Expression of the sweet protein brazzein in maize for production of a new commercial sweetener

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.     

Effect of host-tree and environmental variables on honeydew production by scale insects (

Honeydew excreted by phloem-sap sucking scale insects (Ultracoelostoma sp.) living in the bark of beech (Nothofagus solandri:) trees growing at a high elevation (900 m) site in the Craigieburn range of Canterbury, New Zealand, was measured over four days during 1–10 May 1996. Average standing crop of honeydew sugar was 3.1 mg m-2, and ranged from 0.4 to 5.5 mg m-2. Daily production of honeydew sugar ranged from 0.2 to 1.5 mg insect-1 24 h-1, and 4.1 to 45.9 mg m-2 24 h-1. Honeydew production varied significantly between trees, and trees with the highest mean individual rates of production (mg sugar insect-1 24 h-1) tended to be those with the highest numbers of insects per unit bark area. Air temperature averaged over the 24 hours preceding each production sample explained 65% of the variability in 3-hourly honeydew production, showing that honeydew production is controlled by environmental and host-tree variables. Using this data, together with previously published estimates of carbon uptake and seasonal variability in honeydew standing crop, it was estimated that carbon contained in honeydew is equivalent to 1.8% of net primary production of beech trees at Craigieburn. Determining the effects of environmental variables on tree and insect physiology will allow formulation of an environmentally-driven process-based model of honeydew production.     

Mitigating climate change: the role of domestic livestock

Livestock contribute directly (i.e. as methane and nitrous oxide (N2O)) to about 9% of global anthropogenic greenhouse gas (GHG) emissions and around 3% of UK emissions. If all parts of the livestock production lifecycle are included (fossil fuels used to produce mineral fertilizers used in feed production and N2O emissions from fertilizer use; methane release from the breakdown of fertilizers and from animal manure; land-use changes for feed production and for grazing; land degradation; fossil fuel use during feed and animal production; fossil fuel use in production and transport of processed and refrigerated animal products), livestock are estimated to account for 18% of global anthropogenic emissions, but less than 8% in the UK. In terms of GHG emissions per unit of livestock product, monogastric livestock are more efficient than ruminants; thus in the UK, while sheep and cattle accounted for 32% of meat production in 2006, they accounted for 48% of GHG emissions associated with meat production. More efficient management of grazing lands and of manure can have a direct impact in decreasing emissions. Improving efficiency of livestock production through better breeding, health interventions or improving fertility can also decrease GHG emissions through decreasing the number of livestock required per unit product. Increasing the energy density of the diet has a dual effect, decreasing both direct emissions and the numbers of livestock per unit product, but, as the demands for food increase in response to increasing human population and a better diet in some developing countries, there is increasing competition for land for food v. energy-dense feed crops. Recalculating efficiencies of energy and protein production on the basis of human-edible food produced per unit of human-edible feed consumed gave higher efficiencies for ruminants than for monogastric animals. The policy community thus have difficult decisions to make in balancing the negative contribution of livestock to the environment against the positive benefit in terms of food security. The animal science community have a responsibility to provide an evidence base which is objective and holistic with respect to these two competing challenges.     

Formation of Two-Carbon Sugar Fragment at an Early Stage of the Browning Reaction of Sugar with Amine

A product easily converted to glyoxal was found in an early stage of the reaction of sugar with amine in ethanol. Glyoxaldicyclohexylimine was isolated from the reaction mixture of d-glucose with cyclohexylamine. This finding suggested the formation of a similar type glyoxaldialkylimine in other reactions of sugar with amine. This two-carbon compound was assumed to be produced directly from sugar or glycosylamine, and a new pathway for sugar fragmentation was proposed.     

The effect of feedback regulation and in situ product removal on the conversion of sugar to cycloheximide by Streptomyces griseus

An addition of cycloheximide to cycloheximide-producing Streptomyces griseus cultures resulted in reductions in the production rate and in the conversion of sugar into cycloheximide. In situ cycloheximide adsorption was observed to enhance: total cycloheximide titers; productivities; and the conversion of sugar to cycloheximide. During the secondary metabolite-producing phase, sugar consumption was observed to be linearly dependent on cycloheximide productivity. From this analysis a true product yield and maintenance coefficient were estimated to be 0.08 g cycloheximide/g glucose and 0.028 g glucose/g cell-h, respectively. The sixfold difference between this true product yield and a theoretical value obtained from knowledge of the biosynthetic pathway is discussed. Since the maintenance sugar requirement for cycloheximide production is large, stimulation of biosynthesis through in situ adsorption significantly increases the overall efficiency of sugar conversion to this secondary metabolite.     

Stylized facts in microalgal growth: interpretation in a dynamic energy budget context

A dynamic energy budget (DEB) model for microalgae is proposed. This model deviates from the standard DEB model as it needs more reserves to cope with the variation of assimilation pathways, requiring a different approach to growth based on the synthesizing unit (SU) theory for multiple substrates. It is shown that the model is able to accurately predict experimental data in constant and light-varying conditions with most of the parameter values taken directly from the literature. Also, model simulations are shown to be consistent with stylized facts (SFs) concerning NC ratio. These SFs are reinterpreted and the general conclusion is that all forcing variables (dilution rate, temperature and irradiance) impose changes in the nitrogen or carbon limitation status of the population, and consequently on reserve densities. Model predictions are also evaluated in comparison with SFs on chlorophyll concentration. It is proposed that an extra structure, more dependent on the nitrogen reserve, is required to accurately model chlorophyll dynamics. Finally, SFs concerning extracellular polymeric substances (EPSs) production by benthic diatoms are collected and interpreted and a formulation based on product synthesis and rejection flux is proposed for the EPSs production rate.     

Phylogenetic analysis of interspecific variation in nectar of hummingbird-visited plants

We tested whether phylogeny, flower size and/or altitude were significant predictors of interspecific variation in nectar production of hummingbird-visited plants in an assembled database (289 species, in 22 orders, 56 families and 131 genera). Although the study is focused on hummingbird-pollinated plants (241 plant species), plants with different pollinator syndromes (48 species) are also included in the analyses. Nectar volume secreted in a given time period (usually 24 h) by a given flower, its sugar concentration and corolla length were compiled mainly from the literature. Altitude was also obtained from the original references. Sugar production was computed basically as the product of nectar secretion and sugar concentration, and expressed on a per 24-h basis. All nectar traits and corolla length (all log transformed), as well as altitude, showed statistically significant phylogenetic signal. Both nonphylogenetic and phylogenetically informed (independent contrasts) analyses indicated a highly significant positive correlation between corolla length and both nectar volume and sugar production. In addition, altitude (which is partially a surrogate for temperature) was significantly negatively correlated with both sugar concentration and production. Possible reasons for coadaptation of nectar production and sugar production with corolla length are discussed.     

Action exerted by atropine sulfate on the luteinization phenomenon induced by a total gonadotropic preparation of prehypophysis at several stages of the estrus cycle in the rat

These experiments explored the effect of 70 mg atropine sulfate, and several doses of Gonadormone Byla, given at 1700 on diestrous I or at 1700 on diestrous II in the strains WI and WII rats derived in the authors' laboratory from Wistar rats. In Experiment 1 300 rats, 30 per group, received 2.5 or 5.0 mouse units of Gonadormone per 100 gm body weight at 1700 of diestrous I, with or without atropine, and were killed for serial ovarian sections at 1100 of proestrus. The 2.5 unit experime nt generated significant differences in frequency of luteinization between season of the year (p less than .001), between atropine and no atropine treatment (p less than .001), and season of atropine administration (p less than .05). Atropine decreased frequency of luteinization defined as proportion of a group having luteinized with or without retained ova. There were no differences in mean coefficients of ovulation, i.e., mean proportion of ovulated corpora lutea in each rat among all luteinized follicles, between rat strains or atropine treatments. The 5 unit dose of gonadotropin per 100 gm body weight increased luteinization 100% over the 2.5 unit dose. In the 2nd series of 180 rats, the frequency of luteinization induced by 1.25 units of gonadotropin was decreased by atropine (p less than .01), but the frequency of ovulation and response in the 2 rat strains did not differ. The results were interpreted as due in part to endogenous gonadotropin release, although atropine was thought to act directly on the ovary.     

Optimal design and experimental validation of a three-zone simulated moving bed process based on the Amberchrom-CG161C adsorbent for continuous removal of acetic acid from biomass hydrolyzate

In the production process of bio-ethanol from biomass, acetic acid is recognized as the key impurity to be removed from the sugar components that are generated by hydrolyzing biomass. In regard to this issue, it has recently been confirmed that the Amberchrom-CG161C resin was highly qualified as the adsorbent of a simulated moving bed (SMB) process for continuous separation of acetic acid from the biomass hydrolyzate, i.e., sugars. However, the previous study on the Amberchrom-CG161C SMB with the aforementioned separation goal has been limited to only a theoretical work, including some batch-chromatography tests. The experimental validation of such an Amberchrom-CG161C SMB process, including its optimal design, was attempted in this article. This task began by assembling the experimental unit of the SMB process with three zones. Its operating conditions were then optimized by using genetic algorithm. Under the optimized operating conditions, the relevant three-zone SMB experiment was conducted. The assay of all the resultant product samples verified that the SMB separation of interest was performed successfully as designed. The experimental data were also found to agree closely with the model predictions. Finally, a partial-discard strategy was applied to maintain the sugar product concentration as high as possible.     

Studies on Oxygen Transfer in Submerged Fermentations

In conventional shaken culture system, control of oxygen supply is performed by changing liquid volume in flasks and it necessarily introduces variation in the effectiveness of agitation and in the partial pressure of carbon dioxide. In jar or tank culture system, also, the changes in mechanical agitation and in the flow rate of air for control of aeration induce similar problems. It is impossible, therefore, to isolate the effects of oxygen on microbial metabolism from these accompanying ones. Hence, there is a basic requirement of making clear distinction among them, and in this paper the effects of agitation and carbon dioxide on product formation are presented in glutamic acid fermentation using the apparatus of controlling the level of dissolved oxygen throughout the fermentation.

To obtain fundamental knowledge required for attaining adequate aeration, the rate of oxygen demand in glutamic acid fermentation was discussed in connection with its fermentation rates. On the basis of specific rates, rates of change per unit mass of cells, glutamic acid fermentation was found to fall in the process pattern of Gaden’s type II, in which a constant rate of oxygen demand was sustained for a considerable time. On the basis of volumetric rates, rates of change per unit volume of broths, oxygen demand was recognized to be correlated with growth, sugar utilization and product formation, and it was pointed out particularly that the oxygen demand was closedly related with sugar utilization. In the particular cases where rapid utilization of sugar occurred, therefore, oxygen deficiency was liable to be evoked being unable to fill the growing oxygen demand. This finding might be useful for scale-up studies or process design.     

Nomenclature and guideline to express the amount of a membrane protein synthesized in animal cells in view of bioprocess optimization and production monitoring

Studies of a bioprocess optimization and monitoring for protein synthesis in animal cells face a challenge on how to express in quantitative terms the system performance. It is possible to have a panel of calculated variables that fits more or less appropriately the intended goal. Each mathematical expression approach translates different quantitative aspects. We can basically separate them into two categories: those used for the evaluation of cell physiology in terms of product synthesis, which can be for bioprocess improvement or optimization, and those used for production unit sizing and for bioprocess operation. With these perspectives and based on our own data of kinetic S2 cells growth and metabolism, as well as on their synthesis of the transmembrane recombinant rabies virus glycoprotein, here indicated as P, we show and discuss the main characteristics of calculated variables and their recommended use. Mainly applied to a bioprocess improvement/optimization and that mainly used for operation definition and to design the production unit, we expect these definitions/recommendations would improve the quality of data produced in this field and lead to more standardized procedures. In turn, it would allow a better and easier comprehension of scientific and technological communications for specialized readers.     

Mitochondrial superoxide and coenzyme Q in insulin-deficient rats: increased electron leak

Mitochondrial superoxide is important in the pathogeneses of diabetes and its complications. However, there is uncertainty regarding the intrinsic propensity of mitochondria to generate this radical. Studies to date suggest that superoxide production by mitochondria of insulin-sensitive target tissues of insulin-deficient rodents is reduced or unchanged. Moreover, little is known of the role of the Coenzyme Q (CoQ), whose semiquinone form reacts with molecular oxygen to generate superoxide. We measured reactive oxygen species (ROS) production, respiratory parameters, and CoQ content in mitochondria from gastrocnemius muscle of control and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between mitochondria isolated from vehicle- or STZ-treated animals. CoQ also was unaffected by weight loss in the absence of diabetes (induced by caloric restriction). Under state 4 or state 3 conditions, both respiration and ROS release were reduced in diabetic mitochondria fueled with succinate, glutamate plus malate, or with all three substrates (continuous TCA cycle). However, H(2)O(2) and directly measured superoxide production were substantially increased in gastrocnemius mitochondria of diabetic rats when expressed per unit oxygen consumed. On the basis of substrate and inhibitor effects, the mechanism involved multiple electron transport sites. More limited results using heart mitochondria were similar. ROS per unit respiration was greater in muscle mitochondria from diabetic compared with control rats during state 3, as well as state 4, while the reduction in ROS per unit respiration on transition to state 3 was less for diabetic mitochondria. In summary, ROS production is, in fact, increased in mitochondria from insulin-deficient muscle when considered relative to electron transport. This is evident on multiple energy substrates and in different respiratory states. CoQ is not reduced in diabetic mitochondria or with weight loss due to food restriction. The implications of these findings are discussed.     

Selection on ultrasonic call rate in neonatal rats affects low frequency,but not ultrasonic,vocalizations in adults

In this experiment, we studied a rodent model selected over 57 generations for high or low rates of ultrasonic vocalizations (USVs) during maternal separation as pups. We investigated the influence of this breeding on the adult animals’ subsequent vocal output, comparing acoustic variables across developmental stages. We hypothesized that selection on pup USV rate would impact adult USV production without affecting lower frequency calls. Contrary to this hypothesis, we found neither number of USV calls or other acoustic variables to differ among selected adult lines. Instead, we found that pup USV selection mainly affected adults’ low-frequency (human-audible) calls. Furthermore, low-frequency vocalizations did not fully fit a predicted correlation between body weight and fundamental frequency: high line males, although the heaviest on average, did not produce the lowest fundamental frequencies. Our findings suggest that selection for early ultrasonic vocal behaviour pleiotropically results in changes in anatomical production mechanisms and/or neural control affecting low-frequency calls.     

Energy metabolism, brain size and longevity in mammals

The mathematical relations between basal energy metabolism, brain size, and life span in mammals have been investigated. The evolutionary level of brain development, or encephalization (c), is a function both of brain weight (E) and of body weight (P) according to (formula; see text) Brain weight was found to be a linear function of the product of encephalization and basal metabolic rate. The oxygen consumption of the brain (Mbrain) is proportional to both encephalization and body weight according to (formula; see text) The ratio of metabolic rate in the cerebral cortex to that in the brain as a whole depends solely upon the degree of encephalization and is independent of the size of the animal. The maximum potential life span of a mammal was found to be proportional to the product of its degree of encephalization and the reciprocal of its metabolic rate per unit weight. Life span may be regarded as the algebraic sum of two components: (1) a deduced somatic component (Lb) inversely related to the basal metabolic rate per unit weight, and (2) an encephalization component (Le) related directly to the evolutionary increase of relative brain size.     

By-products resulting from lignocellulose pretreatment and their inhibitory effect on fermentations for (bio)chemicals and fuels

Lignocellulose might become an important feedstock for the future development of the biobased economy. Although up to 75 % of the lignocellulose dry weight consists of sugar, it is present in a polymerized state and cannot be used directly in most fermentation processes for the production of chemicals and fuels. Several methods have been developed to depolymerize the sugars present in lignocellulose, making the sugars available for fermentation. In this review, we describe five different pretreatment methods and their effect on the sugar and non-sugar fraction of lignocellulose. For several pretreatment methods and different types of lignocellulosic biomass, an overview is given of by-products formed. Most unwanted by-products present after pretreatment are dehydrated sugar monomers (furans), degraded lignin polymers (phenols) and small organic acids. Qualitative and quantitative effects of these by-products on fermentation processes have been studied. We conclude this review by giving an overview of techniques and methods to decrease inhibitory effects of unwanted by-products.     

Growth and water-use efficiency of 10 Triticum aestivum cultivars at different water availability in relation to allocation of biomass

In environments where the amount of water is limiting growth, water-use efficiency (biomass production per unit water use) is an important trait. We studied the relationships of plant growth and water use efficiency with the pattern of biomass allocation, using 10 wheat cultivars, grown at two soil moisture levels in a growth chamber. Allocation pattern and relative growth rate were not correlated, whereas allocation pattern and water use efficiency were. Variation in transpiration per plant resulted from variation in the rate of transpiration per unit leaf area or root weight, rather than from differences in leaf area or root weight per plant. Transpiration per unit leaf area or root weight was lower when the leaf area or root weight per unit plant weight was larger. Also, the efficiency of water use at the plant and leaf levels was higher for plants with a higher leaf area per unit plant weight, and it was not correlated with the plant's growth rate. Differences in water-use efficiency at the leaf level were related to variation in stomatal conductance, rather than in the rate of photosynthesis. A high photosynthetic water-use efficiency was associated with a low efficiency of nitrogen use for photosynthesis.     

A convenient synthesis of C-galactofuranosylic compounds (C-galactofuranosides)

Galactofuranose sugar units are essential for the production of the cell coat of many pathogenic microorganisms. This sugar is not found in mammals, and so compounds that may interfere with the biosynthetic processing of this sugar unit provide interesting targets for drug design. This paper describes the use of a cyanation reaction for the production of a one-carbon extension of a galactofuranosylic unit at C-1, giving 2,5-anhydro-3,4,6,7-tetra-O-benzoyl-D-glycero-L-manno-heptononitrile. A procedure for the efficient hydrolysis of the introduced nitrile group to produce the methyl ester is reported, along with procedures for the synthesis of both the corresponding alpha,beta-unsaturated, and 3-deoxy ester derivatives.     

Optimization of polyhydroxybutyrate production by experimental design of combined ternary mixture (glucose,xylose and arabinose) and process variables (sugar concentration,molar C:N ratio)

Conversion of lignocellulosic feedstocks to polyhydroxybutyrate (PHB) could make lignocellulosic biorefineries more profitable and sustainable. Glucose, xylose and arabinose are the main sugars derived from pretreatment and hydrolysis of herbaceous feedstocks. Burkholderia sacchari DSM 17165 is a bacterium that can convert these sugars into PHB. However, the effects of sugar ratio, sugar concentration, and molar C:N ratio on PHB production have not been studied. In this study, a seven-run mixture design for sugar ratio combined with a 3² full factorial design for process variables was performed to optimize PHB production. A polynomial model was built based on experimental data, and optimum conditions for different sugar streams were derived and validated. The highest PHB production (3.81 g/L) was achieved with arabinose at a concentration of 25.54 g/L and molar C:N ratio of 74.35. Results provide references for manipulation of sugar mixture and process control to maximize PHB production.

     

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1‐checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2015;112: 141–155. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.