Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Addition of stimulatory chemical such as glycerol was found to increase recombinant protein production in Chinese hamster ovary (CHO) cells. However, glycerol influenced cell mitosis and reduced cell growth rate. We developed a controlled proliferation strategy to utilize the stimulation of glycerol on recombinant protein production and mitigate the problem of growth inhibition. The approach is to apply a two-stage process, where cells are cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol is added to achieve high specific productivity. In addition, a model for predicting the profiles of cell proliferation and recombinant protein production was developed and validated. A two-stage process, addition of 1% glycerol after 1 day of growth, could increase the final production of macrophage-colony stimulating factor (M-CSF) by 38% compared with the value obtained without addition of glycerol.     

Glycerol metabolism in the extremely halophilic bacterium Salinibacter ruber

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by glycerol. In contrast to glucose consumption, which starts only after more easily degradable substrates present in yeast extract have been depleted, glycerol is consumed during the earliest growth phases. When U-(14)C-labeled glycerol was added to the culture, up to 25% of the radioactivity was incorporated by the cells. Glycerol kinase activity was detected only in cells grown in the presence of glycerol (up to 90 nmol mg protein(-1) min(-1)). This enzyme functioned over salt concentrations from 0.6 to 2.8 M KCl. No significant activity of NAD-dependent glycerol dehydrogenase was found. It is suggested that Salinibacter may use glycerol as one of its principal substrates in its habitat, the saltern crystallizer ponds.     

Production of PHB from Crude Glycerol

Crude glycerol – a by‐product of the large scale production of diesel oil from rape – is examined for its possible use as a cheap feedstock for the biotechnological synthesis of poly(3‐hydroxybutyrate) (PHB). The glycerol samples of various manufacturers differ in their contamination with salts (NaCl or K₂SO₄), methanol or fatty acids. At high cell density fermentation these pollutants could possibly accumulate to inhibiting concentrations. The bacteria used were Paracoccus denitrificans and Cupriavidus necator JMP 134, which accumulate PHB from pure glycerol to a content of 70 % of cell dry mass. When using crude glycerol containing 5.5 % NaCl, a reduced PHB content of 48 % was observed at a bacterial dry mass of 50 g/L. Furthermore the PHB yield coefficient was reduced, obviously due to osmoregulation. The effect of glycerol contaminated with K₂SO₄ was less pronounced. The molecular weight of PHB produced with P. denitrificans or C. necator from crude glycerol varies between 620000 and 750000 g/mol which allows the processing by common techniques of the polymer industry.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

Isolation and characterization of salt-sensitive mutants of a salt-tolerant yeast Zygosaccharomyces rouxii

Abstract Twenty salt-sensitive (ss) mutants were isolated from the salt-tolerant yeast Zygosaccharomyces rouxii by treatment with N -methyl- N '-nitro- N -nitrosoguanidine. The mutants were divided into five classes on the basis of their ability to grow in media containing various high concentrations of NaCl. The mutant with the greatest sensitivity to NaCl of all the mutants tested was able to grow very slowly with a longer lag phase in medium containing 2 M NaCl, in contrast to the wild strain which had the capacity to grow in medium containing 3.5 M NaCl. Most of the ss mutants exhibited, to some extent, less tolerance to high concentrations of glucose than the wild strain. It appeared from the characterization of the ss mutants that the following factors are necessary for growth of Z. rouxii in high concentrations of NaCl: (a) the ability to produce glycerol under these conditions; (b) the ability to maintain a defined concentration of glycerol within the cells; (c) the ability to take up glycerol that has leaked into the medium, and to assimilate glycerol; and (d) unknown factor(s).     

酵母细胞对高渗环境的适应与胞内甘油累积

甘油是包括酿酒酵母在内的许多种酵母细胞中的主要相容性溶质。为适应在高渗环境下的生存,酵母细胞将在胞内累积甘油。胞内甘油累积的增加可由甘油合成的增强,甘油利用的减弱,细胞膜通透性下降导致的胞内甘油流失的减少以及从环境中吸取更多的甘油而产生。本文综述了酵母细胞对环境渗透压变化的信号传导,高渗诱导的基因表达,环境渗透压升高时酵母细胞内甘油的累积以及甘油合成的限速步骤。     

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

甘油脱水酶再激活因子提高重组大肠杆菌3-羟基丙酸合成能力

甘油脱水酶是甘油转化3-羟基丙酸生物合成途径中的关键性限速酶,然而底物甘油的存在会抑制该酶的活性,从而引起3-羟基丙酸合成量的下降.因此解除底物甘油对甘油脱水酶活性的抑制作用,是提高生物合成3-羟基丙酸产量的方法之一.克隆来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶编码基因dhaB、甘油脱...     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.     

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.     

Dual pathways of glycerol assimilation in Klebsiella aerogenes NCIB 418

Klebsiella aerogenes NCIB 418 assimilates glycerol via alternative pathways: one involves a glycerol kinase with a high affinity for glycerol (apparent K _m=1–2×10^–6 M), and the second a glycerol dehydrogenase with a much lower affinity for its substrate (apparent K _m=2–4×10^–2 M).In variously-limited chemostat cultures, one or the other pathway predominated. Thus, aerobic carbonlimited organisms contained only the glycerol kinase pathway whereas aerobic sulphate-limited or ammonia-limited organisms (grown on glycerol) used only the glycerol dehydrogenase pathway. Anaerobic cultures invariably contained glycerol dehydrogenase, and glycerol kinase was absent.Washed suspensions of aerobically-grown organisms oxidized glycerol with kinetics similar to that of the particular enzyme (the primary enzyme of the assimilatory pathway) which they possessed, thus indicating a close association between these two enzymes and the uptake process. But a supply of exogenous glycerol was not a prerequisite for the synthesis of either glycerol kinase or glycerol dehydrogenase, and nor was molecular oxygen the key factor in effecting modulation between the alternative pathways of glycerol metabolism, as had been previously suggested.The physiological significance of dual pathways of glycerol assimilation is discussed.     

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch

Abstract The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.     

Intracellular glycerol in Dunaliella is depleted by intracellular metabolism in response to hypoosmotic stress by dilution

Abstract When Dunaliella tertiolecta cells are subjected to a dilution stress (hypoosmotic shock) the intracellular glycerol is metabolised biochemically, but it does not leak into the medium. However, Dunaliella cells have a certain 'threshold' to withstanding a hypoosmotic shock beyond which cell damage occurs and then glycerol is leaked into the medium. Both in the light or the dark, the glycerol metabolism was inhibited by micromolar concentration of carbonylcyanide m - chlorophenyl - hydrazone (CCCP) suggesting that the required ATP for the glycerol dissimilation is dependent upon an energized membrane, and most of which can be supplied through the oxidative phosphorylation.     

Enzymatic production of glycerol acetate from glycerol

In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.     

Mannitol production from glycerol by resting cells of Candida magnoliae

Production of mannitol from glycerol by resting cells of Candida magnoliae under aerobic condition was investigated. The resting cells were suspended in aqueous solution of glycerol in Erlenmeyer flasks and incubated on rotary shaker. The samples were analyzed by ion exclusion–HPLC equipped with refractive index and UV detector. The resting cells of C. magnoliae produced mannitol from fructose, sucrose and glycerol but not from glucose. Addition of yeast extract and/or potassium phosphate to the glycerol solution adversely affected its conversion to mannitol. The conversion of glycerol to mannitol was dependent on oxygen availability. Using resting cells, the yield of mannitol was as high as 45%. This is probably the first report of conversion of glycerol to mannitol by osmophilic yeast.     

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.     

鲁氏酵母3-磷酸甘油脱氢酶基因（ZrGPD1）在粉状毕赤酵母中的表达

为探索在野生型粉状毕赤酵母（Pichia farinosa）中整合表达来源于耐高渗鲁氏酵母（Zygosacharomyces rouxii）的3-磷酸甘油脱氢酶基因（ZrGPD1）以提高产甘油能力的可行性,应用PCR方法从P. farinosa的染色体中扩增出乳清苷酸脱羧酶基因（URA3）片段,以此作为同源整合的靶序列,构建了整合型表达载体pUR-ZG。电击转化粉状毕赤酵母,以抗生素Zeocin为筛选标记,获得转化子pfa-gu,摇瓶发酵结果表明：以P. farinosa作为对照菌株,发酵72h后,转化子pfa-gu的生物量和甘油含量均高于对照菌株,其中甘油含量达到37g/L,比对照提高了30%。结论：在P. farinosa中异源表达ZrGPD1能够提高细胞的产甘油能力和对渗透压的调节能力。