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1.
BMC Biol ; 18(1): 63, 2020 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-32552824

RESUMEN

BACKGROUND: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. RESULTS: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. CONCLUSIONS: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.


Asunto(s)
Vías Biosintéticas/genética , Cafeína/biosíntesis , Carotenoides/metabolismo , Evolución Molecular , Gardenia/genética , Duplicación de Gen , Gardenia/metabolismo , Genoma de Planta
2.
Int J Mol Sci ; 22(23)2021 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-34884439

RESUMEN

Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2'-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2'-O-glucopyranoside, midecamycin 2'-O-GlcNAc and midecamycin 2'-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2'-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.


Asunto(s)
Antibacterianos/química , Glicosiltransferasas/genética , Leucomicinas/química , Antibacterianos/metabolismo , Biocatálisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Variación Genética , Glicosilación , Glicosiltransferasas/metabolismo , Leucomicinas/metabolismo , Modelos Moleculares , Ingeniería de Proteínas , Azúcares/química
3.
J Asian Nat Prod Res ; 22(3): 201-216, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31497993

RESUMEN

As the continuous scientific research, seven new 1-oxygenated cholestane glycosides named osaundersiosides 1 A - 1 G were isolated from an EtOH extract of the bulbs of Ornithogalum saundersiae. Their structures were deduced by means of spectroscopic data, chemical evidence and the results of hydrolytic cleavage. The cytotoxicity and anti-inflammatory effects of osaundersiosides 1 A - 1 G were evaluated, but none of them displayed significant activities. [Formula: see text].


Asunto(s)
Antineoplásicos Fitogénicos , Colestanos , Ornithogalum , Glicósidos , Estructura Molecular
4.
Molecules ; 25(3)2020 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-31979165

RESUMEN

Steroidal glycosides are important sources of innovative drugs. The increased diversification of steroidal glycosides will expand the probability of discovering active molecules. It is an efficient approach to diversify steroidal glycosides by using steroidal glycosyltransferases. OcUGT1, a uridine diphosphate-d-glucose (UDP-Glc)-dependent glycosyltransferase from Ornithogalum caudatum, is a multifunctional enzyme, and its glycodiversification potential towards steroids has never been fully explored. Herein, the glycodiversification capability of OcUGT1 towards 25 steroids through glucosylation and transglucosylation reactions were explored. Firstly, each of 25 compounds was glucosylated with UDP-Glc. Under the action of OcUGT1, five steroids (testosterone, deoxycorticosterone, hydrocortisone, estradiol, and 4-androstenediol) were glucosylated to form corresponding mono-glucosides and biosides. Next, OcUGT1-mediated transglucosylation activity of these compounds with another sugar donor ortho-nitrophenyl-ß-d-glucopyranoside (oNPGlc) was investigated. Results revealed that the same five steroids could be glucosylated to generate mono-glucosides and biosides by OcUGT1 through transglucosylation reactions. These data indicated that OcUGT1-assisted glycodiversification of steroids could be achieved through glucosylation and transglucosylation reactions. These results provide a way to diversify steroidal glycosides, which lays the foundation for the increase of the probability of obtaining active lead compounds.


Asunto(s)
Glucósidos/metabolismo , Glicósidos/metabolismo , Glicosiltransferasas/metabolismo , Esteroides/metabolismo , Glicosilación , Ornithogalum/química
5.
BMC Plant Biol ; 19(1): 195, 2019 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-31088366

RESUMEN

BACKGROUND: Flavonol synthase (FLS) is the key enzyme responsible for the biosynthesis of flavonols, the most abundant flavonoids, which have diverse pharmaceutical effects. Flavonol synthase has been previously found in other species, but not yet in Ornithogalum caudatum. RESULTS: The transcriptome-wide mining and functional characterisation of a flavonol synthase gene family from O. caudatum were reported. Specifically, a small FLS gene family harbouring two members, OcFLS1 and OcFLS2, was isolated from O. caudatum based on transcriptome-wide mining. Phylogenetic analysis suggested that the two proteins showed the closest relationship with FLS proteins. In vitro enzymatic assays indicated OcFLS1 and OcFLS2 were flavonol synthases, catalysing the conversion of dihydroflavonols to flavonols in an iron-dependent fashion. In addition, the two proteins were found to display flavanone 3ß-hydroxylase (F3H) activity, hydroxylating flavanones to form dihydroflavonols. Unlike single F3H enzymes, the F3H activity of OcFLS1 and OcFLS2 did not absolutely require iron. However, the presence of sufficient Fe2+ was demonstrated to be conducive to successive catalysis of flavanones to flavonols. The qRT-PCR analysis demonstrated that both genes were expressed in the leaves, bulbs, and flowers, with particularly high expression in the leaves. Moreover, their expression was regulated by developmental and environmental conditions. CONCLUSIONS: OcFLS1 and OcFLS2 from O. caudatum were demonstrated to be flavonol synthases with iron-independent flavanone 3-hydroxylase activity.


Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Ornithogalum/enzimología , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Electroforesis en Gel de Poliacrilamida , Flavonoles/metabolismo , Perfilación de la Expresión Génica , Genes de Plantas/genética , Genes de Plantas/fisiología , Hierro/metabolismo , Redes y Vías Metabólicas , Ornithogalum/genética , Ornithogalum/metabolismo , Análisis de Secuencia de ADN , Transcriptoma
6.
J Asian Nat Prod Res ; 20(7): 662-674, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29852779

RESUMEN

Herein, a flavonoid glycosyltransferase (GT) OcUGT1 was determined to be able to attack C-8 position of 7,8-dihydroxyflavone (7,8-DHF) via both glycosylation and transglycosylation reactions. OcUGT1-catalyzed glycosylation of 7,8-DHF resulted in the formation of two monoglycosides 7-O-ß-D-glucosyl-8-hydroxyflavone (1a), 7-hydroxy-8-O-ß-D-glucosylflavone (1b), as well as one diglycoside 7,8-di-O-ß-D-glucosylflavone (1c). Under the action of OcUGT1, inter-molecular trans-glycosylations from aryl ß-glycosides to 7,8-DHF to form monoglycosides 1a and 1b were observable.


Asunto(s)
Flavonas/biosíntesis , Glicosiltransferasas/metabolismo , Catálisis , Escherichia coli/enzimología , Escherichia coli/genética , Glicósidos , Glicosilación , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular
7.
Protein Expr Purif ; 130: 63-72, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27725246

RESUMEN

As the first step of ongoing efforts to investigate the genes responsible for the biosynthesis of steroidal saponins in the medicinal plant Ornithogalum caudatum, this investigation reported the cDNA isolation, prokaryotic expression and functional characterization of squalene synthase (SQS) gene from O. caudatum for the first time. Specifically, two unigenes showing high sequence identity to SQS were retrieved from RNA-Taq data, and then a full-length OcSQS1 corresponding to the two unigenes was isolated from O. caudatum genome by a nested PCR assay. The open reading frame of OcSQS1 was 1230 bp and encoded a polypeptide of 409 aa. OcSQS1 was predicted to be a membrane-bound protein with at least four conserved motifs associated with binding, regulatory and catalytic activities of OcSQS1 and two transmembrane domains. Next, many attempts to generate soluble OcSQS1 in heterologous Escherichia coli were made, including optimization of expression conditions, application of varied expression plasmids with different tags, secretory peptides and molecular chaperones, and truncated mutation of OcSQS1. Finally, the successful availability of a soluble, truncated OcSQS1 mutant was achieved by combinational use of the utensils from the vast genetic toolbook. Moreover, this truncated OcSQS1 mutant retained the folding capability as well as its catalytic activity, converting FPP to form squalene. Importantly, the present research tentatively verified the involvement of the second transmembrane domain in the proper folding of the recombinant OcSQS1 protein.


Asunto(s)
Clonación Molecular , ADN Complementario , Escherichia coli/metabolismo , Farnesil Difosfato Farnesil Transferasa , Ornithogalum/genética , Proteínas de Plantas , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Escherichia coli/genética , Farnesil Difosfato Farnesil Transferasa/biosíntesis , Farnesil Difosfato Farnesil Transferasa/química , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/aislamiento & purificación , Ornithogalum/enzimología , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
8.
Microb Cell Fact ; 15: 27, 2016 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-26846670

RESUMEN

BACKGROUND: (2S)-Pinocembrin is a chiral flavanone with versatile pharmacological and biological activities. Its health-promoting effects have spurred on research effects on the microbial production of (2S)-pinocembrin. However, an often-overlooked salient feature in the analysis of microbial (2S)-pinocembrin is its chirality. RESULTS: Here, we presented a full characterization of absolute configuration of microbial (2S)-pinocembrin from engineered Escherichia coli. Specifically, a transcriptome-wide search for genes related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum, a plant rich in flavonoids, was first performed in the present study. A total of 104,180 unigenes were finally generated with an average length of 520 bp. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping assigned 26 unigenes, representing three enzyme families of 4-coumarate:coenzyme A ligase (4CL), chalcone synthase (CHS) and chalcone isomerase(CHI), onto (2S)-pinocembrin biosynthetic pathway. A total of seven, three and one full-length candidates encoding 4CL, CHS and CHI were then verified by reverse transcription polymerase chain reaction, respectively. These candidates were screened by functional expression in E. coli individual or coupled multienzyme reaction systems based on metabolic engineering processes. Oc4CL1, OcCHS2 and OcCHI were identified to be bona fide genes encoding respective pathway enzymes of (2S)-pinocembrin biosynthesis. Then Oc4CL1, OcCHS2 and MsCHI from Medicago sativa, assembled as artificial gene clusters in different organizations, were used for fermentation production of (2S)-pinocembrin in E. coli. The absolute configuration of the resulting microbial pinocembrin at C-2 was assigned to be 2S-configured by combination of retention time, UV spectrum, LC-MS, NMR, optical rotation and circular dichroism spectroscopy. Improvement of (2S)-pinocembrin titres was then achieved by optimization of gene organizations, using of codon-optimized pathway enzymes and addition of cerulenin for increasing intracellular malonyl CoA pools. Overall, the optimized strain can produce (2S)-pinocembrin of 36.92 ± 4.1 mg/L. CONCLUSIONS: High titre of (2S)-pinocembrin can be obtained from engineered E. coli by an efficient method. The fermentative production of microbial (2S)-pinocembrin in E. coli paved the way for yield improvement and further pharmacological testing.


Asunto(s)
Vías Biosintéticas/genética , Flavanonas/metabolismo , Ingeniería Metabólica/métodos , Ornithogalum/enzimología , Ornithogalum/genética , Transcriptoma/genética , Espectroscopía de Resonancia Magnética con Carbono-13 , Cromatografía Líquida de Alta Presión , Dicroismo Circular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Fermentación , Flavanonas/química , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Espectroscopía de Protones por Resonancia Magnética , Proteínas Recombinantes/metabolismo
9.
Plant Cell Rep ; 35(11): 2403-2421, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27591771

RESUMEN

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.


Asunto(s)
Carboxiliasas/genética , Genes de Plantas , Familia de Multigenes , Ornithogalum/enzimología , Ornithogalum/genética , Transcriptoma/genética , Azúcares de Uridina Difosfato/metabolismo , Uridina Difosfato Xilosa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Compuestos de Amonio/farmacología , Biocatálisis/efectos de los fármacos , Tampones (Química) , Calcio/farmacología , Carboxiliasas/química , Carboxiliasas/metabolismo , Cromatografía Líquida de Alta Presión , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Ornithogalum/efectos de los fármacos , Espectroscopía de Protones por Resonancia Magnética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Temperatura , Transcriptoma/efectos de los fármacos , Azúcares de Uridina Difosfato/química , Uridina Difosfato Xilosa/química
10.
Molecules ; 21(11)2016 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-27834878

RESUMEN

d-Galacturonic acid (GalA) is an important component of GalA-containing polysaccharides in Ornithogalum caudatum. The incorporation of GalA into these polysaccharides from UDP-d-galacturonic acid (UDP-GalA) was reasonably known. However, the cDNAs involved in the biosynthesis of UDP-GalA were still unknown. In the present investigation, one candidate UDP-d-glucuronic acid 4-epimerase (UGlcAE) family with three members was isolated from O. caudatum based on RNA-Seq data. Bioinformatics analyses indicated all of the three isoforms, designated as OcUGlcAE1~3, were members of short-chain dehydrogenases/reductases (SDRs) and shared two conserved motifs. The three full-length cDNAs were then transformed to Pichia pastoris GS115 for heterologous expression. Data revealed both the supernatant and microsomal fractions from the recombinant P. pastoris expressing OcUGlcAE3 can interconvert UDP-GalA and UDP-d-glucuronic acid (UDP-GlcA), while the other two OcUGlcAEs had no activity on UDP-GlcA and UDP-GalA. Furthermore, expression analyses of the three epimerases in varied tissues of O. caudatum were performed by real-time quantitative PCR (RT-qPCR). Results indicated OcUGlcAE3, together with the other two OcUGlcAE-like genes, was root-specific, displaying highest expression in roots. OcUGlcAE3 was UDP-d-glucuronic acid 4-epimerase and thus deemed to be involved in the biosynthesis of root polysaccharides. Moreover, OcUGlcAE3 was proposed to be environmentally induced.


Asunto(s)
Carbohidrato Epimerasas , ADN Complementario , Ornithogalum , Proteínas de Plantas , Raíces de Plantas , Carbohidrato Epimerasas/biosíntesis , Carbohidrato Epimerasas/genética , Expresión Génica , Ornithogalum/enzimología , Ornithogalum/genética , Pichia , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Azúcares de Uridina Difosfato/genética , Azúcares de Uridina Difosfato/metabolismo
11.
Molecules ; 21(6)2016 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-27294908

RESUMEN

M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, ß-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-Δ4-steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-Δ5-steroids. This result indicates that the BM3 mutants prefer 3-keto-Δ4-steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-Δ5-steroids and three 3-keto-Δ4-steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-Δ4-steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-Δ5-steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-Δ4-steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants.


Asunto(s)
Bacterias/metabolismo , Familia 1 del Citocromo P450/metabolismo , Enzimas/metabolismo , Esteroides/metabolismo , Bacterias/química , Bacterias/genética , Familia 1 del Citocromo P450/química , Familia 1 del Citocromo P450/genética , Enzimas/química , Enzimas/genética , Hidroxilación , Mutación , Noretindrona/química , Noretindrona/metabolismo , Oxidación-Reducción , Esteroides/química , Especificidad por Sustrato , Testosterona/química , Testosterona/metabolismo
12.
Yao Xue Xue Bao ; 50(5): 627-32, 2015 May.
Artículo en Zh | MEDLINE | ID: mdl-26234148

RESUMEN

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.


Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos/metabolismo , Ciclización , Cinética , Staphylococcus aureus/enzimología
13.
Z Naturforsch C J Biosci ; 69(5-6): 259-70, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25069165

RESUMEN

Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10) catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form farnesyl pyrophosphate (FPP), a key precursor of sesquiterpenoids, triterpenoids, sterols, and farnesylated proteins. Here we report the molecular cloning and functional identification of a new full-length cDNA encoding FPPS from Ornithogalum saundersiae, a potential medicinal plant that produces a promising antitumour sterol glycoside, OSW-1. An 1327 bp long unigene with an open reading frame of 1044 bp was retrieved from the transcriptome sequencing of O. saundersiae. The full-length FPPS cDNA, designated OsaFPPS, was isolated from O. saundersiae with gene-specific primers. The resultant OsaFPPS encodes a 347-amino acids protein with a calculated molecular mass of 40,085.6 Da, and a theoretical isoelectric point of 5.01. Phylogenetic tree analysis indicated that OsaFPPS belongs to the plant FPPS super-family. Expression of soluble OsaFPPS in E. coli was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Functional analysis of the purified OsaFPPS protein was carried out using IPP and DMAPP as substrates, and the product was unambiguously determined by gas chromatography-mass spectrometry (GC-MS) analyses.


Asunto(s)
ADN Complementario/genética , ADN de Plantas/genética , Geraniltranstransferasa/genética , Ornithogalum/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Cromatografía de Gases y Espectrometría de Masas , Geraniltranstransferasa/química , Datos de Secuencia Molecular , Ornithogalum/clasificación , Ornithogalum/genética , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido
14.
Molecules ; 19(2): 1608-21, 2014 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-24476601

RESUMEN

OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5' non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD), HPLC-MS and nuclear magnetic resonance (NMR) analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.


Asunto(s)
Clonación Molecular , Ornithogalum/enzimología , Saponinas/biosíntesis , Transcinamato 4-Monooxigenasa/genética , Colestenonas/química , Colestenonas/aislamiento & purificación , Cinamatos/química , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Hidroxilación , Raíces de Plantas/enzimología , Saccharomyces cerevisiae/genética , Saponinas/química , Saponinas/aislamiento & purificación , Transcinamato 4-Monooxigenasa/biosíntesis , Transcinamato 4-Monooxigenasa/aislamiento & purificación
15.
Yao Xue Xue Bao ; 49(6): 905-12, 2014 Jun.
Artículo en Zh | MEDLINE | ID: mdl-25212039

RESUMEN

Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VY1 against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.


Asunto(s)
Antivirales/farmacología , Ciclotidas/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Viola/química , Antivirales/aislamiento & purificación , Espectrometría de Masas en Tándem
16.
Int J Biol Macromol ; 254(Pt 1): 127721, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37913883

RESUMEN

Glycosylation at C3-OH is the favorable modification for pharmaceutical activities and diversity expansion of 20(R)-dammarane ginsenosides. The 3-O-glycosylation, exclusively occurring in 20(R)-PPD ginsenosides, has never been achieved in 20(R)-PPT ginsenosides. Herein, 3-O-glycosylation of 20(R)-PPT enabled by a glycosyltransferase (GT) OsSGT2 was achieved with the combined assistance of AlphaFold 2 and molecular docking. Firstly, we combined AlphaFold2 algorithm and molecular docking to predict interactions between 20(R)-PPT and candidate GTs. A catalytically favorable binding geometry was thus identified in the OsSGT2-20(R)-PPT complex, suggesting OsSGT2 might act on 20(R)-PPT. The enzymatic assays demonstrated that OsSGT2 reacted with varied sugar donors to form 20(R)-PPT 3-O-glycosides, exhibiting donor promiscuity. Additionally, OsSGT2 displayed acceptor promiscuity, catalyzing 3-O-glucosylation of 20(R/S)-PPT, 20(R/S)-PPD and 20(R/S)-Rh1, respectively. Protein engineering on OsSGT2 was thus performed to probe its catalytic mechanism underlying its stereoselectivity. The W207A mutant preferred 20(S)-dammarane aglycons, while F395Q/A396G(QG) displayed a conversion enhancement towards both 20(R/S)-dammarane aglycons. The QG mutant was then used to synthesize 20(R)-PPT 3-O-glucoside, which displayed a moderate angiotensin-converting enzyme inhibitory effect with an IC50 of 27.5 ± 4.7 µM, superior to that of its 20(S)-epimer, with the combined assistance of target fishing and reverse docking. The water solubility of 20(R)-PPT 3-O-glucoside increased as well.


Asunto(s)
Ginsenósidos , Glicosilación , Ginsenósidos/farmacología , Simulación del Acoplamiento Molecular , Damaranos , Glucósidos
17.
Yao Xue Xue Bao ; 48(2): 193-205, 2013 Feb.
Artículo en Zh | MEDLINE | ID: mdl-23672015

RESUMEN

Abstract: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.


Asunto(s)
Artemisininas/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/metabolismo , Biología Sintética , Familia de Aldehído Deshidrogenasa 1 , Antimaláricos/metabolismo , Antimaláricos/provisión & distribución , Artemisininas/provisión & distribución , Vías Biosintéticas , Dosificación de Gen , Ingeniería Genética , Isoenzimas/genética , ARN Nucleotidiltransferasas/genética , Retinal-Deshidrogenasa/genética
18.
Yao Xue Xue Bao ; 48(2): 187-92, 2013 Feb.
Artículo en Zh | MEDLINE | ID: mdl-23672014

RESUMEN

The synthetic biology matures to promote the heterologous biosynthesis of the well-known drug paclitaxel that is one of the most important and active chemotherapeutic agents for the first-line clinical treatment of cancer. This review focuses on the construction and regulation of the biosynthetic pathway of paclitaxel intermediates in both Escherichia coli and Saccharomyces cerevisiae. In particular, the review also features the early efforts to design and overproduce taxadiene and the bottleneck of scale fermentation for producing the intermediates.


Asunto(s)
Alquenos/metabolismo , Diterpenos/metabolismo , Escherichia coli/metabolismo , Paclitaxel/biosíntesis , Saccharomyces cerevisiae/metabolismo , Biología Sintética , Alquenos/química , Antineoplásicos Fitogénicos/biosíntesis , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/metabolismo , Vías Biosintéticas , Diterpenos/química , Fermentación , Ingeniería Metabólica , Paclitaxel/química , Paclitaxel/metabolismo , Profármacos
19.
Yao Xue Xue Bao ; 48(2): 228-35, 2013 Feb.
Artículo en Zh | MEDLINE | ID: mdl-23672019

RESUMEN

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.


Asunto(s)
Proteínas Luminiscentes/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Biología Sintética , Secuencia de Aminoácidos , Secuencia de Bases/genética , Clonación Molecular , Cartilla de ADN/biosíntesis , Regulación Fúngica de la Expresión Génica , Vectores Genéticos , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , Transformación Genética , Proteína Fluorescente Roja
20.
Z Naturforsch C J Biosci ; 67(3-4): 195-207, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22624336

RESUMEN

Amorpha-4,11-diene is the precursor of the antimalarial compound artemisinin. The effect of Vitreoscilla hemoglobin (VHb) and its yeast-conform variant (VHbm) on amorpha-4,11-diene production in engineered Saccharomyces cerevisiae was investigated. First, the VHb gene was mutated to the yeast-conform variant VHbm based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial VHbm gene contained codons preferred by the yeast translation machinery. Two yeast expression vectors containing VHb or VHbm gene were constructed and introduced into the amorpha-4,11-diene-producing strain S. cerevisiae WK1 to form WK1[VHb] and WK1[VHbm], respectively. Western blot and CO-difference spectrum absorbance assay showed that VHb and VHbm were successfully expressed. In shake flasks, VHbm expression conferred higher cell growth than VHb expression. GC-MS results indicated the amorpha-4,11-diene production in WK1[VHbm] and WK1[VHb] was 3- and 2-fold higher than that in WK1, respectively. This suggests that VHb might improve the amorpha-4,11-diene production in engineered S. cerevisiae.


Asunto(s)
Proteínas Bacterianas/metabolismo , Saccharomyces cerevisiae/genética , Sesquiterpenos/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Western Blotting , Cartilla de ADN , Datos de Secuencia Molecular , Plásmidos , Sesquiterpenos Policíclicos , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Hemoglobinas Truncadas/genética
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