Production enhancement of the glycopeptide antibiotic A40926 by an engineered Nonomuraea gerenzanensis strain

Biotechnology Letters - To improve the production of A40926, a combined strategy of constructing the engineered strain and optimizing the medium was implemented. The engineered strain lcu1 with the...     

Synthesis and antibacterial activity of alkyl derivatives of the glycopeptide antibiotic A40926 and their amides

New derivatives of the glycopeptide antibiotic A40926 were synthesized and evaluated for antimicrobial activity against VRE. Deacylated A40926 was obtained by microbial transformation of the parent antibiotic with the use of Actinoplanes teichomyceticus ATCC 31121. Regioselective synthesis of alkylated derivatives of Deacyl A40926 was carried out using lipophilic aliphatic and aromatic halides or aldehydes. Further modification of the two carboxylic acids was performed to increase antibiotic activity. Poor antimicrobial activity was observed for the derivatives obtained by lipophilic mono- or dialkylation of the amino groups present on the molecule, while simultaneous condensation of both carboxylic groups, in hydrophobic derivatives, with dibasic amines led to a strong increase in antibiotic activity.     

A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.     

The glycopeptide domain of the rat vasopressin precursor

The vasopressin precursor is composed of 3 domains, namely vasopressin, MSEL-neurophysin and a glycopeptide. Processing occurs during axonal transport from hypothalamus to neurohypophysis from which the 3 fragments can be isolated. The glycopeptide fragment of the rat vasopressin precursor has been purified and sequenced. Despite the fact that rat MSEL-neurophysin is shortened (93 residues instead of 95 for other mammals), rat glycopeptide has 39 residues, as do the other mammalian glycopeptides, suggesting a similar processing. Fifteen substitutions are however observed when compared to ox glycopeptide. The C-terminal part of MSEL-neurophysin (residues 77-93) and the glycopeptide are encoded by the same exon and the homologies when compared with their bovine counterparts are 58% and 62% respectively. In contrast, the central part of rat MSEL-neurophysin (residues 10-76), which is encoded by a separate exon, displays 96% of homology; vasopressin and the N-terminal part of MSEL-neurophysin (residues 1-9), encoded by a third exon, are nearly invariant.     

Continuous biotransformation of glycopeptide antibiotic A40926 in a cascade of three airlift bioreactors using immobilized Actinoplanes teichomyceticus cells

Immobilized cells of Actinoplanes teichomyceticus ATCC 31121 were used to selectively cleave the acyl group of A40926 yielding the deacylated form of the molecule. The feasibility of this particular biotransformation in a series of three perfectly mixed airlift bioreactors with immobilized cells was examined. A continuously operated airlift cascade was designed using a model for a series of reactors with immobilized biocatalyst beads obeying Michaelis–Menten kinetics. In independent experimental runs the cascade bioreactor system was operated continuously for 56 days with an overall conversion of 99%. Model estimates for reactor volumes and relative conversions were found to be in a good agreement with the experimental results.     

Guinea pig copeptin. The glycopeptide domain of the vasopressin precursor

The vasopressin precursor is composed of 3 domains in line, namely vasopressin, MSEL-neurophysin and a glycopeptide referred to as copeptin, which are separated during the processing. In guinea pig neurohypophysis, the precursor is partially processed so that a two-domain fragment, MSEL-neurophysin--copeptin, can be found along with free MSEL-neurophysin adn copeptin. Guinea pig copeptin has been sequenced. It is a glycopeptide composed of 38 amino acid residues rather than the 39 found in other mammalian copeptins. Compared with other copeptins, that from guinea pig shows a few substitutions and the deletion of one acidic residue, probably in position 32. This deletion might be responsible for incomplete cleavage by the trypsin-like processing enzyme.     

Stem cell maintenance by manipulating signaling pathways: past,current and future

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676]     

The glycopeptide moiety of vasopressin-neurophysin precursor is neurohypophysial prolactin releasing factor

All of the classically-described hypothalamic, hypophysiotropic factors that regulate anterior pituitary hormone secretion have now been isolated and identified except for prolactin releasing factor. We report here that the 39-amino acid glycopeptide comprising the carboxyterminus of the neurohypophysial vasopressin-neurophysin precursor stimulates prolactin release from cultured pituitary cells as potently as does thyrotropin releasing hormone but has no effect on the secretion of other pituitary hormones. Furthermore, antisera to the glycopeptide administered to lactating rats attenuated suckling-induced prolactin secretion. Thus, this glycopeptide appears to be the neurohypophysial prolactin releasing factor.     

Biotransformation of the lipoglycopeptide antibiotic A40926 byActinoplanes teichomyceticus cells

Compound A40926, produced byActinomadura ATCC 39727, is a lipoglycopeptide antibiotic complex which inhibits Gram-positive bacteria andNeisseria species. Individual components of the complex have an identical glycopeptide core but differ in the acid chains attached to the amino group of the glucuronic moiety. Suspension cultures and resting cells ofActinoplanes teichomyceticus ATCC 31121 were able to deacylate compound A40926 factors to yield the glycopeptide nucleus, which can be then synthetically reacylated to form new analogs. In an optimized fedbatch deacylation process, 0.5 g L^–1 of compound A40926 was almost completely converted into the deacyl derivative. Under the same conditions, deacylation was also accomplished withtert-butoxycarbonyl (tert-BOC) A40926, in which the amino group at C15 was blocked to prevent formation of diacyl analogs during reacylation. The deacylase is an endoenzyme whose preliminary characterization is presented.     

Structure guided approaches toward exploiting and manipulating nonribosomal peptide and polyketide biosynthetic pathways

Nonribosomal peptide and polyketide natural products are structurally diverse small molecules synthesized on complex enzyme assemblies. The ability to rationally engineer secondary metabolic pathways is a promising approach to novel therapeutics. Atomic resolution structures of biosynthetic enzymes provide information on active site architecture and macromolecular assembly that can aid in the engineering of new compounds. This review surveys recent applications toward biosynthetic engineering of natural products guided by structural biology.     

Electroporation: A general phenomenon for manipulating cells and tissues

Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (μs to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5–1.5 V. In the case of isolated cells, the pulse magnitude is 10³–10⁴ V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.     

Improvement of bacterial cellulose production by manipulating the metabolic pathways in which ethanol and sodium citrate involved

Nowadays, bacterial cellulose has played more and more important role as new biological material for food industry and medical and industrial products based on its unique properties. However, it is still a difficult task to improve the production of bacterial cellulose, especially a large number of byproducts are produced in the metabolic biosynthesis processes. To improve bacterial cellulose production, ethanol and sodium citrate are added into the medium during the fermentation, and the activities of key enzymes and concentration of extracellular metabolites are measured to assess the changes of the metabolic flux of the hexose monophosphate pathway (HMP), the Embden–Meyerhof–Parnas pathway (EMP), and the tricarboxylic acid cycle (TCA). Our results indicate that ethanol functions as energy source for ATP generation at the early stage of the fermentation in the HMP pathway and the supplementation of ethanol significantly reduces glycerol generation (a major byproduct). While in the EMP pathway, sodium citrate plays a key role, and its supplementation results in the byproducts (mainly acetic acid and pyruvic acid) entering the gluconeogenesis pathway for cellulose synthesis. Furthermore, by adding ethanol and sodium citrate, the main byproduct citric acid in the TCA cycle is also reduced significantly. It is concluded that bacterial cellulose production can be improved by increasing energy metabolism and reducing the formation of metabolic byproducts through the metabolic regulations of the bypasses.     

Quantitative elementary mode analysis of metabolic pathways: the example of yeast glycolysis

Background  

Elementary mode analysis of metabolic pathways has proven to be a valuable tool for assessing the properties and functions of biochemical systems. However, little comprehension of how individual elementary modes are used in real cellular states has been achieved so far. A quantitative measure of fluxes carried by individual elementary modes is of great help to identify dominant metabolic processes, and to understand how these processes are redistributed in biological cells in response to changes in environmental conditions, enzyme kinetics, or chemical concentrations.     

Intracellular pathways of folded and misfolded amyloid precursor protein degradation

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein beta-amyloid (Abeta). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when alpha-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.     

Cross-regulation of signaling pathways: An example of nuclear hormone receptors and the canonical Wnt pathway

Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.