Effect of oxygen partial pressure on bialaphos synthesis,sugar metabolism and the activity of tricarboxylic acid cycle enzymes in submerged culture of Streptomyces hygroscopicus

Bialaphos [a herbicide, 2-amino-4-(hydroxy)(methyl)-phospinoylbutyryl-alanyl-alanine] produced by Streptomyces hygroscopicus increased significantly as the oxygen supply decreased during both growth and production phases of batch culture. Under low oxygen partial pressure, there were decreases in sugar consumption by the cells, cell concentration and the activity of tricarboxylic acid cycle enzymes in the cells. On the other hand, the activity of glyoxylic acid cycle enzymes in the cells increased. These phenomena were more prominent in high producing than in low producing strains. From these results, it is suggested that in high bialaphos producing strains, low oxygen partial pressure suppresses the activity of tricarboxylic acid enzymes so that both acetyl-CoA and pyruvate (the substrates for bialaphos synthesis) are used mainly for secondary metabolism. This results in a high production of bialaphos.     

Breeding of bialaphos producing strains from a biochemical engineering viewpoint

A breeding study was performed from the viewpoint of biochemical engineering to obtain strains which produce bialaphos (a herbicide) with high productivity and high yield from inexpensive substrates under low oxygen supply, with good filtration efficiency and easy downstream treatment. After induing mutagenesis using conventional chemical mutagens, first selection on agar culture was made to obtain strains which exhibited a large inhibition zone with a small colony, without being influenced by carbon catabolite regulation and with suppression of product decomposition. Subsequently, in a liquid culture system, selection was made to obtain strains which exhibited high product concentrations under low oxygen supply, with good filtration efficiency. A strain thus obtained was found to have about five hundred times higher product concentration than the wild strain. It also enabled us to use an inexpensive carbon source and to produce bialaphos under low oxygen supply, with good filtration efficiency.     

Studies on the biosynthesis of bialaphos (SF-1293) Part 3. Production of phosphinic acid derivatives,MP-103, MP-104 and MP-105, by a blocked mutant of Streptomyceshygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos

During biosynthetic studies on bialaphos to reveal the formation mechanisms of carbon-phosphorous bonds in detail, three new metabolites containing a HPC bond structure were isolated from the fermentation broth of a mutant of $\text{Streptomyces}\text{hygroscopicus}$ SF-1293. Based on the spectroscopic analysis, the structures of these compounds have been determined as shown in Fig. 1. Transformation experiments of these metabolites to bialaphos suggested that the reduction of the phosphorous atom in phosphate will take place at an early biosynthetic stage.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair — Direct alkylation vs. metabolic activation and breakdown. I. Butonate,vinylbutonate, trichlorfon,dichlorvos, demethyl dichlorvos and demethyl vinylbutonate

The following organophosphates were tested for their ability to induce DNA damage in a rec-type repair test with Proteus mirabilis strains PG713 (rec^? hcr^?) and PG273 (wild type) and point mutations in his^? strain TA100 of Salmonella typhimurium — butonate: O,O-dimethyl-(1-n-butyryloxy-2,2,2-trichloroethyl)-phosphonate; vinylbutonate: O,O-dimethyl-(n-butyryloxy-2,2-dichlorovinyl)-phosphonate; trichlorfon: O,O-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate; dichlorvos: O,O-dimethyl-O-(2,2-dichlorovinyl)-phosphate; the demethylated derivatives — demethyldichlorvos: O-methyl-O-(2,2-dichlorovinyl)-phosphoric acid; demethyl vinylbutonate: O-methyl-(1-n-butyryloxy-2,2-dichlorovinyl)phosphonic acid. Of the six compounds tested, dichlorvos and trichlorfon induced base pair substitutions and DNA damage. No mutagenicity and DNA damage were found in experiments with butonate, vinylbutonate, demethyl vinylbutonate and demethyl dichlorvos. Genotoxic activity for dichlorvos and the absence of both mutagenic and DNA damaging properties for its non-alkylating demethyl derivative favors the hypothesis that alkylation of DNA is the essential step for mutation induction by this organophosphate. Furthermore, the absence of genetic effects after treatment with vinylbutonate and demethyl dichlorvos does not support a crucial role of vinyl or allyl groups in side chains of organophosphates for genetic activity. Microsomal enzymes decreased genetic activity of dichlorvos and trichlorfon in vitro. No evidence for a role of metabolic activation in the mutagenic activity of any of these compounds was found.     

Expression of cloned bialaphos resistance gene (bar) in Streptomyces strains

The object of the study was a strain of Streptomyces hygroscopicus producing bialaphos, an antibiotic used as a herbicide, which is promising and ecologically safe. Molecular cloning of a bialaphos resistance gene (bar) was performed in the recipient strain, S. lividans TK64, within the 2.0-kb DNA fragment with the plasmid pIJ699. Introduction of a bar gene into another strain producing bialaphos, i.e. S. viridochromogenus Tu494, led to its higher constitutive resistance to bialaphos. The results confirmed the data on different regulation of bar (S. hygroscopicus) and pat (S. virido-chromogenus) resistance genes.     

Growth of Chlorella sorokiniana at Hyperbaric Oxygen Pressures

The growth rate of Chlorella sorokiniana decreased in a linear fashion as the partial pressure of oxygen was increased from 711 to 1,478 mm of Hg. Under two atmospheres of oxygen pressure, growth ceased after 10 to 12 hr. This cessation of growth was not due to any permanent injury, as growth resumed when oxygen partial pressure was reduced to ambient levels. The inhibition occurred under both autotrophic and heterotrophic growth conditions and was not accompanied by an increase in cell size. The results indicated that the tolerance of Chlorella cells to elevated oxygen pressures was not an absolute immunity, and that inhibition of growth at very high oxygen pressures cannot be accounted for by an inhibition of photosynthesis alone.     

Cuauthemone sesquiterpenoids from Blumea alata

The investigation of five Blumea species afforded, in addition to known compounds, several cuauthemone derivatives, most of them being 7,11-epoxides, some thymol derivatives, an eremophilane epoxide and a himachalane diol. From Pterocaulon virgatum a further himachalane derivative was isolated. The structures were elucidated by spectroscopic methods and in part by partial synthesis.     

EFFECTS OF INCREASED PARTIAL PRESSURES OF OXYGEN, NITROGEN AND HELIUM ON CELLS IN CULTURE

The effects of gas-phase oxygen, nitrogen and helium at superatmospheric as well as at atmospheric partial pressures have been studied, using both normal and malignant cells. The ability of certain types of cells in monolayer cultures to proliferate when exposed to high or low oxygen tensions, and to recover when cultured further under more optimal conditions, has been assessed. The presence of an inert gas at high partial pressure permits survival of various types of cells in culture at much higher oxygen tensions than would otherwise be possible. A tentative explanation for this phenomenon is provided and its significance discussed.     

The influence of oxygen supply on metabolism of neural cells cultured on a gas‐permeable PTFE foil

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen‐permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma‐derived cells (GOS‐3) and rat pheochromacytoma cells (PC12) were cultured on the gas‐permeable PTFE foil as well as a conventional non oxygen‐permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Mutasynthesis of pyrrole spiroketal compound using calcimycin 3-hydroxy anthranilic acid biosynthetic mutant

The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.     

Genetically engineered herbicide resistance in cyanobacterium Synechococcus sp. by bialaphos resistance gene from Streptomyces hygroscopicus

A novel cyanobacterial vector, pTT201, containing the bar gene encoding resistance to herbicides, bialaphos and phosphinothricin, was constructed. In Synechococcus sp. strain PCC7942-SPc, the bar gene was successfully expressed. Plasmid pTT201 increased a minimum inhibitory concentration for bialaphos 16-fold over Synechococcus sp. strain PCC7942-SPc without pTT201. The combination of the bialaphos as a selective agent and the transformation by bar gene serves as a photostable selection system for Synechococcus.     

Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

Effect of environmental factors on degradation of the CP bond of methylphosphonate by Escherichia coli cells

Biodegradation of the chemically resistant but bacterially degradable CP bond of alkylphosphonates is a pressing problem of environmental protection biotechnology. However, the environmental factors and physiological conditions for degradation of these compounds by bacteria are still obscure. The current work shows that adaptation of Escherichia coli cells to methylphosphonate (Pn) is more intensive under cell growth at 30 °C and does not depend on substrate concentration. Its degradation is optimal under low partial pressure of oxygen, pH of the medium 8.0, and logarithmic phase of culture growth. The increased synthesis of proteins of CP lyase complex degrading Pn and encoded by the genes cloned in plasmids does not increase the efficiency of Pn degradation, which indicates the presence of limiting factors of unknown nature.     

Stromal Phosphate Concentration Is Low during Feedback Limited Photosynthesis

It has been hypothesized that photosynthesis can be feedback limited when the phosphate concentration cannot be both low enough to allow starch and sucrose synthesis at the required rate and high enough for ATP synthesis at the required rate. We have measured the concentration of phosphate in the stroma and cytosol of leaves held under feedback conditions. We used non-aqueous fractionation techniques with freeze-clamped leaves of Phaseolus vulgaris plants grown on reduced phosphate nutrition. Feedback was induced by holding leaves in low O₂ or high CO₂ partial pressure. We found 7 millimolar phosphate in the stroma of leaves in normal oxygen but just 2.7 millimolar phosphate in leaves held in low oxygen. Because 1 to 2 millimolar phosphate in the stroma may be metabolically inactive, we estimate that in low oxygen, the metabolically active pool of phosphate is between negligible and 1.7 millimolar. We conclude that halfway between these extremes, 0.85 millimolar is a good estimate of the phosphate concentration in the stroma of feedback-limited leaves and that the true concentration could be even lower. The stromal phosphate concentration was also low when leaves were held in high CO₂, which also induces feedback-limited photosynthesis, indicating that the effect is related to feedback limitation, not to low oxygen per se. We conclude that the concentration of phosphate in the stroma is usually in excess and that it is sequestered to regulate photosynthesis, especially starch synthesis. The capacity for this regulation is limited by the coupling factor requirement for phosphate.     

Dihydrostilbene phytoalexins from Dioscorea rotundata

2′,3-Dihydroxy-5-methoxybibenzyl (Batatasin IV), its demethyl derivative and 3,5-dihydroxybibenzyl (dihydropinosylvin) were isolated only from flesh of Dioscorea rotundata infected with Botryodiplodia theobromae and may therefore be considered phytoalexins. These compounds were found to be antifungal using bioassays with Cladosporium cladosporioides, Botryodiplodia theobromae, Aspergillus niger and Penicillium schlerotgenum. Dihydro-pinosylvinin also exhibited strong antibacterial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.     

Effect of partial oxygen pressure on survival, proliferation, and differentiation of mouse bone marrow mesenchymal stem cells

Effects of partial oxygen pressure in a gas mixture surrounding the culture medium on survival, proliferation and differentiation of mesenchymal stem cells (MSC) isolated from mouse bone marrow was studied. It was found that 3% oxygen increased the survival of cells seeded at low density; the rate and duration of the MSC proliferation were also elevated. Effect of oxygen concentration on replicative activity of MSC was manifested as early as the first few days after the onset of cell growth. Studies of colony formation revealed that preincubation of cells with 21% oxygen had a negative effect on cell growth under 3% oxygen, while preincubation with 3% oxygen stimulated MSC proliferation in the presence of 21% oxygen. Notwithstanding, this effect of oxygen cannot be interpreted as unequivocally deleterious. Low partial oxygen pressure inhibited osteogenic differentiation of MSC, but adipogenic differentiation was insensitive to oxygen concentration. It is concluded that proliferation and differentiation of MSC depend critically on oxygen content in the culture medium.