Large-scale Production Systems Research Articles

Large-scale production of GDP-fucose and Lewis X by bacterial coupling

A large-scale production system of GDP-fucose (GDP-Fuc) and fucosylated oligosaccharides was established by the combination of recombinant Escherichia coli cells overexpressing GDP-Fuc biosynthetic genes and Corynebacterium ammoniagenes cells. E. coli cells overexpressed the genes for glucokinase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose (GDP-Man) dehydratase, and GDP-4-keto-6-deoxy-mannose (GKDM) epimerase/reductase as well as phosphoglucomutase and phosphofructokinase. C. ammoniagenes contributed to the formation of GTP from GMP. GDP-Fuc accumulated to 29 mM (18.4 g l−1) after a 22-h reaction starting with GMP and mannose through introducing the two-step reaction to overcome the inhibition of GDP-Fuc on GDP-Man dehydratase activity. When E. coli cells overexpressing the α1,3-fucosyltransferase gene of Helicobacter pylori were put into the GDP-Fuc production system, Lewis X [Galβ1–4(Fucα1–3)GlcNAc] was produced at an amount of 40 mM (21 g l−1) for 30 h from GMP, mannose, and N-acetyl lactosamine. The production system through bacterial coupling can be applied to the industrial manufacture of fucosylated oligosaccharides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 213–217.

Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli.

The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLlambda, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from pRA96. Expression of L-leucine dehydrogenase was controlled by its constitutive B. cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition. After cell cultivation at 30 degrees C and shifting to 41 degrees C to induce plasmid replication, E. coli BL21[pIET98] yielded 200 U LeuDH/mg protein, which corresponds to >50% of the soluble cell protein. Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure.

Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling.

A large-scale production system of cytidine 5'monophospho-N-acetylneuraminic acid (CMP-NeuAc) and sialyloligosaccharides was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammonia-genes. For the production of CMP-NeuAc, two recombinant E. coli strains were generated that overexpressed the genes of CMP-NeuAc synthetase and CTP synthetase, respectively. C. ammoniagenes contributed to the formation of UTP from orotic acid. CMP-NeuAc was accumulated at 27 mM (17 g/l) after a 27-h reaction starting with orotic acid and N-acetylneuraminic acid. When E. coli cells that overexpressed the alpha-(2-->3)-sialyltransferase gene of Neisseria gonorrhoeae were put into the CMP-NeuAc production system, 3'-sialyllactose was accumulated at 52 mM (33 g/l) after an 11-h reaction starting with orotic acid, N-acetylneuraminic acid, and lactose. Almost no oligosaccharide byproducts other than 3'-sialyllactose were observed after the reaction. The production of 3'-sialyllactose at a 5-l jar fermenter scale was almost the same as that at a beaker scale, which indicated the high potential of the 3'-sialyllactose production on an industrial scale.

Future renewable resource needs: will genomics help?

With world demand for consumer goods, materials, and energy continuing to spiral upwards, man has become increasingly reliant on finite reserves of fossil fuels. Moreover, as consumption accelerates there is a danger that the net carbon emissions to the atmosphere may result in detrimental effects in future years. Currently, bio-based inputs account for about 2% of all basic building blocks in manufacturing, excluding wood furniture. A goal of a five-fold increase (to 10%) in the use of bio-based renewable resources, by 2020, has been created for the US. One of the proposed priority research components, to help achieve such an increase, is the further application of genomics to the development of novel renewable resources. The concept is that structural and functional genomics should provide gene discovery methods plus an improved ability to understand the genetic base for the regulation of particular natural molecular structures. Additional biotechnology tools may then be used for metabolic engineering of existing pathways, or to transfer discovered genes into large-scale production systems or to transform high-yielding crops. © 2000 Society of Chemical Industry

Adaptation opportunities to climate variability and change in the exploitation and utilisation of marine living resources

Because they contribute little to climate change, fisheries, aquaculture and other uses of marine renewable resources and environment have limited means to mitigate climate impacts. Adaptation is, therefore, critical. Though likely effects on oceans and fisheries can be identified, few can be quantified and, thus, priorized. Consequently, adaptation strategies should aim at enhancing the resilience of marine renewable resources and their uses and the current capacity to respond to surprises. Already, these uses are characterized by massive over-capacities, excessive resource exploitation, and pervasive conflicts within and between uses. Two complementary adaptation strategies are available. The first consists in adjusting conventional management systems to the new conditions of resource scarcity. The second aims at reducing the current resource constraint by promoting the development of aquaculture and a better utilization of fishery and aquaculture harvests. In this respect, small-scale and large-scale production systems, and developing and developed countries, have different capabilities. In summary, climate change does not modify, but enhances, existing priorities of environment and fisheries management and aquaculture development.

Plants as bioreactors for protein production: avoiding the problem of transgene silencing.

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

Justification of continuous packed-bed reactor for retroviral vector production from amphotropic PsiCRIP murine producer cell.

To indentify a plausible large-scale production system forretroviral vector, three culture systems, i.e., batch culturewith medium exchange, microcarrier culture, and packed-bedreactor culture were compared. In batch cultures with mediumexchange, high cell concentrations were maintained for about amonth, and the harvested retroviral titer remained constant. Inmicrocarrier cultures, although cell growth was rapid, theretroviral titer was unexpectedly low, suggesting that the lowtiter was due either to serious damage to the retroviral vectoror to a reduction in the production rate of retroviral vector,caused by mechanical shear forces. Although the retroviral titer(maximum titer, 1.56 x 10(6)) in the packed-bedreactor was a little bit lower than that obtained in the batchculture with medium exchange (maximum titer, 1.91 x10(6)), continuous production made it possible to increasethe cumulative titer up to 16-fold of that from the batchculture with medium exchange. Moreover, as the packed-bedreactor system requires less labor and shows excellentvolumetric productivity in comparison to batch cultures withmedium exchanges, it will be an appropriate production systemfor retroviral vector in large quantities.

Non-differentiable optimisation for solution of large scale planning problems

In large-scale production systems, there are many complex decisions relating to a variety of asset utilisation and cost implications. Efficient and optimisation based planning systems can be beneficial in deriving feasible and low cost plans. A production-planning task usually involves the allocation of resources to products/packs and also the determination of product paths through the plant. In most cases, the mathematical models result in a high dimensional mixed integer linear problem formulation. For larger problems, the resulting mixed integer models involve several thousands of integer and continuous variables. Usually, to solve such problems, explicit consideration is given to the large-scale nature of the problem. The solution methodology usually consists of a linear programming embedded within a branch and bound procedure. For realistic and commercial scale problems, these methods are hopelessly time-consuming. In this work, an efficient non-differentiable optimiser in co-operation with a linear programming system have been used to optimise the integer decisions in planning problems. The current approach is quite general and is capable of representing a very wide range of planning systems. The methodology outlined in this paper has been implemented in a medium term planning system for solution of large-scale industrial problems.

Scheduling grouped jobs on single machine with genetic algorithm

Production scheduling of grouped jobs has been an active research area since GT (Group Technology) was widely applied in practical manufacturing systems. To minimize the total flowtime of grouped jobs on a single machine, we combine jobs into fundamental runs based upon the necessary condition of the optimal solution. It is proved that the optimal solution is a combination of fundamental runs. A genetic algorithm is designed based on studies on the combinatorial rules of fundamental runs. The numerical results show that the computational performance of the algorithm depends on the number of ‘fundamental’ runs, not on the number of jobs. In general, the number of fundamental runs is far less than the number of jobs. Therefore, the algorithm has potential for practical application in large scale production systems.

Large-scale production of N-acetyllactosamine through bacterial coupling

A large-scale production system of N-acetyllactosamine, a core structure of various oligosaccharides, was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains over-expressed the UDP-Gal biosynthetic genes and the β-(1→4)-galactosyltransferase gene of Neisseria gonorrhoeae, respectively. C. ammoniagenes contributed the production of UTP from orotic acid. N-Acetyllactosamine was accumulated at 279 mM (107 g L −1) after a 38 h reaction (2.5 L in volume) starting from orotic acid, d-galactose, and 2-acetamido-2-deoxy- d-glucose.

Modeling of InGaN MOVPE in AIX 200 Reactor and AIX 2000 HT Planetary Reactor

Multiwafer Planetary Reactor is a promising system for large-scale production of heterostructures for LED's based on III-group nitrides. Analysis of chemical processes occurring in the reactor allows one to get insight into specific mechanisms governing growth of nitride based heterostructures. In the present paper results of modeling analysis of MOVPE of InxGa1−xN layers in AIX-200 Reactor and AIX 2000 HT Planetary Reactor are reported. The model used for MOVPE process analysis accounts for gas flow, heat transfer, and multicomponent mass transport along with gas phase and surface chemical reactions. Results of the modeling analysis of In transport and incorporation into the solid phase are compared with experimental data. It is shown that the model predicts reasonably well the In incorporation during MOVPE of InGaN under In/(In+Ga) ratio in the gas phase less than 20%.

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells.

Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large-scale production systems can parallel those that develop within fast-growing solid tumors such as small cell lung cancer (SCLC). Ammonia's specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane-bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH4Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose-dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI-N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4-day culture with 10 mM NH4Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N41 7 cells is a global rather than glycosylation-specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression.

Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria.

A large-scale production system of uridine 5'-diphospho-galactose (UDP-Gal) has been established by the combination of recombinant Escherichia coli and Corynebacterium ammoniagenes. Recombinant E. coli that overexpress the UDP-Gal biosynthetic genes galT, galK, and galU were generated. C. ammoniagenes contribute the production of uridine triphosphate (UTP), a substrate for UDP-Gal biosynthesis, from orotic acid, an inexpensive precursor of UTP. UDP-Gal accumulated to 72 mM (44 g/L) after a 21 h reaction starting with orotic acid and galactose. When E. coli cells that expressed the alpha1,4-galactosyltransferase gene of Neisseria gonorrhoeae were coupled with this UDP-Gal production system, 372 mM (188 g/L) globotriose (Galalpha1-4Galbeta1-4Glc), a trisaccharide portion of verotoxin receptor, was produced after a 36 h reaction starting with orotic acid, galactose, and lactose. No oligosaccharide by-products were observed in the reaction mixture. The production of globotriose was several times higher than that of UDP-Gal. The strategy of producing sugar nucleotides by combining metabolically engineered recombinant E. coli with a nucleoside 5'-triphosphate producing microorganism, and the concept of producing oligosaccharides by coupling sugar nucleotide production systems with glycosyltransferases, can be applied to the manufacture of other sugar nucleotides and oligosaccharides.

Sustainability as Applied to Farm Animal Systems

Sustainable concepts have been applied to livestock and dairy production and this application is being expanded. However, the use of sustainable concepts probably will not progress as rapidly as some individuals and agencies may suggest or desire. Most of the changes in production and management being researched or applied are important in either conventional or sustainable animal agriculture. The recycling of waste materials as livestock feeds, research and application of performance or production enhancers, reducing construction, operating and maintenance requirements of enclosed housing systems, application of preventive medicine, expanded use of grazing-forage systems, and other management alternatives are just as appropriate (and necessary) in sustainable agriculture as in the “modern” large-scale animal production systems that we have always known and strived to improve.

Assay and structural determination of fructo-oligosaccharides synthesized by an enzymatic system from Penicillium rugulosum

The structure and the fructo-oligosaccharides (FOS) content of a fructan mixture synthesized with an enzymic system from a new strain of rugulosum isolated in our laboratory were investigated by both HPLC and 13 NMR spectrometry. The oligomers were identified as 1-kestose, 1-nystose and 1-fructofuranosyl-nystose. The FOS content of the crude mixture and different commercial products were compared. Results confirmed the interest of such a system for large scale production of FOS.

Human insulin-like growth factor I (IGF-I) produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic activity, and effects on IGF-binding proteins.

Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions offering a range of important therapeutic applications. To evaluate a system for large-scale production of this peptide in the mammary glands of transgenic livestock, we generated transgenic rabbits carrying fusion genes in which a synthetic DNA coding for human IGF-I (hIGF-I) was placed under the transcriptional control of regulatory elements isolated from the bovine alpha S1-casein (alpha S1-cas) gene. Western blot analysis of milk from alpha S1-cas-hIGF-I transgenic rabbits demonstrated production of high amounts of mature hIGF-I peptide (7.6 kDa). Quantitative analysis by RIA revealed hIGF-I levels between 50 and 300 micrograms/ml milk. Recombinant hIGF-I purified from the milk of alpha S1-cas-hIGF-I transgenic rabbits bound to IGF-I receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I produced in Escherichia coli. Ligand blot analysis of milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa IGF-binding proteins (IGFBPs). The 30-kDa IGFBP was shown to be IGFBP-2 by immunoprecipitation using an antiserum raised against human IGFBP-2. Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction in alpha S1-cas-hIGF-I transgenic rabbits. The latter displayed slightly increased milk yield, but no significant changes in total protein content or overall milk protein composition, and reared their offspring without any problems or clinical signs of impaired welfare, even after multiple lactations. Our results indicate that high amounts of biologically active hIGF-I can be produced in the mammary glands of alpha S1-cas-hIGF-I transgenic rabbits. Local production of hIGF-I in mammary tissue is associated with increased secretion of IGFBP-2, which may prevent major biological effects by high levels of hIGF-I on the mammary gland.

High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris.

The early conceptus (embryo and associated membranes) of domestic ruminats signals its presence to the maternal uterus through production of interferon-tau (IFN-tau). Production of IFN-tau ensures continued production of progesterone, the hormone of pregnancy, by the ovarian corpus luteum. This paper reports the high-level expression and efficient secretion of biologically active recombinant ovine IFN-tau (rOvIFN-tau) by Pichia pastoris. The developed method produces more than 80% pure recombinant ovine IFN-tau, obviating the need for further purification for many purposes. Initial fermentation studies produced IFN-tau at 280 mg/liter and demonstrate the potential of this system for large-scale production of IFN-tau.

Integrated management of insect pests, diseases and weeds of cotton in Africa

Integrated pest management systems were developed originally in response to the appearance of insect populations with resistance to common insecticides. Cotton with its heavy dependence on insecticidal sprays was one of the first crops in which the effectiveness of control declined due to resistance in the target pests. Although insecticide resistance is more of a problem in large scale production systems, the IPM approach can also benefit the smallholder by reducing the number of sprays required with consequent cost savings. Where crop growth is adversely affected by diseases, competition from weeds or poor management, the full benefit of insecticide spray programmes cannot be realised. To be fully effective, insect control should therefore be integrated with other crop protection activities. This paper reviews the insect pests, diseases and weed problems of cotton in Africa and discusses the possibilities for extending the principles of IPM to cover all the crop protection activities with practical examples drawn from both small scale and larger scale production systems.

Sensitivity Analysis for Base-Stock Levels in Multiechelon Production-Inventory Systems

Effective management of inventories in large-scale production and distribution systems requires methods for bringing model solutions closer to the complexities of real systems. Motivated by this need, we develop simulation-based methods for estimating sensitivities of inventory costs with respect to policy parameters. These sensitivity estimates are useful in adjusting optimal parameters predicted by a simplified model to complexities that can be incorporated in a simulation. We consider capacitated, multiechelon systems operating under base-stock policies and develop estimators of derivatives with respect to base-stock levels. We show that these estimates converge to the correct value for finite-horizon and infinite-horizon discounted and average cost criteria. Our methods are easy to implement and experiments suggest that they converge quickly. We illustrate their use by optimizing base-stock levels for a subsystem of the PC assembly and distribution system of a major computer manufacturer.

Large-scale production of thermostable alanine dehydrogenase from recombinant cells

We have cloned the thermostable alanine dehydrogenase gene from a thermophile, Bacillus stearothermophilus into Escherichia coli C600 with a vector plasmid pKK 223-3. The recombinant cells produced a large amount of the enzyme, which corresponded to about 10% of the total amount of cellular proteins. In order to prevent the cells from leaking to the environment, cell culture and disruption were consecutively carried out in the same fermentor equipped with a HEPA filter, which served as a biological barrier. The cells grown in the culture broth were treated with Triton X-100, lysozyme, and EDTA, whose final concentrations were 0.1%, 100 mg/ l, and 10 mM, respectively, for 30 min, followed by heating at 60°C for 30 min. Almost all of the enzyme (about 98%) was found in the broth, whereas no living recombinant cells were found in the broth, by these treatments. Based on these we established a large-scale production system of thermostable enzyme from recombinant cells.