Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

广叶绣球菌不同生长阶段栽培基质中代谢物差异分析

【背景】栽培基质的利用是广叶绣球菌(Sparassis latifolia)栽培中重要的生理过程，但栽培过程中基质代谢物的变化尚不清楚。【目的】通过不同生长阶段栽培基质中差异代谢物分析挖掘关键代谢物，为广叶绣球菌基质利用机理研究提供理论参考。【方法】利用UHPLC-MS/MS技术分析广叶绣球菌菌丝(Myc)、原基(Pri)和子实体(FB)生长阶段栽培基质中代谢产物的变化，通过不同数据库进行代谢物注释并进行KEGG通路富集分析。利用LC-MS/MS技术检测不同发育阶段绣球菌中植物类激素含量。【结果】三个不同栽培阶段基质中共鉴定出代谢产物1 360个。不同比较组(Pri vs. Myc、FB vs. Myc和FB vs. Pri)间共有的差异代谢产物179个，含量最高的50个代谢物主要包括氨基酸、脂质、吡喃酸、吡喃酮和植物类激素等物质。其中氨基酸含量在Myc、Pri和FB阶段基质中逐渐降低，而吡喃酸和吡喃酮类化合物含量逐渐升高。植物类激素中的赤霉素在Pri和FB阶段基质中含量较高，茉莉酸在Myc阶段基质中含量较高。对不同发育阶段绣球菌植物类激素进行检测，发现赤霉素GA7仅在原基中检测到，1...     

Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated π pulses

An improved expression protocol is proposed for amino acid type-specific [¹³C], [¹⁵N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449–456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.     

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log₁₀ lower than in ZoBell marine broth 2216. The fates of ¹⁴C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.     

Tea waste as a supplement for the cultivation of <Emphasis Type="Italic">Ganoderma</Emphasis> <Emphasis Type="Italic">lucidum</Emphasis>

Tea waste (TW) was investigated as a new supplement for substrate mixtures in Ganoderma lucidum cultivation. The effects of sawdust (S) based substrates supplemented with TW at the various levels (75S:25TW, 80S:20TW, 85S:15TW, and 90S:10TW) and Ganoderma lucidum strains on yield, biological efficiency (BE) and the chemical composition of fruiting bodies were determined in solid-state fermentation. Significant differences were found among substrates regarding yield and BE, while yield and BE of the strains were not different. The substrate formulations producing highest yield and BE were 80S:20TW (87.98 g/kg substrate and 34.90%) and 75S:25TW (82.30 g/kg substrate and 31%). Yield and BE of substrates containing TW were generally higher than that of the control (80sawdust:18wheat bran:1sucrose:1 CaCO₃). Nitrogen, potassium, iron, and manganese contents and C:N ratios of substrates were strongly correlated with yield. BE showed positive and significant correlations with potassium, iron and manganese. Moisture content, potassium, magnesium, calcium, iron, and zinc contents of the fruiting bodies were affected by both strain and substrate. It was concluded that TW can be used as a supplement for substrate preparation in G. lucidum cultivation.     

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids

Summary Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of l-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.     

Relationship between the Molecular Structure of the Nitrogen Source and the Activity of the Extracellular Lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] upon Submerged Cultivation

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca²⁺ and Mn²⁺ ions in the medium. Quantum-chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.     

<Emphasis Type="Italic">Polyporus tenuiculus</Emphasis>: a new naturally occurring mushroom that can be industrially cultivated on agricultural waste

Polyporus tenuiculus is a naturally occurring species from Central and South America that is consumed by different ethnic groups in the region. To determine the optimal conditions for fruiting body production, two strains were assayed on wheat straw and sawdust with or without supplements. Sixty days of incubation at 25°C were needed to produce a solid block. The highest yield was obtained with strain ICFC 383/00 grown on supplemented willow sawdust. In a second experiment the strain ICFC 383/00 and different supplements were used to improve the biological efficiency (BE) and to determine the quality traits and its biodegradation capacity. The highest yields were obtained on sawdust with 25% of supplements reaching 82.7% of BE. Supplements raised the number of flushes, generally from four to five, contributing to increased yields. The type of substrate had a significant effect on fruiting body diameters of P. tenuiculus, and the largest mushrooms were harvested on supplemented substrate with the highest BE coinciding with the highest dry matter loss in substrates. P. tenuiculus showed a capacity to degrade sawdust, causing a decrease of 67.2–74.5% in cellulose, 80.4–85.7% in hemicellulose, and 60.6–66.2% in lignin content at the end of the cultivation cycle. The decrease in hemicellulose was relatively greater than that of cellulose and lignin on supplemented substrates. This is the first report of the cultivation of P. tenuiculus on lignocellulosic waste, and it is a promising species both for commercial production and for its potential use in the degradation of other biowastes.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Investigation of substrate specificity of wildtype and mutant BphK (a glutathione S-transferase) from Burkholderia LB400

The bphK gene located in the bph operon of Burkholderia LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione-S-transferases (GST), a group of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Comparison of the amino acid sequence of BphK^LB400 with GST from other polychlorinated biphenyl (PCB)-degrading bacteria identified a number of highly conserved amino acids in the C-terminal region of the protein that may be associated with substrate specificity. In this study, two of these conserved amino acids in BphK^LB400 (amino acids 152 and 180) were selected for mutation, using site-directed mutagenesis, and substrate specificity assays. BphK^LB400 (wildtype and mutant) was over-expressed in Escherichia coli where the bphK gene (wildtype and mutant) is under the expression of a lac promoter and is induced by isopropyl thiogalactoside, and bacterial cell extracts were prepared for GST activity assays. Mutations at amino acids 152 and 180 were shown to affect GST activity of BphK^LB400 using 1-chloro-2,4-dinitrobenzene, the model substrate for GST activity assays; 4-chlorobenzoate and 3-chlorobenzoate, intermediates in the polychlorinated biphenyl (PCB) degradation pathway, and 2,4-dichlorophenoxyacetate and atrazine, commonly used herbicides; as substrates. A BphK^LB400 mutant (Ala180Pro) is identified in this study as having increased activity towards all substrates tested. This mutant may have potential in bioremediation.     

Ontogenesis of catabolic and energy metabolism capacities during the embryonic development of spotted wolffish (Anarhichas minor)

The catabolic and energy metabolism capacities during spotted wolffish (Anarhichas minor) embryogenesis were investigated. We assessed the embryo's ability to catabolize proteins (trypsin-like proteases) and lipids (triglyceride lipase) and examined the development of metabolic capacities using enzymatic assays: ability to use carbohydrates (pyruvate kinase), amino acids (aspartate aminotransferase) and fatty acids (hydroxyacyl-CoA dehydrogenase) for energy production, and aerobic (citrate synthase) and anaerobic (lactate dehydrogenase) energy production. Functional enzymatic systems were detected from the eyed stage (350 degree-days), except for fatty acids, which was detected from 540 degree-days. To compare the development of 1) aerobic and anaerobic pathways and 2) the capacity to mobilize the different energy substrates, enzymatic ratios were calculated. Anaerobic capacity appeared to increase at a significantly higher rate than the aerobic capacity. Ratios revealing the relative capacity to use specific energy substrates showed a significantly slower increase during development in the capacity to use carbohydrates than amino acids and fatty acids. The end of embryogenesis was characterized by a significant decrease in the use of carbohydrates for aerobic energy production but an increasing capacity to use amino acids. Egg survival as affected by the variability in metabolic parameters is discussed.     

Comparative use of amino acids by three auxotypes of Neisseria gonorrhoeae

Auxotypes of Neisseria gonorrhoeae are usually distinguishable by their particular requirements for growth; these requirements often include amino acids. It is possible that strains needing particular substrates to grow can be distinguished not merely by their growth requirements but also by their metabolism of these particular substrates. In this work amino acid utilization and oxidation studies were performed enabling prototype, pro- and thia-strains to be distinguished. The metabolism study also underlined the importance of proline as an energy source and pointed to the probability of distinct relationships with the metabolism of the key amino acids, glutamic and aspartic acids, for the three auxotypes.It is proposed that the specific amino acid required by the naturally occurring auxotype serves as an energy source at the site of infection and has important implications with respect to particular auxotypes at various sites.     

Overexpression of a cyanobacterial phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase with diminished sensitivity to feedback inhibition in<Emphasis Type="Italic"> Arabidopsis</Emphasis> changes amino acid metabolism

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, l-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C₄-like pathway is proposed.Abbreviations CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kan Kanamycin - 2-ME 2-Mercaptoethanol - MS/G medium 1/2 Murashige–Skoog and 1/2 Gamborg mixed medium - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - Sv Synechococcus vulcanus - ZmPEPC Maize PEPC involved in C₄ photosynthesis     

Carboxylic acid consumption and production by Corynebacterium glutamicum

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l -lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P_tuf-ldcC^opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability. Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase. Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or l -lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g_{cell dry weight}⁻¹ hr⁻¹, respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids.     

Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Minimal Defined Media for Vegetative Growth of the Acrasian Polysphondylium pallidum WS-320*

SYNOPSIS. Polysphondylium pallidum WS-320 grows indefinitely as vegetative amebae in a liquid medium where (a) substrates comprise sucrose, glycerol, acetate, lactate, citrate, and glutamate; (b) essential nutrients (riboflavin, lysine, glycine, and possibly several other amino acids that may be essential) are supplied. The growth thus supported (2 × 10⁶ cells/ml) is more than doubled by provision of a mixture of crude fatty acids, an acid hydrolysate of casein supplemented with B vitamins, purines, pyrimidines, and fat-soluble antioxidants.     

Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis

Aims: Three precursor amino acids and adenosine triphosphate ( ATP) are necessary for fermentative production of glutathione. In this study, our aims were to develop a strategy to enhance glutathione production by adding three precursor amino acids coupled with ATP in high cell density (HCD) cultivation of Candida utilis. Methods and Results: A high-glutathione yeast strain, C. utilis WSH 02-08, was used in this study. Whole fermentative process for glutathione production was divided into two phases of cell growth and glutathione synthesis. Cells concentration was increased by HCD cultivation. Meanwhile, intracellular glutathione content was enhanced by the addition of three precursor amino acids. Concentrations of three precursor amino acids added at stationary phase were optimized by response surface methodology. Moreover, the addition of ATP 15 h after the addition of the three amino acids can further enhance glutathione production. Based on aforementioned phenomenon, a strategy of adding three precursor amino acids coupled with ATP was developed to enhance glutathione production. Conclusion: Without the addition of three precursor amino acids and the ATP, a total glutathione of 1123 mg l⁻¹ was achieved after 60-h cultivation. In comparison, addition of three precursor amino acid counterparts resulted in a total glutathione of 1841 mg l⁻¹. Moreover, by adding amino acids combined with ATP, a total glutathione of 2043 mg l⁻¹ was achieved after 72-h cultivation, increased by 81·9% and 11%, respectively, as compared with the control and the one without ATP addition. Significance and Impact of the Study: This is the first report on investigating changes of the intracellular three precursor amino acids and ATP, and γ-glutamylcysteine synthase activity in HCD cultivation of C. utilis for glutathione production. A strategy of combining addition of three precursor amino acids with ATP was developed to enhance glutathione production in C. utilis.     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

Wistar Rats and Premature Infants

The molar proportions of essential amino acids in the fasting plasma of Wistar rats were shown to have utility in: (i) Determining the limiting essential amino acid for peanut butter and whole egg protein; (ii) Ranking in proper order peanut butter protein supplemented with two levels of lysine and whole egg protein according to their Biologic value and net protein utilization.

The molar proportions of the essential amino acids in the fasting plasma of adult humans were shown to have utility in ranking five rations according to their Biol.ogic value and net protein utilization by premature infants.