Bioproducts and Bioprocesses 2

Engineering research related to the life sciences is becoming more important as an increasing number of products from genetic engineering and cell fusion technology reach the market place. Novel bioprocess engineering, both up-stream and downstream, is also needed to provide a fundamental engineering basis for the economical manufacturing of substances of biological origin. Research linking the expertise of engineers and life scientists is crucial to providing such a fundamental basis, and requires individuals who are broadly competent in each of their fields and who are also willing to collaborate on research projects. This presentation intends to provide an overview of how the Engineering Directorate within the National Science Foundation plans to provide support for the engineering research needed to address the problems of the economic production of products obtained from the most recent advances in the life sciences.

On-line monitoring and control of bioprocesses is one of the most challenging research areas in biochemical engineering. Sterilizable dissolved oxygen (DO) and pH probes have been available since the 1960s. No other biosensors developed since then have proved to be as good as the DO and pH probes. Dissolved oxygen, pH and temperature along with off-gas analysis are the only parameters which are monitored on-line in industrial fermentations. None of the present on-line measurement techniques involves intracellular parameters. This limitation has hampered the development of bioprocess control since our ability to control and perform on-line optimization of fermentation processes is limited by our ability to monitor what is happening in the fermentation.

The reaction mechanism in a microorganism is much more complicated than that of an ordinary chemical reactor. The mechanism is fundamentally programmed by the DNA sequences of the organism involved, which can change from their original situation with time. It is difficult to keep all of the characteristics of a microorganism constant for a long period, and the improvement of strains by genetic engineering and/or screening techniques is often tried in order to improve industrial fermentation processes. As a result, models of a real fermentation process usually work only for a limited period, and it is virtually impossible to construct a comprehensive and robust model. Furthermore, only a few kinds of sensors are available for monitoring the culture states in fermentation processes, and we cannot measure the state inside cells directly. Therefore, a deterministic model for the application to the control or the simulation of fermentation processes cannot easily be constructed.

The Engineering Centers Division supports university-based research centers aimed at enhancing industrial competitiveness by strengthening university/industry coupling in research and education. The division supports two major programs, Engineering Research Centers (ERC) and Industry /University Cooperative Research Centers (IUCRC).

The glutamine synthetase (GS) system is a novel gene amplification system in which vectors containing GS coding sequences are used as dominant markers amplifiable by selection with methionine sulfoximine (MSX). DNA encoding the desired protein is combined and co-amplified with the GS gene. Transfected cells are cultured in glutamine-free medium with 500 uM MSX to potentiate the amplification.

It has been previously observed that in a semi-solid air fluidized bed bioreactor, with a potato providing the sugar substrate, and defined media providing the minerals and vitamins [2], certain proteins produced by the bakers’ yeast growing on this aerated semi-solid are carried out of the bioreactor by the effluent air stream [3]. Since proteins can be separated from solution in traditional submerged fermentation processes at their isoelectric points by bubble fractionation [4], it seemed reasonable to propose that the isoelectric point may be an important variable in the air fluidized bed protein synthesis and separation process [5–7], as well. It turned out that for an initial 100 mg/L invertase concentration in an experimental model batch system (a shake flask), the separation between the air phase (or more precisely, micro-water droplets in the air phase) and the bulk liquid phase is not affected by changes in the initial pH concentration over the range 2 < (pH)₀ ≤ 8. This indicated that the separation between the media surface and the water droplet surface in the fluidized bed may not be sensitive to the bulk liquid pH level [8].

Chymosin (calf rennin), an enzyme obtained from the calf stomach has been used as a milk-coagulant in the cheese industry. Chymosin, a member of the aspartic proteinases, cleaves at a specific position (Phel05-Metl06) of κ-casein, as a result of which, milk micelles are destabilized, leading to the clotting of milk. Chymosin is characterized by its high milk-clotting activity and very weak proteolytic activity. Recent success in X-ray crystallographic analysis has revealed its bilobal structure composed of two topologically similar domains rich in β-structures [1]. At their junction is located the substrate-binding cleft and at the bottom two catalytic aspartyl residues, Asp32 and Asp215, are contained. Despite a wide variety of catalytic properties in many aspects, members of the aspartic proteinases possess well-conserved tertiary structure and well-conserved amino acid sequences covering the two catalytic aspartyl residues.

Most previous research on genetic improvement of metabolic activities in industrial organisms has focused on improvement or modification or biosynthesis pathways. This strategy is reasonable because enhancement of key steps and alteration of feedback controls can increase the intracellular allocation of required intermediates and cofactors into production of the desired compound. Ultimately, however, any such manipulation is limited by the upstream kinetic and regulatory network which controls the concentration and the fluxes to biosynthetic intermediates, ATP, and redox cofactors. Although manipulation and improvement of the central pathways of carbon and energy metabolism is complicated by the extensive regulation which pervades these networks and by their interconnection with all other cellular activities, genetic restructuring of carbon and energy metabolism is an important arena for future research. Efforts in this area complement synthesis pathway engineering activities. Furthermore, successful improvements in carbon and energy metabolism may be beneficial over a broad spectrum of organisms and products.

As the result of the development of recDNA techniques, it has become possible to manipulate genes and breed useful microorganisms. Among various transformants bred in our laboratory to produce large amounts of useful compounds 2 topics will be described in this article. One is the breeding of a transformant of E. coli which accumulates tuna growth hormone. The other is the breeding of a jumbo yeast and production of S-lactoylglutathione.

Clostridium acetobutylicum, a natural producer of butanol, acetone and ethanol, is of interest as a potential commercial source of these solvents. The biochemistry by which C. acetobutylicum initially produces acetate and butyrate and subsequently the above solvents, in batch culture, is well understood. The genetics of this anaerobic, highly regulated, branched, primary metabolism is, however, poorly understood despite the fact that many of the genes responsible for the formation of the various acids and solvents have recently been cloned [1–5].

Thermophilic bacteria produce various useful enzymes. Some of them have been purified and characterized, and their structural genes have already been cloned and sequenced [1–3]. Bacillus stearothermophilus NCA 1503 was found to produce a thermostable alcohol dehydrogenase (ADH-T) amounting to 1–2% of soluble cell protein. By using this strain, ethanol was produced from sucrose or glucose as a carbon source under anaerobic condition at high temperatures [4, 5]. Alcohol dehydrogenases (ADH) were also isolated from both eucaryotes [6–9] and procaryotes [10, 11], and they exhibited various features. The ADH reaction mechanism was originally studied using horse liver ADH based on X- ray crystallographic analysis and kinetic studies [6, 12, 13]. The catalysis with horse liver ADH is performed by the proton release system consisting of a zinc atom, water molecule, and serine and histidine residues. By a series of intensive studies, the system including the 2’-hydroxyl group of the nicotinamide ribose was proposed for horse liver and human liver ADH [14–16]. Threonine and histidine of human liver ADH corresponded to serine and histidine of horse liver ADH, respectively, both of which functioned as catalytic sites [16, 17]. The amino acid residues responsible for substrate specificity in human liver ADH and yeast ADH were also proposed based on three-dimensional information on the horse liver ADH [18–20].

When a recombinant fermentation process is to be optimized and/or improved on a rational basis, many important parameters closely related to the cellular physiology and metabolism, genetic characteristics of the recombinant having foreign DNA, bioreactor environmental and operational conditions and all of their effects on productivity must be well understood and appropriate bioprocess strategy must be developed. In recombinant fermentation systems, there is a heterogeneous cell population consisting of productive plasmid-harboring cells and non-productive plasmid-free cells. Their cell population dynamics and fraction of productive cell population must be understood. Determination of key genetic parameters and assessment of their effects on the heterogeneous cell population and the productivity of genetically engineered recombinant organisms are very important to the design, control, and optimization of large- scale recombinant fermentation processes [1].

Cellulose and xylan are two main components of plant cell mass and are polysaccharides polymerized mainly by β-l,4-glycosylic linkage. The major difference between the two is that the sugar unit of cellulose is glucose while that of xylan is xylose. The chemical difference of these two sugar moieties is that one of the hydrogen atom of xylose at carbon 5 is replaced by a hydroxymethyl group in glucose. From the similarity of chemical structure of xylan and cellulose, it is interesting to compare the enzymes acting on these two polysaccharides. We have cloned and sequenced the genes coding xylanase of Bacillus pumilus IPO and cellulase of Aspergillus acleatus and have succeeded in solving the tertiary structure of these enzymes by X-ray crystallographic analysis. In this paper, I will discuss the homology of the tertiary structures though no significant homology in the amino acid sequence is observed.

In recent years, the most significant development in the field of synthetic chemistry has been the application of biological systems to chemical reactions. Reactions catalyzed by enzymes or enzyme systems display far greater specificities than more conventional forms of organic reactions; some have been shown to be useful for synthetic or biotechnological applications.

Enzymatic catalysis in non-aqueous media has progressed from using enzymes in aqueous solutions containing relatively low fractions of water-miscible organic cosolvents to that in biphasic aqueous-organic mixtures, to that in microemulsions and reversed micelles, to that in monophasic organic media containing small amounts of water, to that in anhydrous organic solvents. It is easy to understand why enzymes retain catalytic activity in the first three types of reaction media, for in all of them the enzyme molecules are located in aqueous environments (and therefore the inherent enzymatic properties in such systems are usually not significantly different from those in water). Conversely, the phenomenon of enzymes vigorously functioning in organic solvents with no water goes against the conventional wisdom and universally accepted dogmas. Nevertheless, it has now been firmly established (see Ref. [1] for a review) that this phenomenon exists, that it is quite general and highly beneficial for bioprocessing [2], and that enzymes in organic solvents exhibit remarkable novel properties, e.g., greatly enhanced thermal stability and dramatically altered substrate specificity and stereoselectivity. In this presentation, fundamental questions concerning enzymatic catalysis in organic solvents will be addressed: How does enzymatic activity depend on the nature of the solvent and why? What physicochemical rules govern substrate, inhibitor, enantiomeric, and positional specificities of enzymes in organic solvents? What is the enzyme’s structure and mechanisms of action in anhydrous media? and What additional new properties do enzymes acquire when placed in non-aqueous solvents?

In eukaryotic cells, some supramolecular systems composed of associated biomolecules are assembled into cellular organelles surrounded by membranes, for example mitochondria, chloroplasts, peroxisomes, lysosomes, vacuoles, endoplasmic reticula and nuclei. These organelles may become attractive models of novel biocatalysts for carrying out multistep and multifunctional reactions efficiently, although the organelles themselves seem to be inferior to enzymes and intact cells as biocatalysts because of their labile membraneous systems.

Although enzymes possess many unique catalytic properties, combining substrate and reaction specificity and high activity at ambient temperatures and pressures, they have not been widely applied in industrial chemical processes. Enzymes generally require an aqueous environment to function and thus cannot be used effectively to synthesize organic chemicals which have limited solubility in water. They may also be inhibited by reaction substrates or products, and in some cases production of the desired product requires the enzyme to operate in the “reverse” direction. Recently enzymes have been shown to function in selected organic solvents, to a large extent overcoming these obstacles. Several examples of enzyme reactions under conditions of low water content will be discussed.

There are three main characters in an enzyme reaction: a substrate-binding site, a catalytic site, and a substrate. If we prepare a new set of these players, the set will show a new catalytic activity and become an artificial enzyme. The investigation of the kinetic properties of these artificial enzymes will enable us to understand the rate-acceleration mechanisms used in natural enzyme reactions.

An on-line diagnosing system for a fed-batch fermentation process has been developed. The system is based on the physiological activities of a micro-organism which are represented by specific rates like cellular growth, substrate consumption and production of an aiming substance. These process variables can be measured by an on-line system named Bio Advanced Control System (BIOACS) which we developed in 1985.

The need for robust sensors for bioprocess monitoring leading to effective control strategies is well recognized by biochemical engineers. Firstly, the ability to monitor on-line cell mass concentrations has been the goal of many re¬searchers. Secondly, the added complexity of many fermentation media contain¬ing suspended solids renders the cell concentration measurement in situ in a fermenter even more challenging. Lastly, the ability to differentiate the total cell mass from the viable fraction in situ could lead to some very exciting on-line control and optimization strategies.

Neurotransmitters play important roles in the brain since they are the key link in communication between neurons. Acetylcholine is the transmitter of motor neurons in the spinal cord as well as in all the nerve-sketal junctions in vertebrates. It is located diffusely throughout the brain. The activity of the enzyme choline acetyltransferase in the brain decreases often significantly with age. This is the enzyme responsible for synthesis of acetylcholine. Glutamate is also the transmitter which is related to expression of long term memory. Mechanisms of long-term potentiation (LTP) in the hippocampus and long-term depression (LTD) in the cerebellar cortex are unclear because of difficulty in measuring glutamate release from the presynaptic membrane and the sensitivity of the glutamate receptor in the postsynaptic membrane. In vivo acetylcholine and glutamate sensors are powerful tools for elucidating the sites of action in the brain where they operate. A smaller electrode does less damage to tissue during insertion into brain or nerve tissue. An extremely small environment can be examined with a microelectrode whose diameter is of the order of a few micrometers. The carbon fiber electrode is considered to be one of the most useful transducers for in vivo biosensors, because carbon fibers provide us with ultramicroelectrodes with high strength and electrochemically pretreated fibers have a much greater sensitivity to dopamine and catecholamine. Acetylcholine and glutamate cannot normally be directly oxidized on the electrode, however the application of carbon fibers has proven otherwise using immobilized enzymes. Several acetylcholine sensors have been developed based on enzyme electrodes with a potentiometric detector or amperometric detection of oxygen and hydrogen peroxide. However these sensors are not suitable for in vivo analysis. The detection of hydrogen peroxide is an established approach to the construction of biosensors based on electrodes containing immobilized oxidases. In a report by Akiyama it was shown that it is possible to obtain a high sensitivity and an excellent reproducibility to dopamine using microcomputer-controlled potentiostatic pulse polarization techniques [1].

Most industrial bioprocesses are generally conducted in batch or fed batch modes with limited process control capabilities. The cell is essentially treated as a black box which takes up nutrients and produces the desired product. Improvements in product yield and productivity have been achieved primarily by random media manipulation and shotgun mutation programs. Although traditional biochemical and genetic methods have resulted in dramatic improvements in production since the late 1940s, they are not based on fundamental understanding of cellular metabolism. Attempts to optimize product yield and nutrient availability have met with only limited success because of a lack of knowledge concerning regulatory metabolism in these organisms. Environmental factors which trigger the onset of bioproducts production are not completely understood, and means to sustain biosynthesis of these bioproducts are usually unknown. Catabolite repression, feedback regulation of product synthesis, degeneration of synthesis, and degradation of the extracellular product are some problems frequently associated with these industrial fermentations [3]. In addition, the diversity of industrial significant metabolites and the organisms which produce them makes it even more difficult to draw general conclusions about cellular regulation. A general and convenient means is needed to study and to monitor intracellular events of cellular metabolism associated with product biosynthesis of industrial significant organisms at a fundamental level. Such a method would allow meaningful comparisons among different industrial significant bioproduct producing cells.

Already for two decades interest in the automatic control of fermentation processes has been growing rapidly because of its theoretical and practical importance. However, despite some achievements, there are still many unsolved problems ensuing mainly from the great complexity and the unusual characteristics of the living systems [1–5]. The serious difficulties encountered call for revision of the methods and techniques used in modeling and control of fermentation processes [6–8]. To compensate for the shortcomings of the traditional control concepts, some new trends have recently arisen; among which most significant is the application of modern AI methods to the process control [9–14]. Our work has been dedicated to the development and application of a system for control of fermentation processes utilizing such techniques, particularly the knowledge-based control approach.

Gradient elution, invented by Tiselius and his co-workers [1], employs a mobile phase whose composition at the column inlet varies with time. This is in contrast to isocratic elution, where the mobile phase composition remains constant at every point in the column throughout the separation. In fact, one of the reasons gradient elution was introduced was to hasten isocratic separations, by reducing tailing and by moving widely separated compounds closer together. It is also well-suited to the separation of macromolecules, whose retention tends to vary strongly with mobile phase composition [2], and is consequently a widely used technique in biotechnology.

Plant cell tissue culture has two primary applications: micropropagation of elite plants and production of chemicals (e.g. medicinals, flavors, pigments, fragrances, and pesticides). In both cases bioreactor designs and operating strategies that can direct cellular physiology towards more productive states are required. The focus of this article will be on the production of chemicals.

Despite our efforts in the last two decades to improve yields of useful secondary products in plant cell cultures, the compounds that are now, or about to be, produced commercially are very limited in number. Past experiences have taught us that selection of stable, high-producing cell lines and optimization of culture conditions are both important. We know that elicitation of plant cells can induce or hasten the timing of biosynthesis of some compounds. Organized cultures, especially root cultures, have been found to be useful in some cases when undifferentiated cell cultures fail to synthesize compounds of interest. Most of these approaches, however, are empirical and underlying cellular mechanisms are usually left unexplored.

A central issue in modern biotechnology is the control of mammalian cell proliferation by hormones and growth factors. The bioprocessing industry is attempting to produce a wide range of peptide growth factors for mammalian cells, for applications in human and animal health care such as wound healing and immune response stimulation. Roughly 60% of sales in the bioprocessing industry over the next decade are projected to be in human therapeutics, and over 3/4 of the current or planned therapeutic products are peptide growth factors [1]. At the same time, use of mammalian cell themselves in bioreactors to produce pharmaceutical agents is gaining favor, due to their specialized capability to synthesize proteins with appropriate conformation and activity [2, 3]. Proliferation of these cells require growth factors or complex mixtures of regulatory proteins. Finally, growth factor interactions with their cellular targets are critically implicated in many issues arising in the investigation of cancer [4]. Clearly, an improved understanding of the mechanisms by which growth factors regulate mammalian cell proliferation would have tremendous beneficial consequences for both production and application aspects of growth factor-based biotechnology.

Plant cell cultures have been investigated for the production of valuable secondary metabolites such as pharmaceuticals, pigments and flavors. Since plant tissue cells are known to be capable of growing in vitro, the use of plant tissue cultures for production of useful bioproducts has become popular in bioengineering research. The use of plant tissue cultures has the following merits: (1) production is not affected by seasons and climate; (2) the area for plants to produce materials using cell culture is much smaller than that necessary for production in the field; (3) the duration of production is shorter in the case of the tissue culture production; (4) some cultured plant cells are able to accumulate the useful products in higher concentrations than intact-plant cells. Thus, plant cell cultures have apparently greater productivity than cultivation of whole plants for the production of biochemicals. However, only a few examples [1, 2] of the industrial application of plant cell cultures have been reported because of their high costs.

With the employment of genetic engineering techniques to alter the genetic makeup of plants, the ability to regenerate whole plants through tissue culture becomes crucial. Plantlet micropropagation based on shoot and somatic embryo cultures are promising methods for commercial production [1]. However, flask shoot culture is labor intensive since the mature shoots often need to be dissected, transferred to a rooting medium, hardened in a greenhouse, and eventually, transferred to the field. Bioreactor shoot cultures [2] also requiremany of these high cost procedures. Somatic embryogenesis presents definite conceptual advantages for large-scale plantlet production [3]. Millions of embryos can be produced in a laboratory scale bioreactor initiated from a small number of cells [4]. The use of artificial seeds [5] minimizes the necessity of disassembly and renders the automation of large-scale manipulation and planting more feasible. Since the discovery of somatic embryogenesis in carrot (Daucus carota (L) [6], this developmental pathway has been demonstrated in many significant crop and woody species.

A novel immobilization method for animal cells, in which the alginate gel was covered with urethane polymer was developed. By this method, gel particles became resistant to physical stress, cell leakage was minimized and a high concentration of cells could be obtained. Use of the immobilized hybridoma cells in a fluidized-bed reactor improved the production of the monoclonal antibody. The production of monoclonal antibody increased with culture time and finally reached 300 mg/ml gel per day on day 17, which was eight times higher than the value obtained by the repeated-batch culture in spinner flasks. Using this method, we were able to introduce air by bubbling, which is the most effective oxygen transfer method. This bubbling caused free cells to die. In the case of the alginate gels, cell leakage was evident but the free cells soon died. In contrast, cells did not leak from the polymer layer and thus gave very good results which were obtained for hybridoma growth and monoclonal antibody production. In the immobilized cell culture with gel entrapment, diffusion of oxygen seems to be poor compared with the suspension culture. Therefore, we attempted to improve the oxygen supply by using pure oxygen gas instead of air. After reaching a stationary phase, oxygen gas containing 0–5% carbon dioxide was introduced at 10 d. By this change, although glucose concentration in the bioreactor was almost the same the lactate concentration decreased to about two-thirds of that under air. Furthermore, the antibody production rate increased 2-fold. It became feasible to obtain a high concentration of monoclonal antibody continuously for a long period.