Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.     

Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels

Microalgal biomass seems to be a promising feedstock for biofuel generation. Microalgae have relative high photosynthetic efficiencies, high growth rates, and some species can thrive in brackish water or seawater and wastewater from the food- and agro-industrial sector. Today, the main interest in research is the cultivation of microalgae for lipids production to generate biodiesel. However, there are several other biological or thermochemical conversion technologies, in which microalgal biomass could be used as substrate. However, the high protein content or the low carbohydrate content of the majority of the microalgal species might be a constraint for their possible use in these technologies. Moreover, in the majority of biomass conversion technologies, carbohydrates are the main substrate for production of biofuels. Nevertheless, microalgae biomass composition could be manipulated by several cultivation techniques, such as nutrient starvation or other stressed environmental conditions, which cause the microalgae to accumulate carbohydrates. This paper attempts to give a general overview of techniques that can be used for increasing the microalgal biomass carbohydrate content. In addition, biomass conversion technologies, related to the conversion of carbohydrates into biofuels are discussed.     

Growth and biomass production with enhanced β‐glucan and dietary fibre contents of Ganoderma australe ATHUM 4345 in a batch‐stirred tank bioreactor

In this study we maximized biomass production by the basidiomycete Ganoderma australe ATHUM 4345, a species of pharmaceutical interest as it is a valuable source of nutraceuticals, including dietary fibers and glucans. We used the Biolog FF MicroPlate to screen 95 different carbon sources for growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint, which is useful in selecting components for media optimization of maximum biomass production. Response surface methodology, based on the central composite design was applied to explore the optimum concentrations of carbon and nitrogen sources of culture medium in shake flask cultures. When the improved culture medium was tested in a 20‐L stirred tank bioreactor, using 13.7 g/L glucose and 30.0 g/L yeast extract, high biomass yields (10.1±0.4 g/L) and productivity of 0.09 g L^?1 h^?1 were obtained. The yield coefficients for total glucan and dietary fibers on biomass formed were 94.82±6 and 341.15±12.3 mg/g mycelium dry weight, respectively.     

Trends in Oil Production from Oleaginous Yeast Using Biomass: Biotechnological Potential and Constraints

In the present scenario of depleting oil reservoir, microbial oil has gained much attention over plant and animal based sources. Among different microorganisms, yeast strains are considered superior source for oil production. The cost of oil produced by yeast could further be lowered using cheaper agro-waste and biomass as substrate. This review focuses on key topics which will help in gaining better understanding to enhance lipid production using yeast strains. The effects of oleaginous yeast co-culturing with microalgae, different cheap carbon sources of biomass, and types of yeast species on oil production were highlighted in the review. An overview of mechanisms of oil production from biomass, viz. pretreatment of biomass, fermentation and oil recovery are also provided. Constraints encountered during the oleogenesis or microbial oil accumulation and their probable solutions along with a section on different by-products obtained during oleo-genesis are also discussed.     

Modeling alginate encapsulation system for biological hydrogen production

Wastewater treatment using encapsulated biomass is a promising approach for high-rate resource recovery. Encapsulation matrices can be customized to achieve desired biomass retention and mass transport performance. This, in turn, facilitates treatment of different waste streams. In this study, a model was developed to describe calcium-alginate beads encapsulating hydrogen-producing biomass, with the goal of enabling appropriate a priori customization of the system. The model was based on a classic diffusion-reaction model, but also included the growth of encapsulated biomass and product inhibition. Experimental data were used to verify the model, which accurately described the effect of hydraulic retention time, bead size, and feed concentration on resource (hydrogen) recovery from brewery wastewater. Sensitivity analyses revealed that the hydrogen production rate was insensitive to substrate diffusivity and bead size, but sensitive to the substrate partition coefficient, initial encapsulated biomass concentration, and the total volume of beads in the reactor, demonstrating that this system was growth-limited rather than diffusion-limited under the tested conditions. Because the model quantifies the relationship between the hydrogen production rate and various input and operating parameters, it should be possible to extend the model to determine the most cost-effective system for optimal performance with a given waste stream.     

Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1beta by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1beta production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode.     

Soybeans as bioreactors for biopharmaceuticals and industrial proteins

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.     

Efficient screening of potential cellulases and hemicellulases produced by <Emphasis Type="Italic">Bosea</Emphasis> sp. FBZP-16 using the combination of enzyme assays and genome analysis

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.     

The role of resource imbalances in the evolutionary ecology of tropical arboreal ants

In numbers and biomass, ants (Hymenoptera, Formicidae) often dominate arthropod faunas of tropical rainforest canopies. Extraordinary ant abundance is due principally to one or a few species able to tap the high productivity of canopy foliage by feeding on plant and homopteran exudates. Prior studies of nitrogen isotopic ratios show that exudate-feeders derive much of their nitrogen (N) by processing large quantities of N-poor, but carbohydrate (CHO)-rich, exudates. CHOs in excess of those that can be coupled with protein for growth and reproduction (postulated as the colony's first priorities) may be directed at little cost and some profit to functions that increase access to limiting protein. High dietary CHO:protein ratios for exudate-feeders appear to subsidize 'high tempo' foraging activity, defence of absolute (level III) territories, and production of N-free alarm/defence exocrine products that enhance ecological dominance in contests with other ants. Among organisms (e.g. plants and Lepidoptera) symbiotic with ants, CHO:protein ratios of ant rewards may control both the identities of ant associates and the quality of ant-rendered services. Dietary ratios of CHO:protein play an important and previously unrecognized role in the ecology and evolution of ants generally. Modifications of worker digestive systems in certain ant subfamilies and genera represent key innovations for handling and processing large volumes of liquid food. The supreme tropical dominants are species released from nest site limitation and able to place their nests in the vicinity of abundant exudate resources. Polydomy appears to be typical of these species and should produce energetic savings by taking colony fragments to the resource.     

Bioenergy from permanent grassland – A review: 2. Combustion

The aim of this review is to summarize current knowledge on suitability and sustainability of grassland biomass for combustion. In the first section grassland management for solid biofuel as well as information on harvest, postharvest and firing technology are described. An extensive grassland management system with one late cut and low level of fertilization is favored for grass as a solid biofuel. The grass harvest usually involves drying in the field and clearing with conventional farm machinery. Pelleting or briquetting improves the biofuel quality. Grass combustion is possible as stand-alone biomass-firing or co-firing with other fuels. Firing herbaceous biomass requires various specific adaptations of the different combustion technologies. In the second section economic and environmental aspects are discussed. Costs for biomass supply mainly depend on yields and harvesting technologies, while combustion costs are influenced by the size and technical design of the plant. Market prices for grass and possible subsidies for land use are crucial for profitability. Regarding biogeochemical cycles a specific feature of combustion is the fact that none of the biomass carbon and nitrogen removed at harvest is available for return to the grassland. These exports can be compensated for by fixation from the air given legumes in the vegetation and sufficient biomass production. Greenhouse gas emissions can be considerably reduced by grass combustion. Solid biofuel production has a potential for predominantly positive impacts on biodiversity due to the extensive grassland management.     

Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor.In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered.Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities.The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.     

On maintenance models in severely and long-term limited membrane bioreactor cultivations

Membrane bioreactors (MBRs) are combinations of common bioreactors and membrane separation units for biomass retention. Through increased biomass concentration, they allow increased productivity (or smaller reactor volume, respectively). Besides high biomass concentrations, operation at very low growth rates is typical for MBRs. In this regime, maintenance metabolism where substrate uptake only yields energy for cell survival becomes of higher importance than in processes run at higher growth rates. While thermodynamically based correlations for the prediction of maintenance coefficients are available for chemostat or other medium growth rate processes, some authors have mentioned a change in energy demand in MBRs and a dependence of maintenance parameters on operating conditions. Due to the fact that often mixed cultures are used and resulting from the different evaluation methods used by different authors, views on the possible influences on maintenance parameters differ. However, it is accepted that common models describing microbial growth and production of metabolites or degradation of pollutants do not consider the effects caused by severe limitations and therefore cannot sufficiently be applied to MBRs. In this study, maintenance parameters were determined for a model organism (Ustilago maydis) and results from different evaluation methods were compared. A continuous fit of respiration data gave more consistent results than the traditional method of plotting specific uptake versus growth rate. They suggest that below micro = 10% micro(max) the maintenance coefficient drops to a third of the value in short-term limited cultures.     

Balancing the amino acid needs of the dairy cow

In order to maximize milk protein production, one must present sufficient amounts of the essential amino acids to the intestinal tract in forms that can be absorbed. We do not know the specific tissue-level amino acid requirements of lactating cows, but they are likely to be similar to the amino acid content of milk protein with requirements for other metabolic functions similar to those in nonruminants. Formulating diets to meet these amino acid requirements is complicated because much of the dietary crude protein is converted to rumen microbial protein. Knowing the amount of dietary crude protein converted to ruminal microbial protein and the amino acid content of the rumen microbes; and the proportion of ruminally undegradable protein, its postruminal digestibility and amino acid content will allow one to make a reasonable estimate of the quality of protein presented for gastrointestinal digestion and absorption. Hypothetical calculations indicate potential dietary differences in quality of protein presented for absorption. Many of these differences correspond very well with production responses observed in research trials. Failure of this system to explain production results in other studies points to areas where additional information is still needed.     

Sustainable Cultivation Concepts for Domestic Energy Production from Biomass

Referee: Dr. J. Grant McLeod, Semiarid Prairie Agricultural Research Centre, Research Branch, Agriculture and Agri-Food Canada, P.O. Box 1030 Swift Current, Saskatchewan S9H 3X2, Canada According to the European Union, biomass will play a major role in the substitution of fossil fuels with renewable resources. Biomass will contribute 83% to the increased use of renewable resources by the year 2010. In contrast to other solar energy sources, plant biomass is always available and can be converted into energy continuously. An important objective in the production of energy crops on arable farm land should be to realize a high net energy yield and fulfill obligations in the field of environmental protection. The “double cropping system” was developed to meet these obligations. Silaging as a conservation method for wet biomass makes this sustainable cultivation system possible. It includes a diverse array of crops and provides the opportunity to integrate rural organic wastes into this energy concept. The model presented, “the energy self supplying farm”, shows that it is possible to meet the energy consumption requirements of a livestock farming operation with energy crop production on 10 to 18% of the arable farm land. According to a new rape energy concept, a land resource requirement of roughly 10% is feasible if biomass residues from rape oil production for liquid fuels are also utilized for energy production. For a farm with livestock, anaerobic digestion technology is an appropriate technique to deliver heat and electricity for the farmstead. Digestion residues, used as fertilizer in energy crop production, results in an almost complete nutrient recycling. Energy output can be increased above the demand of the farm via the biogas reactor, using the total biomass produced with double cropping. Surplus electricity is supplied to the grid at a guaranteed price. Biomass is a domestic energy resource, and farmers have the chance to extend their function from a supplier of raw material to managers of domestic energy resources. Under the currently established framework, monetary return per hectare could be more than double with biomass energy production via biogas. This will allow the agricultural economy to recover and promote a sustainable regional development. In addition to being a convenient method of waste management, sustainable energy crop production can make a significant contribution to environmental protection and the improvement of the social and economic cohesion within a community.     

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.     

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat’s pellets

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.     

Does dietary P affect feeding and performance in the mixed-feeding grasshopper (Acrididae) Melanoplus bivitattus?

Although consequences of limited dietary protein and carbohydrate to performance are well studied for terrestrial insect herbivores, the importance of phosphorus (P) remains poorly understood. We examined the significance of dietary P to performance in fifth-instar nymphs of the grasshopper Melanoplus bivittatus fed artificial diets. Consumption, digestion, developmental rate, and growth in response to different levels of P nested within standard-Protein and carbohydrate diets were determined. Developmental rate was slowest on high-P diets; protein:carbohydrate concentration and P in diets affected frass production and consumption. Approximate digestibility and conversion of digested food were primarily influenced by the protein:carbohydrate quality of the diet but not P. Mass gain was marginally lower in the low-Protein:high carbohydrate diet used in this study. At the individual level, other than small effects to developmental rate at high concentrations for M. bivittatus, dietary P otherwise seems to have little effect on nymphal performance. To the degree that it is important, effects of dietary P depend on the concentrations of protein and carbohydrate in the diet.     

Microalgae mass culture: the constraints of scaling-up

There is overwhelming evidence that microalgae would be the logical source of oils for biodiesel production, the best option for CO₂ sequestration and numerous other applications. However, this apparent lucrative approach is still in its infancy. In order to impact on global energy needs, bioremediation and other potential applications, vast quantities of biomass must be produced at a reliable rate and as cost-effective as possible. When extrapolating volumetric rates from laboratory or small-scale outdoor cultures to large-scale outdoor areal production rates, it becomes apparent that many of the potential claims are either misleading or still only a dream. Open raceway ponds are at present the only feasible culture system for the production of millions of tons of biomass. To date, at best photosynthetic efficiencies of around 1.2% have been achieved, but with present understanding and know-how efficiencies of double that should be achievable, especially when vertical mixing is increased in raceway ponds.     

Effects of dietary protein types on immune responses and levels of infection with Eimeria vermiformis in mice

The present study reports the dietary effects of bovine alpha whey fraction, bovine casein and soy protein isolate on the immune responsiveness of C57BL/6J mice infected with Eimeria vermiformis. During the patent period, mice fed alpha whey fraction had significantly higher blood total white cell, CD4+ and CD8+ lymphocyte counts and higher Con A-stimulated IFN-gamma production by spleen cells than those fed other protein sources, but there was no significant difference in output of faecal oocysts. Casein-fed mice had significantly higher levels of Con A- stimulated IFN-gamma production and a lower output of faecal oocysts than soy-fed mice. The study demonstrated that dietary proteins have different impacts on immune responsiveness and level of parasitic infection in the gut; however, the mechanisms affecting level of infection are not clear. These effects appeared not to be solely related to nutritional properties of the diets. Further research into the underlying immune mechanisms and possible direct interactions between bioactive proteins and the parasite E. vermiformis should be fruitful.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.