Metabolic regulation of polyamines and γ‐aminobutyric acid in relation to spermidine‐induced heat tolerance in white clover

• Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance.
• Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ‐aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT‐PCR, were applied.
• Results showed that exogenous Spd significantly alleviated heat‐induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up‐regulating genes encoding Chl synthesis (PBGD and Mg‐CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up‐regulated HSP70, HSP70B and HSP70‐5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress.
• In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

     

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135–1148, 2016     

Yield response of field‐grown soybean exposed to heat waves under current and elevated [CO2]

Elevated atmospheric CO₂ concentration ([CO₂]) generally enhances C₃ plant productivity, whereas acute heat stress, which occurs during heat waves, generally elicits the opposite response. However, little is known about the interaction of these two variables, especially during key reproductive phases in important temperate food crops, such as soybean (Glycine max). Here, we grew soybean under elevated [CO₂] and imposed high‐ (+9°C) and low‐ (+5°C) intensity heat waves during key temperature‐sensitive reproductive stages (R1, flowering; R5, pod‐filling) to determine how elevated [CO₂] will interact with heat waves to influence soybean yield. High‐intensity heat waves, which resulted in canopy temperatures that exceeded optimal growth temperatures for soybean, reduced yield compared to ambient conditions even under elevated [CO₂]. This was largely due to heat stress on reproductive processes, especially during R5. Low‐intensity heat waves did not affect yields when applied during R1 but increased yields when applied during R5 likely due to relatively lower canopy temperatures and higher soil moisture, which uncoupled the negative effects of heating on cellular‐ and leaf‐level processes from plant‐level carbon assimilation. Modeling soybean yields based on carbon assimilation alone underestimated yield loss with high‐intensity heat waves and overestimated yield loss with low‐intensity heat waves, thus supporting the influence of direct heat stress on reproductive processes in determining yield. These results have implications for rain‐fed cropping systems and point toward a climatic tipping point for soybean yield when future heat waves exceed optimum temperature.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Photosynthetic acclimation of Chlorella saccharophila to heat stress

Photosynthesis is one of the most important metabolic processes of algae; which is altered as a stress response. During mass cultivation of algae, temperature rise and high light are major factors that affect biomass productivity. High temperature affects photosystem II (PSII) complex irreversibly, damaging intermolecular interactions in it. However, the impact of high temperature on photosynthesis is highly variable among different algal species, depending on the prior acclimation to environmental conditions they were exposed to. The acclimation plays an important role in combating high temperature stress via regulation of photosynthetic responses. Chlorophyll a fluorescence is a highly sensitive, non‐destructive and reliable tool for such measurements of photosynthetic parameters, which provides information about algal photosynthetic performance under given conditions. To understand the effect of heat stress on the responses of high light acclimated alga Chlorella saccharophila, chlorophyll a fluorescence transients were measured after heat exposure at 40°C. Our study demonstrates that rise in temperature for short duration; during open field cultivation reversibly affects the efficiency of PSII in light acclimated alga C. saccharophila. The effects of heat stress on chlorophyll a fluorescence in this alga, grown under high light (max‐1600 μmol photons m^?2 s^?1) are presented here; they are used to infer changes in photosynthetic process during its exposure to heat, as well as their recovery after 72 h. We speculate that heat resistance may have been acquired due to prior exposures to high light.     

A comparative study of Taxol production in liquid and solid‐state fermentation with Nigrospora sp. a fungus isolated from Taxus globosa

Aims: To determine in liquid (LF) and solid‐state fermentation (SSF) the effect of medium concentration on growth and Taxol produced by Nigrospora sp., a fungus isolated from the Mexican yew. Methods and Results: Nigrospora sp. was grown at different concentrations of the base culture medium M1D, i.e. two (2×), four (4×), six (6×) and eight times (8×) the base concentration. The titres of Taxol determined by competitive inhibition enzyme immunoassay increased with increasing medium concentration in LF and SSF but were higher in SSF in every medium concentration. The Taxol produced in SSF and LF with 8× medium was 221 and 142 ng l^?1. The SSF gave also higher biomass, growth and sugar utilization than LF in every medium. The growth and sugar consumption were modelled by the logistic and the Pirt models, respectively. However, the Luedeking–Piret model was unsuitable for Taxol. Conclusions: The SSF surpassed LF in terms of Taxol, growth and sugar utilization; thus, it has significant advantages over LF. Significance and Impact of the Study: This is the first report on Taxol production by SSF and the first contribution to evaluate the influence of the medium on Taxol production in LF and SSF.     

Gene expression profiles during short‐term heat stress in the red sea coral Stylophora pistillata

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide‐spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic ‘r’ strategist. It can thrive in relatively unstable environments and is considered a stress‐tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short‐term (13‐day) heat stress. The results allowed us to identify a two‐step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential ‘early warning genes’ and ‘severe heat‐related genes’. Our findings suggest that during short‐term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER‐unfolded protein response (UPR) and ER‐associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.     

Effect of temperature up-shift on fermentation and metabolic characteristics in view of gene expressions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces heat shock genes to the temperature up-shift, and changes the metabolism by complicated mechanism. The heat shock response is of practical importance for the variety of applications such as temperature-induced heterologous protein production, simultaneous saccharification and fermentation (SSF) etc. However, the effect of heat shock on the metabolic regulation is not well investigated. It is strongly desired to understand the metabolic changes and its mechanism upon heat shock in practice for the efficient metabolite production by temperature up-shift. In the present research, therefore, we investigated the effect of temperature up-shift from 37°C to 42°C on the metabolism in view of gene expressions.     

Out‐of‐season production of 17,20β‐dihydroxypregn‐4‐en‐3‐one in the roach Rutilus rutilus

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) and 17,20β,21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5–10 ng ml^?1 in blood; and a rate of release of 5–20 ng fish^?1 h^?1 into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11‐ketotestosterone (11‐KT) in males and 17β‐oestradiol and VTG in females. The 3 months with the highest production of 11‐KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17β‐oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.     

Comprehensive proteome profiling of the Fe(III)‐reducing myxobacterium Anaeromyxobacter dehalogenans 2CP‐C during growth with fumarate and ferric citrate

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta‐proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2‐D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2‐DE and MALDI‐TOF‐MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi‐quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.     

Real‐time measurements of human chondrocyte heat production during in vitro proliferation

Isothermal microcalorimeters (IMC) are highly sensitive instruments that allow the measurement of heat flow in the microwatt range. Due to their detection of minute thermal heat, IMC techniques have been used in numerous biological applications, including the study of fermentation processes, pharmaceutical development, and cell metabolism. In this work, with the ultimate goal of establishing a rapid and real‐time method to predict the proliferative capacity of human articular chondrocytes (HAC), we explored to use of IMC to characterize one of the crucial steps within the process of cartilage tissue engineering, namely the in vitro expansion of HAC. We first established an IMC‐based model for the real‐time monitoring of heat flow generated by HAC during proliferation. Profiles of the heat and heat flow curves obtained were consistent with those previously shown for other cell types. The average heat flow per HAC was determined to be 22.0 ± 5.3 pW. We next demonstrated that HAC proliferation within the IMC‐based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. HAC growth and HAC heat over time appeared correlated for cells derived from particular donors. However, based on the results from 12 independent donors, no predictive correlation could be established between the growth rate and the heat increase rate of HAC. This was likely due to variability in the biological function of HAC derived from different donors, combined with the complexity of tightly couple metabolic processes beyond proliferation. In conclusion, IMC appears to be a promising technique to characterize cell proliferation. However, studies with more reproducible cell sources (e.g., cell lines) could be used before adding the complexity associated with primary human cells. Biotechnol. Bioeng. 2011;108: 3019–3024. © 2011 Wiley Periodicals, Inc.     

Online monitoring of the respiratory quotient reveals metabolic phases during microaerobic 2,3‐butanediol production with Bacillus licheniformis

Microaerobic cultivation conditions are often beneficial for the biotechnological production of reduced metabolites like 2,3‐butanediol. However, due to oxygen limitation, process monitoring based on oxygen transfer rate, or dissolved oxygen measurement provides only limited information. In this study, online monitoring of the respiratory quotient is used to investigate the metabolic activity of Bacillus licheniformis DSM 8785 during mixed acid‐2,3‐butanediol production under microaerobic conditions. Thereby, the respiratory quotient provides valuable information about different metabolic phases. Based on partial reaction stoichiometries, the metabolic activity in each phase of the cultivation was revealed, explaining the course of the respiratory quotient. This provides profound information on the formation or consumption of glucose, 2,3‐butanediol, ethanol and lactate, both, in shake flasks and stirred tank reactor cultivations. Furthermore, the average respiratory quotient correlates with the oxygen availability during the cultivation. Carbon mass balancing revealed that this reflects the increased formation of reduced metabolites with increasing oxygen limitation. The results clearly demonstrate that the respiratory quotient is a valuable online signal to reveal and understand the metabolic activity during microaerobic cultivations. The approach of combining respiratory quotient monitoring with stoichiometric considerations can be applied to other organisms and processes to define suitable cultivation conditions to produce the desired product spectrum.     

Microbial production of d‐lactic acid from dried distiller's grains with solubles

d ‐Lactic acid production is gaining increasing attention due to the thermostable properties of its polymer, poly‐d ‐lactic acid . In this study, Lactobacillus coryniformis subsp. torquens, was evaluated for its ability to produce d ‐lactic acid using Dried Distiller's Grains with Solubles (DDGS) hydrolysate as the substrate. DDGS was first subjected to alkaline pretreatment with sodium hydroxide to remove the hemicellulose component and the generated carbohydrate‐rich solids were then subjected to enzymatic hydrolysis using cellulase mixture Accellerase® 1500. When comparing separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) of L. coryniformis on DDGS hydrolysate, the latter method demonstrated higher d ‐lactic acid production (27.9 g/L, 99.9% optical purity of d ‐lactic acid), with a higher glucose to d ‐lactic acid conversion yield (84.5%) compared to the former one (24.1 g/L, 99.9% optical purity of d ‐lactic acid). In addition, the effect of increasing the DDGS concentration in the fermentation system was investigated via a fed‐batch SSF approach, where it was shown that the d ‐lactic acid production increased to 38.1 g/L and the conversion yield decreased to 70%. In conclusion, the SSF approach proved to be an efficient strategy for the production of d ‐lactic acid from DDGS as it reduced the overall processing time and yielded high d ‐lactic acid concentrations.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

The synthesis of N‐benzoylindoles as inhibitors of rat erythrocyte glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase

Glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N‐benzoylindoles 9a‐ ‐ 9d , which form the main framework of many natural indole derivatives such as indomethacin and N‐benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N‐benzoylindoles inhibited the enzymatic activity with IC₅₀ in the range of 3.391505 μM for G6PD and 2.19–990 μM for 6PGD.     

L‐2,3‐diaminopropionate generates diverse metabolic stresses in Salmonella enterica

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L‐2,3‐diaminopropionate (Dap) is a non‐protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia‐lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL‐dependent degradation of Dap to pyruvate proceeds through an unbound 2‐aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaL‐dependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification.     

Determination of trace α‐fetoprotein variant by affinity adsorption solid substrate‐room temperature phosphorimetry

The 3.5‐generation dendrimers (3.5G‐D)–porphyrin (P) dual luminescent molecule (3.5G‐D–P) was used to label concanavalin agglutinin (Con A); the product of the reaction is 3.5G‐D–P–Con A. A new method for the determination of trace AFP‐V by affinity adsorption solid substrate–room temperature phosphorimetry (AA‐SS–RTP) was established, based on the room temperature phosphorescence (RTP) property of the product on polyamide membrane (PAM) substrate and the specific affinity adsorption (AA) reaction between 3.5G‐D–P–Con A and α‐fetoprotein variant (AFP‐V), which caused the RTP of the system to be sharply enhanced, the ΔIp was linearly correlated to the content of AFP‐V. The sensitivity of the method was obviously high. It could accurately detect the content of AFP‐V in serum. The results were tallied well with those obtained by the ELISA method. Copyright © 2008 John Wiley & Sons, Ltd.     

Economic analysis of pilot‐scale production of B‐phycoerythrin

β‐Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B‐phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two‐phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two‐phase systems in a scaled‐up process. This study analyzed the production of B‐Phycoerythrin using real data obtained during the scale‐up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two‐phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10‐year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B‐phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472–1479, 2016