Effect of culture media on protease and oxytetracycline production with mycelium and protoplasts of Streptomyces rimosus

For the simultaneous production of protease and oxytetracycline, mycelium and protoplasts of Streptomyces rimosus TM-55 were cultivated in basal medium containing soluble starch, corn steep liquid, ammonium sulphate, calcium carbonate, sodium chloride and soybean oil. Protease and oxytetracycline production increased with decreasing in ratio of culture broth to vessel volume from 1:2 to 1:5. Each ml of broth with 0.286 mg fresh mycelia yielded 168–204 units of protease and 785–972 g of oxytetracycline after replacement of corn steep liquor, sodium chloride and soybean oil with beef extract and sunflower oil, while each ml of broth with 7.5 × 10⁷ protoplasts produced 141–153 units of protease and 504–615 g of oxytetracycline. Protease and oxytetracycline production were low when the pH was 5.1 or 9.0. Soluble starch and ammonium sulphate were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease and oxytetracycline production. The productivity of protoplasts decreased sharply when the incubation temperature increased from 28 to 34 °C, while the productivity of mycelium was almost unchanged.     

Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5 % yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.     

Aminonitriles: Possible role in chemical evolution

The formation of HCN, ammonium cyanide, alkylnitriles, aminoacetonitrile and its C-and N-methyl homologs was demonstrated earlier in a simulated Jovian atmosphere. The polymeric material resulting in these experiments was shown to give glycine, alanine, sarcosine, aspartic acid and someimiono dibasic acids on acid hydrolysis suggesting thereby the participation of the monomeric nitriles into the formation of the polymeric products(s). Further examination of products resulting from semi-corona and are discharge through a mixture of methane and ammonia has provided evidence for the formation of alkylaminopropionitriles as a complex mixture and also some pyridyl and pyrimidyl type heterocyclic compounds. A GC-MS examination of the heterocyclics showed resemblance with those found in some carbonaceous chondrites. The significance of these findings in relation to chemical evolution will be discussed.     

Isolation and description ofHaloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophile common to Great Salt Lake sediments

A new halophilic species is described that was isolated from the hypersaline (>20%) surface sediments of Great Salt Lake, Utah, via transfer from MPN end-dilution tubes that contained a complex organic medium. The organism was an obligate anaerobe that proliferated optimally at approximately 13% salt, but did not grow significantly at <2% or ≥30% salt. It stained Gram-negative, was nonmotile, nonsporing, and contained an outer-wall membranous layer. The complex lipids of the organism were fatty acid esters that did not change dramatically during growth at 5% or 25% NaCl. The DNA base composition was 27.0±1 mol% guanosine plus cytosine. The temperature range for growth was >5°C and <60°C, the pH range was between 6.0 and 9.0. The doubling time for growth in complex medium with 25% NaCl was 7 h. The organism utilized carbohydrates, peptides, and amino acids. Butyrate, acetate, propionate. H₂, and CO₂ were the major fermentation end products formed. Glucose, mannose, fructose,n-acetyl glucosamine, and pectin were used as energy sources for growth. Methylmercaptan was produced from methionine degradation. The nameHaloanaerobium praevalens gen. nov. sp. nov. is proposed for the type strain GSL which has been deposited as DSM 2228. The taxonomic relationships ofH. praevalens to other obligate halophiles and anaerobes, as well as its biological role in the Great Salt Lake microbial ecosystem, are discussed.     

Relationship between the composition of the regulatory complex of esterases and the age ofMycobacterium phlei culture

During submerged cultivation ofMycobacterium phlei a mixture of macromolecular compounds ia released into the medium. Concentrated filtrates of cultures of different ages were separated on Sephadex G-25 fine and polyanions were found to predominate in the young culture. During further days of fermentation the proportion of polycations significantly increases. The results are discussed with respect to the regulatory complex of esterases.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.     

One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21 %, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.     

The reduction of aldehydes by broad bean and maize alcohol dehydrogenases and a study of the substrate binding to the enzyme protein

Alcohol dehydrogenase was prepared from 2-day germinating maize and 3-day germinating broad-bean seeds by ammonium sulphate fractionation of sodium phosphate extracts, chromatography onDEAE cellulose and Sephadex G-200. The activity of the broad beanADH amounted to182 800 units per mg protein, that of maizeADH 79 000 units per mg protein. Besides oxidation of a series of alcohols at pH optimum in the alkaline region and with KM equalling 10-2M, alcohol dehydrogenases isolated from both plants catalyze the reduction of acetaldehyde, n-propanal, n-butanal, isobutanal and crotonal at pH optimum in the neutral region with KM equalling 10-3M. The inhibition studies using fatty acids and chloride ions revealed that the oxidation of alcohols is inhibited competitively by both types of inhibitors, with inhibition constants of 10-2M and 10-1M, respectively. The inhibition in the presence of acetaldehyde is non-competitive since the inhibitors do not compete with acetaldehyde and do not form an enzyme-NADH-inhibitor complex, yet they obviously react with the enzyme-NAD product only, thus giving rise to an enzyme-NAD-inhibitor complex. These differences in the behaviour of inhibitors may be interpreted in the sense that the binding sites of ethanol and acetaldehyde as substrates for broad bean and maize alcohol dehydrogenases are non equivalent. The nonequivalency discussed in the text.     

Marine actinobacteria associated with marine organisms and their potentials in producing pharmaceutical natural products

Actinobacteria are ubiquitous in the marine environment, playing an important ecological role in the recycling of refractory biomaterials and producing novel natural products with pharmic applications. Actinobacteria have been detected or isolated from the marine creatures such as sponges, corals, mollusks, ascidians, seaweeds, and seagrass. Marine organism-associated actinobacterial 16S rRNA gene sequences, i.e., 3,003 sequences, deposited in the NCBI database clearly revealed enormous numbers of actinobacteria associated with marine organisms. For example, RDP classification of these sequences showed that 112 and 62 actinobacterial genera were associated with the sponges and corals, respectively. In most cases, it is expected that these actinobacteria protect the host against pathogens by producing bioactive compounds. Natural products investigation and functional gene screening of the actinobacteria associated with the marine organisms revealed that they can synthesize numerous natural products including polyketides, isoprenoids, phenazines, peptides, indolocarbazoles, sterols, and others. These compounds showed anticancer, antimicrobial, antiparasitic, neurological, antioxidant, and anti-HIV activities. Therefore, marine organism-associated actinobacteria represent an important resource for marine drugs. It is an upcoming field of research to search for novel actinobacteria and pharmaceutical natural products from actinobacteria associated with the marine organisms. In this review, we attempt to summarize the present knowledge on the diversity and natural products production of actinobacteria associated with the marine organisms, based on the publications from 1991 to 2013.     

Generation and characterization of a novel human IgG1 antibody against vascular endothelial growth factor receptor 2

VEGF and its receptors, especially VEGFR2 (KDR), are known to play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies. This study was aimed at developing a fully human IgG1 antibody (mAb-04) constructed from a phage-derived scFv, targeting the VEGF/VEGFR2 pathway. Firstly, an innovative transfection system, containing two recombinant expression vectors (pMH3 and pCApuro), were introduced into CHO-s cells and clones with higher yield selected accordingly. After an optimal fermentation condition was determined, fed-batch fermentation was performed in 5-L bioreactor with a final yield up to 60 mg/L. Further, cell proliferation, wound healing, transwell invasion, tube formation and chick embryo chorioallantoic membrane assays showed significant anti-angiogenic activity of mAb-04 in vitro and in vivo. In addition, the results of Western blotting indicated the ability of mAb-04 to inhibit VEGF-induced VEGFR2 signaling pathway. Finally, ADCC assay demonstrated that mAb-04 is capable of mediating tumor cell killing in presence of effector cells. This study has therefore proved that the full-length antibody targeting human VEGFR2 has potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Investigation of the X virus inhibitor from potato leaf sap

The inhibitor of the X virus (Jermoljev, Albrechtová 1965) can be separated from other proteins by 60 min centrifugation at 40000 r.p.m. in the Spinco L centrifuge and by subsequent gel filtration of the supernatant on a Sephadex G-50 column. For the elution it is best to use Tris-maleate buffers and 0·2M Tris-HCl of pH 7·2. The inhibitor is present in the first fractions containing proteins, between 30 and 45 ml. It is most suitable to concentrate the inhibitor by salting out with ammonium sulphate to 30–50% saturation. The inhibitor can be preserved by adding ammonium sulphate to the sap. At 3°C it can be kept in this manner for 2 months. If the pH of the sap drops below 5·5, the inhibitor is inactivated. Complete inactivation of the inhibitor takes place after a 10 min heating at 67·5°C. No substantial difference in the amount of inhibitor in the sap from apical leaves and leaves from middle levels has been found. Keeping the plants in the dark has no effect on the inhibitor level.     

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.     

Isolation and identification of an endophytic fungus Pezicula sp. in Forsythia viridissima and its secondary metabolites

In a survey of endophytic fungal biodiversity, an antimicrobial endophytic isolate zjwcf069 was obtained from twigs of Forsythia viridissima, Zhejiang Province, Southeast China. Zjwcf069 was then identified as Pezicula sp. through combination of morphological and phylogenetic analysis based on ITS-rDNA. Zjwcf069 here represented the first endophytic fungus in Pezicula isolated from host F. viridissima. From the fermentation broth, four compounds were obtained through silica gel column chromatography and Sephadex LH-20 under the guide of bioassay. Their structures were elucidated by spectroscopic analysis as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 here stood for the very first time as natural product from microbes. In vitro antifungal assay showed that compound 1 displayed growth inhibition against 9 plant pathogenic fungi, especially Botrytis cinerea and Fulvia fulva with EC₅₀ values below 50 μg/mL. Endophytic fungi in medicinal plants were good resources for bioactive secondary metabolites.     

The mitochondrial complex I of trypanosomatids - an overview of current knowledge

The contribution of trypanosomatid mitochondrial complex I for energy transduction has long been debated. Herein, we summarize current knowledge on the composition and relevance of this enzyme. Bioinformatic and proteomic analyses allowed the identification of many conserved and trypanosomatid-specific subunits of NADH:ubiquinone oxidoreductase, revealing a multifunctional enzyme capable of performing bioenergetic activities and possibly, also of functioning in fatty acid metabolism. A multimeric structure organized in 5 domains of more than 2 MDa is predicted, in contrast to the 1 MDa described for mammalian complex I. The relevance of mitochondrial complex I within the Trypanosomatidae family is quite diverse with its NADH oxidation activity being dispensable for both procyclic and bloodstream Trypanosoma brucei, whereas in Phytomonas serpens the enzyme is the only respiratory complex able to sustain membrane potential. Aside from complex I, trypanosomatid mitochondria contain a type II NADH dehydrogenase and a NADH-dependent fumarate reductase as alternative electron entry points into the respiratory chain and thus, some trypanosomatids may have bypassed the need for complex I. The involvement of each of these enzymes in the maintenance of the mitochondrial redox balance in trypanosomatids is still an open question and requires further investigation.     

Kinetics of the appearance of mRNA for light-harvesting chlorophyll a/b protein in polysomes of barley

Polysomes from dark-grown and illuminated barley seedlings were translated in cell-free systems. The translation products reacting with the antibody against the light-harvesting chlorophyll a/b protein (LHCP) were analyzed by polyacrylamide gel electrophoresis. It was found that, in addition to the precursor protein of LHCP, a product was obtained that co-migrated with the mature protein. Furthermore, the results show that the light-induced proly(A)RNA for LHCP is integrated into the polysomal complex without delay, indicating that the integration of LHCP into the membrane is controlled at a higher level of gene expression.     

Role of organic acid metabolism in the biosynthesis of peptide ergot alkaloids

Regulatory mechanism of primary metabolism responsible for the biosynthesis of peptide ergot alkaloids was proposed on the basis of experimental results from the production phase ofClaviceps purpurea (Fr.) Tul. in parasitic and saprophytic cultures. The production-phase metabolism is characterized by uncoupling of glycolysis from the citric acid cycle and by a break in this cycle at the level of the 2-exoglutarate dehydrogenase complex. This regulation is due to the inhibition by citrate and malate, i.e. the final products continuously taken up from the external medium. The rate of the intact reaction steps of the citric acid cycle is therefore not limited by the actual acetyl-CoA or oxaloacetate pool. The regulation also leads to an excessive synthesis of acetyl-CoA, 2-oxoglutarate and oxaloacetate, which represent the key metabolites of primary metabolism, and to their utilization in the biosynthesis of peptide alkaloids.     

Effect of nitrogen sources on shoot bud differentiation ofDioscorea deltoidea Wall. callus cultures

Tuber callus by subculture ofDioscorea deltoidea Wall, was grown with different sources of nitrogen on N-free Murashige and Skoog’s basal media supplemented with a high level of kinetin (1, 3, 6, 9 and 12 ml 1^-1) and low concentration of auxin (0.01 mg 1^-1 2,4-D) for shoot bud differentiation. Shoot and root differentiation was observed only in the case of ammonium nitrate. Other sources of nitrogen failed to produce shoot bud differentiation except in the case of ammonium citrate where tissues were slight green in colour and were recognizable as pro-embryo.     

Translation of hepatic mRNA in extracts from wheat germ embryos

Extracts from wheat embryos have been used to study the incorporation of amino acids into TCA insoluble products using hepatic mRNA fractions. The properties of this system are described and compared to the incorporation obtained with poly U and rabbit globin mRNA. SDS-acrylamide gel analysis showed that the major polypeptide synthesized with globin mRNA co-migrates with rabbit globin (15 500 daltons). Rat liver products were numerous, with molecular weights from less than 10000 to greater than 65000 daltons. The KCl concentration for maximum incorporation into TCA precipitable polypeptides with hepatic mRNA was not the optimum KCl concentration for synthesis of complete products.     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.