bacillus sp. RP1 yang diisolasi dari sumber air panas ke dalam vektor pGEM-T Easy. E. coli BL21 (DE3) ditransformasikan dengan vektor yang mengandung gen egc tersebut. Setelah kloning, optimasi proses berupa desain medium turut dilakukan untuk mengoptimalkan ekspresi gen egc. Desain medium diawali dengan seleksi komposisi medium menggunakan metode Plackett-Burman. Komponen medium yang diuji adalah kulit beras, molase, amonium klorida, urea dan tepung ikan. Kulit beras dan molase diperoleh sebagai bahan yang paling berpengaruh terhadap aktivitas enzim dan berat kering sel. Tahap selanjutnya melibatkan metode statistik Box-Behnken dan metodologi respons permukaan yang bertujuan mengoptimalkan respons aktivitas enzim dan berat kering sel terhadap konsentrasi molase, konsentrasi kulit beras dan lama fermentasi. Konsentrasi yang diuji adalah 1%, 5,5% dan 10%, sedangkan lama fermentasi yang diuji adalah 24, 36 dan 48 jam. Konsentrasi optimal molase adalah 7,45% dan konsentrasi optimal kulit beras adalah 6,45% dengan lama fermentasi optimal 39,52 jam.Kata Kunci: Bacillus sp. RP1, E. coli BL21 (DE3), egc, Endoglukanase, optimasi

scholarly journals Ferulic Acid production by metabolically engineered Escherichia coli

2021 ◽  
Author(s):  
Huajun Lv ◽  
Ying Zhang ◽  
Jie Shao ◽  
Haili Liu ◽  
Yong Wang
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Caffeic Acid ◽  
Pyridine Nucleotide ◽  
Promoter Strength ◽  
T7 Promoter ◽  
Nadph Regeneration ◽  
E Coli ◽  
Ferulic Acid Production ◽  
K 12

Abstract Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics areas. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding Tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p - coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as Caffeic acid O-methytransferase. Fermentation results revealed JM109(DE3) as more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 ori or p15a ori), and the combination p15a-T5 works best. To further improve FA production, E.coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also over expressed. Based on the flask fermentation data which shows that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E.coli K-12 strains reported to the best of our knowledge.

scholarly journals Influence of sodium chlorate, ferulic acid, and essential oils on Escherichia coli and porcine fecal microbiota

2020 ◽  
Vol 98 (3) ◽  
Author(s):  
Claudio Arzola-Alvarez ◽  
Michael E Hume ◽  
Robin C Anderson ◽  
Elizabeth A Latham ◽  
Oscar Ruiz-Barrera ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Essential Oils ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Gene Sequencing ◽  
Sodium Chlorate ◽  
The Other ◽  
Enterotoxigenic Escherichia Coli ◽  
Similarity Coefficients ◽  
E Coli

Abstract The influence of sodium chlorate (SC), ferulic acid (FA), and essential oils (EO) was examined on the survivability of two porcine diarrhetic enterotoxigenic Escherichia coli (ETEC) strains (F18 and K88) and populations of porcine fecal bacteria. Fecal bacterial populations were examined by denaturing gradient gel electrophoresis (DGGE) and identification by 16S gene sequencing. The treatments were control (no additives), 10 mM SC, 2.5 mg FA /mL, a 1.5% vol/vol solution of an EO mixture as well as mixtures of EO + SC, EO + FA, and FA + SC at each of the aforementioned concentrations. EO were a commercial blend of oregano oil and cinnamon oil with water and citric acid. Freshly collected porcine feces in half-strength Mueller Hinton broth was inoculated with E. coli F18 (Trial 1) or E. coli K88 (Trial 2). The fecal-E. coli suspensions were transferred to crimp top tubes preloaded with the treatment compounds. Quantitative enumeration was at 0, 6, and 24 h. All treatments reduced (P &lt; 0.05) the counts of E. coli F18 at 6 and 24 h. With the exception of similarity coefficient (%SC), all the other treatments reduced (P &lt; 0.05) the K88 counts at 24 h. The most effective treatments to reduce the F18 and K88 CFU numbers were those containing EO. Results of DGGE revealed that Dice percentage similarity coefficients (%SC) of bacterial profiles among treatment groups varied from 81.3% to 100%SC. The results of gene sequencing showed that, except for SC at 24 h, all the other treatments reduced the counts of the family Enterobacteriaceae, while Lactobacillaceae and Ruminococcaceae increased and Clostridiaceae decreased in all treatments. In conclusion, all treatments were effective in reducing the ETEC, but EO mixture was the most effective. The porcine microbial communities may be influenced by the studied treatments.

scholarly journals High-Level Heterologous Production of d-Cycloserine by Escherichia coli

2015 ◽  
Vol 81 (22) ◽  
pp. 7881-7887 ◽  
Author(s):  
Takanori Kumagai ◽  
Tomoki Ozawa ◽  
Momoko Tanimoto ◽  
Masafumi Noda ◽  
Yasuyuki Matoba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Resting Cells ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Biosynthetic Genes ◽  
T7 Promoter ◽  
Α Subunit ◽  
Content Type ◽  
E Coli

ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 11924. In this study, we put fourd-CS biosynthetic genes (dcsC,dcsD,dcsE, anddcsG) in tandem under the control of the T7 promoter in anEscherichia colihost. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl-serine and hydroxyurea (HU), the precursors ofd-CS, were incubated together with theE. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene product, DcsD, formsO-ureido-l-serine fromO-acetyl-l-serine (OAS) and HU, which are intermediates ind-CS biosynthesis. DcsD also catalyzes the formation ofl-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd-CS biosynthetic genes, together withdcsJ, were incubated withl-serine and HU, thed-CS production was 980 ± 57 μM, which is comparable to that ofd-CS-producingS. lavendulaeATCC 11924 (930 ± 36 μM).

scholarly journals Global Role for ClpP-Containing Proteases in Stationary-Phase Adaptation of Escherichia coli

2003 ◽  
Vol 185 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Dieter Weichart ◽  
Nadine Querfurth ◽  
Mathias Dreger ◽  
Regine Hengge-Aronis
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Growth Phase ◽  
Proteome Analysis ◽  
Resting Cells ◽  
Wild Type ◽  
Late Stationary Phase ◽  
E Coli ◽  
In The Wild ◽  
Dps Protein

ABSTRACT To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.

scholarly journals Biosynthesis of fraxetin from three different substrates using engineered Escherichia coli

2020 ◽  
Vol 63 (1) ◽  
Author(s):  
Seung Hoon An ◽  
Gyu-Sik Choi ◽  
Joong-Hoon Ahn
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Iron Homeostasis ◽  
Antibacterial Activities ◽  
Enzymatic Reactions ◽  
Biosynthesis Pathway ◽  
Coumaric Acid ◽  
E Coli ◽  
Coa Ligase ◽  
Key Enzyme

Abstract Fraxetin, which is a simple coumarin, is a phytochemical present in medicinal plants, such as Fraxinus rhynchophylla, and Cortex Fraxini. In plants, it serves as a controller of iron homeostasis. The health-enhancing activities of fraxetin, such as anticancer, neuroprotective and antibacterial activities, are known. Scopoletin 8-hydroxylase (S8H) is a key enzyme involved in the synthesis of fraxetin from scopoletin. Scopoletin can be synthesized either from esculetin by O-methylation or from ferulic acid by feruloyl CoA 6′-hydroxylase (F6′H) and 4-coumaric acid CoA ligase (4CL). To enable fraxetin synthesis, the fraxetin biosynthesis pathway was introduced into Escherichia coli. Three distinct routes, from ferulic acid, esculetin, and scopoletin, were designed for the synthesis of fraxetin. In the first approach, E. coli strain harboring S8H was used and found to synthesize 84.8 μM fraxetin from 100 μM scopoletin. Two E. coli strains were used for the other two approaches because these approaches required at least two enzymatic reactions. Through this approach, 41.4 μM fraxetin was synthesized from 100 μM esculetin, while 33.3 μM fraxetin was synthesized from 100 μM ferulic acid.

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