In vitro reconstitution of the cyclosporine specific P450 hydroxylases using heterologous redox partner proteins

The cytochrome P450 enzymes (CYPs) CYP-sb21 from Sebekia benihana and CYP-pa1 from Pseudonocardia autotrophica are able to hydroxylate the immunosuppressant cyclosporin A (CsA) in a regioselective manner, giving rise to the production of two hair-stimulating agents (with dramatically attenuated immunosuppressant activity), γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH) and γ-hydroxy-N-methyl-l-Leu9-CsA (CsA-9-OH). Recently, the in vitro activity of CYP-sb21 was identified using several surrogate redox partner proteins. Herein, we reconstituted the in vitro activity of CYP-pa1 for the first time via a similar strategy. Moreover, the supporting activities of a set of ferredoxin (Fdx)/ferredoxin reductase (FdR) pairs from the cyanobacterium Synechococcus elongatus PCC 7942 were comparatively analyzed to identify the optimal redox systems for these two CsA hydroxylases. The results suggest the great value of cyanobacterial redox partner proteins for both academic research and industrial application of P450 biocatalysts.     

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.     

Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.     

Identification of a vitamin D3-specific hydroxylase genes through actinomycetes genome mining

We previously completed whole-genome sequencing of a rare actinomycete named Sebekia benihana, and identified the complete S. benihana cytochrome P450 complement (CYPome), including 21 cytochrome P450 hydroxylase (CYP), seven ferredoxin (FD), and four ferredoxin reductase (FDR) genes. Through targeted CYPome disruption, a total of 32 S. benihana CYPome mutants were obtained. Subsequently, a novel cyclosporine A region-specific hydroxylase was successfully determined to be encoded by a CYP-sb21 gene by screening the S. benihana CYPome mutants. Here, we report that S. benihana is also able to mediate vitamin D₃ (VD₃) hydroxylation. Among the 32 S. benihana CYPome mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb3a, failed to show regio-specific hydroxylation of VD₃ to 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃. Moreover, the VD₃ hydroxylation activity in the ΔCYP-sb3a mutant was restored by CYP-sb3a gene complementation. Since all S. benihana FD and FDR disruption mutants maintained VD₃ hydroxylation activity, we conclude that CYP-sb3a, a member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific VD₃ hydroxylation process in S. benihana. Expression of the CYP-sb3a gene exhibited VD₃ hydroxylation in the VD₃ non-hydroxylating Streptomyces coelicolor, implying that the regio-specific hydroxylation of VD₃ is carried out by a specific P450 hydroxylase in S. benihana.     

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> and <Emphasis Type="Italic">Pelobates cultripes</Emphasis>

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (N _e /N _c ) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (N _b ) and the effective population size (N _e ) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the N _e /N _c and N _b /N _c ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl?=?0.51%; P. cultripes?=?1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of N _e and N _b provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.     

Intestinal enterobacteria of the hibernating <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> L. bees

Dynamics of enterobacteria of normal intestinal microflora was studied in Apis mellifera mellifera L. bees hibernating under snow in the Western Urals. The cell numbers (N) of the predominant species Klebsiella oxytoca increased from 10-10⁶ CFU/bee in November 2004 to 10⁴-10⁷ CFU/bee in March 2005; its frequency of occurrence (P) increased from 92 to 100%. Increase of Providencia rettgeri (11.2004: N up to 10⁶, P 25%; 03.2005: N 10²-10⁶, P 80%) was accompanied by the substitution of Morganella morganii (11.2004: N up to 10⁶, P 25%) with Proteus vulgaris (03.2005: N up to 10⁵, P 8%). By spring, Hafnia alvei and Citrobacter sp., which are pathogenic to bees, disappeared (11.2004: N up to 10⁵, P 13 and 10%, respectively). Endophytic species Pantoea agglomerans, Leclecria sp., and other representatives of the “Enterobacter agglomerans” group were present in November and after the first emergence in spring (N up to 10⁵; November: P 15%; April: P 23%). In April, the number of enterobacteria decreased to 10⁵, and P. rettgeri became the predominant species (P 54%) instead of K. oxytoca (P 43%).     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

Ectopic expression and functional characterization of type III polyketide synthase mutants from <Emphasis Type="Italic">Emblica officinalis</Emphasis> Gaertn

Key message

Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase.

Abstract

Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

     

Biodiversity and ecology of flower-associated actinomycetes in different flowering stages of <Emphasis Type="Italic">Protea repens</Emphasis>

Actinomycete bacteria have previously been reported from reproductive structures (infructescences) of Protea (sugarbush/suikerbos) species, a niche dominated by fungi in the genera Knoxdaviesia and Sporothrix. It is probable that these taxa have symbiotic interactions, but a lack of knowledge regarding their diversity and general ecology precludes their study. We determined the diversity of actinomycetes within Protea repens inflorescence buds, open inflorescences, young and mature infructescences, and leaf litter surrounding these trees. Since the P. repens habitat is fire-prone, we also considered the potential of these bacteria to recolonise infructescences after fire. Actinomycetes were largely absent from flower buds and inflorescences but were consistently present in young and mature infructescences. Two Streptomyces spp. were the most consistent taxa recovered, one of which was also routinely isolated from leaf litter. Lower colonisation rates were evident in samples from a recently burnt site. One of the most consistent taxa isolated from older trees in the unburnt site was absent from this site. Our findings show that P. repens has a distinct community of actinomycetes dominated by a few species. These communities change over time and infructescence developmental stage, season and the age of the host population. Mature infructescences appear to be important sources of inoculum for some of the actinomycetes, seemingly disrupted by fire. Increased fire frequency limiting maturation of P. repens infructescences could thus impact future actinomycete colonisation in the landscape. Streptomyces spp. are likely to share this niche with the ophiostomatoid fungi, which merits further study regarding their interactions and mode of transfer.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Physical mapping of NBS-coding resistance genes to the <Emphasis Type="Italic">Me</Emphasis>-gene cluster on chromosome P9 reveals markers tightly linked to the <Emphasis Type="Italic">N</Emphasis> gene for root-knot nematode resistance in pepper

Natural root-knot nematode resistance genes are unique resources to control this major pest in pepper (Capsicum annuum). Although four genes (Me1, Me3, Me7 and N) conferring broad-spectrum resistance were mapped to a cluster in a 28-cm interval on chromosome P9, limited markers targeting this region were available. In the present study, the Me-gene cluster was structurally annotated for resistance genes to develop markers targeting the N gene. As a result, the Me-gene cluster (4.07 Mb in size) was found to contain three resistance gene hotspots. In addition, a SSR maker tightly linked to the N gene (0.8 cM away) was developed for marker-assisted selection in pepper.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Cinnamate 4-hydroxylase (C4H) catalyzes the regioselective para-hydroxylation of trans-cinnamic acid to form p-coumaric acid, the biosynthetic precursor of phenylpropanoid-based polymers. These biopolymers play an essential role in plant structure construction, development, and defense. Herein the open reading frame of CaC4H2 was cloned from Camptotheca acuminata, a deciduous camptothecin-producing tree native to China. CaC4H2 showed 94 % amino acid residues identity with those of reported CaC4H, which suggested that CaC4H2 is an isoform of C4Hs presented in C. acuminata. The intact CaC4H2 was overexpressed in Escherichia coli with its functional reaction partner cytochrome P450 reductase, CamCPR, which transfers electrons from NADPH to CaC4H2 to support the catalytic hydroxylation activity of CaC4H2. Upon incubating trans-cinnamic acid with the recombinant CaC4H2 and tCamCPR, the formation of p-coumaric acid was confirmed by the HPLC–DAD and UPLC-DAD-ESIMS analyses, which indicated the catalytic hydroxylation activity of CaC4H2. Quantitative real-time PCR analyses showed that CaC4H2 was expressed in all tissues of C. acuminata seedlings, which is consistent with the well-known conclusion that the C4H-catalyzed hydroxylation reaction is a key step within the biosynthetic pathway of phenylpropanoids. The functional characterization of CaC4H2 will be useful for molecular breeding and sustainable utilization and protection of the camptothecin-producing plant.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

Antimicrobial properties of endophytic actinomycetes isolated from <Emphasis Type="Italic">Combretum latifolium</Emphasis> Blume,a medicinal shrub from Western Ghats of India

Endophytic actinomycetes were isolated from Combretum latifolium Blume (Combretaceae),Western Ghats of Southern India and identified by its characteristic culture morphology and molecular analysis of 16S rRNA gene sequences. In this survey of endophytic actinomycetes, a total of 117 isolates representing 9 different genera of endophytic actinomycetes were obtained using four different isolation media and several of them seemed to be novel taxa. Streptomyces genera (35%) was the most frequently isolated strains, followed by Nocordiopsis (17%) and Micromonospora (13%). ISP-4 medium recovered more isolates (47%) when compared to rest of the media used. Preliminary antibacterial activity of the isolates was carried out by confrontation test. Ethyl acetate fraction of selected isolates in disc diffusion assay exhibited broad spectrum antimicrobial activity against test human pathogens. All Streptomyces spp. strains displayed significant antimicrobial activity against test pathogens. Strain CLA-66 and CLA-68 which are Nocordipsis spp. inhibited both bacterial and fungal pathogens where as other isolates inhibited atleast three test human pathogens in disc diffusion assay. Antimicrobial screening of endophytic actinomycetes from this host may represent a unique potential niche for antimicrobial compounds of industrial and pharmaceutical applications. This work is the first comprehensive report on incidence of potential endophytic actinomycetes inhabiting C. latifolium Blume.     

<Emphasis Type="Italic">NmEXT</Emphasis> Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis>

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.     

Improved recovery and biological activities of an engineered polyene NPP analogue in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

NPP A1 produced by Pseudonocardia autotrophica is a unique disaccharide-containing polyene macrolide. NPP A1 was reported to have higher water solubility and lower hemolytic toxicity than nystatin A1 while retaining its antifungal activity. An engineered NPP A1 analogue, NPP A2, was generated by inactivation of the nppL gene, encoding a P450 monooxygenase in P. autotrophica. The resulting compound exhibited the corresponding chemical structure of NPP A1 but lacked a C10 hydroxyl group. In this study, newly developed crystallization recovery methods for NPP A2 purification, followed by an evaluation of in vitro antifungal activity and hemolytic activity, were performed. The crystallization methods were designed to eliminate the undesired viscous impurities encountered during the NPP A2 purification process, resulting in improved purity from 5.3 to 83.5% w/w. NPP A2 isolated from the improved purification process also exhibited two times higher antifungal activity and 1.8 times higher hemolytic toxicity than those of NPP A1. These results suggest that the minor structural modification of disaccharide-containing polyene macrolides, such as removing a C10 hydroxyl group, might require an alternative recovery process, such as crystallization, to confirm its improved biological activity.     

Phenotypic and phylogenetic characterization of actinomycetes isolated from <Emphasis Type="Italic">Lasius niger</Emphasis> and <Emphasis Type="Italic">Formica cunicularia</Emphasis> ants

Multiple actinomycete strains were isolated from two ant species, Lasius niger and Formica cunicularia, and their phenotypic properties and phylogenetic position were studied. Partial sequencing of 16S rRNA assigned the greater part of them to the genus Streptomyces, but only one belonged to Nocardia. However, some isolates had significant color and morphological differences from their closest phylogenetic relatives. The abundance and biodiversity of actinomycete communities isolated from L. niger ants greatly exceeded those found for F. cunicularia. All of the actinomycetes associated with F. cunicularia ants demonstrated cellulolytic activity, but only one had such ability among the strains associated with black ants.     

Alteration of a recombinant protein <Emphasis Type="Italic">N</Emphasis>-glycan structure in silkworms by partial suppression of <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase gene expression

Objective

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

Results

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man₃GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

Conclusions

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

     

Characterization of phosphate solubilizing rhizobacteria associated with pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) isolated from two agricultural soils

Phosphorous (P) availability is a major concern in European agriculture where reserves are limited. In the case of pea (Pisum sativum L.), one of the most important legumes in the human diet, P has specific effects on nodulation and N₂ fixation. Therefore, when biofertilization schemes are considered for pea cropping, it is very important to include symbiotic dinitrogen-fixing bacteria as well as phosphate-solubilizing bacteria (PSB). In this study sixteen PSB were isolated from the rhizosphere of two pea cultivars in two French soils with different characteristics. They were phenotypically and genotypically diverse displaying 9 different Two Primers-Random Amplified Polymorphic DNA (TP-RAPD) patterns. The 16S rRNA gene analysis of representative strains showed that they belong to four highly divergent phylogenetic groups. Most of the PSB strains belonged to the genus Pseudomonas and were closely related to Pseudomonas baetica, P. lutea, P. azotoformans, P. jessenii and P. frederiksbergensis. Other strains from the genus Burkholderia were closely related to B. caledonica and those from the genus Rhizobium to R. grahamii. The single strain of genus Bacillus was close to Bacillus toyonensis. Some phylogenetic groups to which our PSB strains belong are widely distributed in plant rhizospheres in different countries and continents. This is particularly interesting in the case of strains from the phylogenetic group of P. fluorescens which includes PSB strains with high ability to solubilize phosphate indicating that they may be used as biofertilizers in many soils.