Protein Engineering of PhUGT, a Donor Promiscuous Glycosyltransferase, for the Improved Enzymatic Synthesis of Antioxidant Quercetin 3-O-N-Acetylgalactosamine.

Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymatically synthesized due to the lack of well-identified N-acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identified flavonoid 3-O-galactosyltransferase from Petunia hybrida, was demonstrated to display quercetin GalNAc-T activity, transferring a N-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc with a low conversion of 11.7% at 40 °C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got an enhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidant activity with other quercetin 3-O-glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase (GT), recognizing seven sugar donors. This finding overturned a previous notion that PhUGT exclusively recognized UDP-galactose (UDP-Gal). The reason why PhUGT was mistaken for a UDP-Gal-specific GT was demonstrated to be a shorter reaction time, in which many quercetin 3-O-glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factory expressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundation for the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.

Glycodiversifying Testosterone with a Promiscuous Glycosyltransferase OsSGT2 from Ornithogalum saundersiae.

The diversity expansion of testosterone17-O-ß-glycosides (TGs) will increase the probability of screening more active molecules from their acetylated derivatives with anticancer activities. Glycosyltransferases (GTs) responsible for the increased diversity of TGs, however, were seldom documented. Herein, a glycosyltransferase OsSGT2 with testosterone glycodiversification capacity was identified from Ornithogalum saundersiae through transcriptome-wide mining. Specifically, OsSGT2 was demonstrated to be reactive with testosterone and eight donors. OsSGT2 displayed both sugar-aglycon and sugar-sugar GT activities. OsSGT2-catalyzed testosterone glycodiversification could be achieved, generating testosterone monoglycosides and disglycosides with varied percentage conversions. Among the eight donors, the conversion of UDP-Glc was the highest, approaching 90%, while the percentage conversions of UDP-GlcNAc, UDP-Gal, helicin, and UDP-Rha were less than 10%. Protein engineering toward F395 was thus performed to improve the conversion of UDP-GlcNAc. Eight variants displayed increased conversions and the mutant F395C got the highest conversion of 72.11 ± 7.82%, eight times more than that of the wild-type. This study provides a promising alternative for diversity expansion of TGs, also significant insights into the molecular basis for the conversion improvement of sugar donors.

Altering the Regioselectivity of Cytochrome P450 BM3 Variant M13 toward Genistein through Protein Engineering and Variation of Reaction Conditions.

The biocatalysts responsible for the enzymatic synthesis of hydroxygenisteins, derivatives of genistein with multiple activities, usually show regioselective promiscuity, hydroxylating genistein to form a mixture of multiple products, which, in turn, results in a cumbersome separation and purification. Hence, it is highly desired to explore the underlying mechanism regulating the regioselectivity of hydroxylases. M13 is a variant of cytochrome P450 BM3 with oxidant activity toward genistein. Herein, genistein was demonstrated to be hydroxylated by M13 to form a mixture of 3'-hydroxygenistein (3'-OHG) and 8-hydroxygenistein (8-OHG), each giving 4% conversion with a ratio of 1:1. Protein engineering toward M13 was thus performed to improve its regioselectivity. When isoleucine at position 86 was mutated into cysteine, the resultant variant M13I86C displayed improved regioselectivity toward 3'-OHG with an increased conversion of 8.5%. The double mutation M13I86CP18W further boosted the conversion of 3'-OHG to 9.6%, and the ratio of 3'-OHG to 8-OHG increased to 12:1. Conversely, both CoCl2 and glucose 6-phosphate (G6P) could lead to more 8-OHG. When Co2+ reached 37.5 mM, M13I86CP18W could give an 8-OHG conversion of 22.4%. The maximal ratio of 8-OHG to 3'-OHG reached 130 when 62.5 mM Co2+ was included in the reaction mixture. With the increase of G6P from 10 to 40 mM, the conversion of M13I86CP18W to 8-OHG gradually increased to 22.6%, while the conversion to 3'-OHG decreased to 6%. Thus, both intrinsic residues and external reaction conditions can affect the regiospecificity of M13, which laid the foundation for the selection of suitable biocatalysts for the hydroxylation of genistein.

Seven new 1-oxygenated cholestane glycosides from Ornithogalum saundersiae.

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].

Structure and bioactivity of cholestane glycosides from the bulbs of Ornithogalum saundersiae Baker.

Eight undescribed cholestane glycosides named osaundersioside A-H, along with three previously known compounds named osaundersioside I-K were isolated from Ornithogalum saundersiae Baker bulbs (Asparagaceae). Their structures were elucidated by extensive spectroscopic analysis and chemical methods. All isolates were evaluated for their cytotoxic activity and inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Osaundersioside C was thus determined to exhibit specific cytotoxicity towards MCF-7 cell line with an IC50 value of 0.20 µM, Osaundersioside H exhibited inhibitory effect on NO production in macrophages at the concentration of 10-5 M, with inhibition rate of 56.81%.

Characterization of two flavonol synthases with iron-independent flavanone 3-hydroxylase activity from Ornithogalum caudatum Jacq.

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.

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity.

Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase (SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (ASGs). Initially, an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3ß- and 17ß-hydroxyl steroids. Unexpectedly, in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA, specifically catalyzing the acetylations of sugar moieties of steroid 17ß-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-ß-glucosides (AT-17ß-Gs) and acetylated estradiol-17-O-ß-glucosides (AE-17ß-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 3'-acetylated testosterone17-O-ß-glucoside towards seven human tumor cell lines were thus observable.

cDNA isolation and functional characterization of squalene synthase gene from Ornithogalum caudatum.

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.

Functional analyses of OcRhS1 and OcUER1 involved in UDP-L-rhamnose biosynthesis in Ornithogalum caudatum.

UDP-L-rhamnose (UDP-Rha) is an important sugar donor for the synthesis of rhamnose-containing compounds in plants. However, only a few enzymes and their encoding genes involved in UDP-Rha biosynthesis are available in plants. Here, two genes encoding rhamnose synthase (RhS) and bi-functional UDP-4-keto-6-deoxy-D-glucose (UDP-4K6DG) 3, 5-epimerase/UDP-4-keto-L-rhamnose (UDP-4KR) 4-keto-reductase (UER) were isolated from Ornithogalum caudatum based on the RNA-Seq data. The OcRhS1 gene has an ORF (open reading frame) of 2019 bp encoding a tri-functional RhS enzyme. In vitro enzymatic assays revealed OcRhS1 can really convert UDP-D-glucose (UDP-Glc) into UDP-Rha via three consecutive reactions. Biochemical evidences indicated that the recombinant OcRhS1 was active in the pH range of 5-11 and over the temperature range of 0-60 °C. The Km value of OcRhS1 for UDP-Glc was determined to be 1.52 × 10-4 M. OcRhS1 is a multi-domain protein with two sets of cofactor-binding motifs. The cofactors dependent properties of OcRhS1 were thus characterized in this research. Moreover, the N-terminal portion of OcRhS1 (OcRhS1-N) was observed to metabolize UDP-Glc to form intermediate UDP-4K6DG. OcUER1 contains an ORF of 906 bp encoding a polypeptide of 301 aa. OcUER1 shared high similarity with the carboxy-terminal domain of OcRhS1 (OcRhS1-C), suggesting its intrinsic ability of converting UDP-4K6DG into UDP-Rha. It was thus reasonably inferred that UDP-Glc could be bio-transformed into UDP-Rha under the collaborating action of OcRhS1-N and OcUER1. The subsequently biochemical assay verified this notion. Importantly, expression profiles of OcRhS1 and OcUER1 revealed their possible involvement in the biosynthesis of rhamnose-containing polysaccharides in O. caudatum.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.

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.

[Enzymatic cyclization of peptides using immobilized sortase A].

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.

cDNA cloning and expression analysis of farnesyl pyrophosphate synthase from Ornithogalum saundersiae.

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.

Molecular cloning and yeast expression of cinnamate 4-hydroxylase from Ornithogalum saundersiae baker.

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.

[The advance in synthetic biology: towards a microbe-derived paclitaxel intermediates].

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.

[Advances in functional studies of nonstructural proteins and development of antiviral agents for enterovirus 71].

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.

[Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis].

The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.

[Recent advances in the study of amorpha-4,11-diene synthase and its metabolic engineering].

Amorpha-4,11-diene synthase (ADS) can convert farnesyl pyrophosphate (FPP) to amorpha-4, 11-diene, a precursor of artemisinin. ADS plays an important role in the biosynthesis of artemisinin. This review summarizes the molecular biology and metabolic engineering study of ADS in recent years. The genomic DNA and its cDNA sequences of amorpha-4, 11-diene synthase were cloned from Artemisia annua L. The cDNA encoding amorpha-4, 11-diene synthase contains a 1 641 bp open reading frame coding for 546 amino acids. ADS shows a broad pH optimum and an absolute requirement for divalent metal ions as cofactors. The specificity of ADS to the substrates and products is not high and the formation of amorpha-4, 11-diene by ADS from FPP is achieved by an initial 1, 6-closure with subsequent 1, 10-closure. The ADS cDNA cloned from Artemisia annua L, or totally synthesized by PCR, was introduced into different hosts including E. coli, S. cerevisiae, Nicotiana tabacum L. Arabidopsis thaliana and A. nidulans resulting in varied engineering microorganisms and cells producing amorpha-4, 11-diene. The way to improve the production of amorpha-4, 11-diene was investigated by two strategies such as improving the supply of substrate and directing FPP flux to amorpha-4, 11-diene production from competing pathways.

[Recent advances in the biosynthesis of Taxol].

Taxol is one of the most potent chemotherapeutic agents known, showing excellent activity against a range of cancers. In addition to anticancer, taxol has the effect of preventing graft arteriosclerosis, antiscaring formation and inhibiting angiogenesis. There are five possible routes to industrialize taxol production: isolation from the bark of the yew species, total synthesis, semisynthesis, tissue or cell culture, endophytic fungal fermentation and metabolism engineering. There are at least 14 genes related to the taxol biosynthesis had been cloned from yews and functionally expressed in different hosts. The combinational expression system of taxol makes progress as the clarification of biosynthetic pathway of taxol.