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Amino acids, the building block of proteins, are used as raw materials in various cellular processes, such as energy generation, nitrogen metabolism, cell wall synthesis and intracellular communication (Stryer et al., 2002). In addition to these, they are widely used in both human and livestock consumption. Amino acids are generally produced by one of four different methods: hydrolysis of natural proteins, chemical synthesis, enzymatic synthesis and bacterial fermentation. However, the fermentative production of amino acids has been established in industry due to its low production cost. In the case of microbial fermentation, cheap carbon and nitrogen sources (molasses and ammonia, respectively) are frequently used as raw materials (Ikeda, 2002). Furthermore, the production of these building blocks by fermentation yields optically active and biologically desired L-form of amino acids. The economic importance of amino acids is enormous since they are used as flavouring agents, food additives, feed supplements and raw materials for the synthesis of cosmetics, shampoos, toothpaste (Eggeling and Sahm, 1999; Leuchtenberger, 1996; Mueller and Huebner, 2002). Due to their high variety of applications, the demand for these amino acids is constantly increasing. Hence, extensive studies on the understanding and improving the metabolic conditions leading to amino acids overproduction have been undertaken in order to increase the yield and productivity (Kramer, 1996; 2004; Sahm et al., 1995).
At present, most of the essential L-amino acids are industrially produced by Corynebacteria fermentation. Corynebacterium glutamicum, a short, aerobic, rod shaped, Gram-positive soil bacterium, is capable of growing on a minimal medium. Taxonomically, Corynebacteria are closely related to Mycobacteria, and they belong to the mycolic acid containing actinomycetes (Kalinowski et al., 2003). The Japanese scientist Kinoshita and his co-workers discovered C. glutamicum (originally named Micrococcus glutamicus) as a potential microorganism for the production of amino acids because of its ability to excrete L-glutamic acid into the surrounding medium under specific growth condition (Kinoshita et al., 1957). Since then, C. glutamicum is regarded as an efficient L-glutamate secreting microorganism. Under favourable growth conditions, this bacterium converts 100g l-1 glucose to 50g l-1 L-glutamic acid. At present, 1,000,000 tons of L-glutamate and 450,000 tons of L-lysine are produced per year by Corynebacteria fermentation. In addition, L-alanine, L-isoleucine and L-proline are also produced industrially (Kalinowski et al., 2003; Kramer, 2004).
During amino acid fermentation by Corynebacteria, an appropriate substrate (glucose, for example) is taken up by cells through the involvement of different uptake systems (phosphotransferase systems, PTSs). The substrate is subsequently entered into the central metabolic pathways (glycolysis, pentose phosphate pathways, tricarboxylic acid cycle) of these bacteria, converted to metabolic intermediates within cells, and is finally branched off to a particular amino acid biosynthetic pathway (Ikeda, 2002). In recombinants of Corynebacteria, the biosynthetic pathways of a particular amino acid are altered in such a way that results in increase of internal amino acid concentration. This is mainly achieved by the following strategies i) by increasing the activity of anabolic enzymes ii) by altering the regulatory enzymes or pathways (loss of feedback control) iii) by blocking the pathways leading to by-products and iv) by blocking the pathways responsible for product degradation in the cytosol. However, the increase of membrane permeability of Corynebacteria is the most important feature for the efficient secretion/production of amino acid, especially L-glutamate. The secretion/excretion of a particular amino acid into the extracellular medium is generally accomplished either by diffusion or by treating the Corynebacteria strains with an agent or with the aid of a carrier system (Kramer, 1994).
It is well known that the cell wall of Corynebacteria has a complex structure since it is formed by thick meso-diaminopimelic acids containing peptidoglycans that are covalently linked to arabinogalactan (Brennan and Nikaido, 1995). Besides the thick peptidoglycan layer, it also contains large amount of lipids in the form of mycolic acid (Lichtinger et al., 2001). The multilayer arrangement of different phospholipids and peptidoglycan contributes extremely low cell wall permeability. However, the wild strains of Corynebacteria are not suitable for the production of amino acids under normal growth conditions. Hence, several treatments that affect the cell membrane by limiting the synthesis of phospholipids and membrane components have been employed in order to induce the membrane permeability of this bacterium (Kramer, 1996; 2004). For L-glutamate efflux, C. glutamicum has been grown on biotin limitation (Shiio et al., 1962); by the addition of surfactant (Duperray et al., 1992; Takinami et al., 1965; 1968); beta-lactam antibiotic, penicillin (Demain and Birnbaum, 1968; Ikeda et al., 1972); ethambutol (Radmacher et al., 2005) and oleic acid or glycerol (Kanzaki et al., 1967; Okazaki et al., 1967). All of these perturbations directly attack to the cell wall of Corynebacteria, result in changes in the composition of cell wall material and eventually increase the L-glutamate efflux (Eggeling and Sahm, 2001; Kramer, 1994). However, it has been mentioned that the main reasons that result in secreting of amino acids in extracellular environment are (i) a dramatic increase of internal amino acid concentration; (ii) a fundamental change in the permeability properties of the cell membrane; or (iii) a defect in the corresponding uptake system that normally counteracts the efflux of a particular amino acid (Kramer, 1994).
Apart from these treatments, several other procedures, such as mutagenesis, screening, specific changes in both genetic and enzymatic levels have been applied in order to develop mutants/recombinants with desirable characteristics (Jetten and Sinskey, 1995b; Nampoothiri and Pandey, 1998; Parekh et al., 2000). In general, the improvement of amino acid producing Corynebacteria strains is carried out by an iterative procedure of mutagenesis and selection. Mutagenic procedures are optimised in terms of mutagen and dose applied. Selection procedures are designed in order to identify the desirable mutants with maximum expression (Nampoothiri and Pandey, 1998). Genetic engineering is also applied to overexpress or repress the characteristics of a particular gene, and thereby new strains with desired genotypes are constructed (Sahm et al., 1995). To obtain high yield and productivity of an amino acid, however, it is necessary to investigate the detailed information of metabolic pathways and their regulation under different environmental conditions. Hence, metabolic engineering and metabolic flux analysis are recently applied in order to quantify the biochemical fluxes leading to the intermediates or metabolites of central metabolic pathways of this organism (Stephanopoulos et al., 1998). In comparison to the modern techniques applied for the characterization and manipulation of metabolic pathways (Sahm et al., 1995; 2000), however, relatively few studies have been conducted in order to elucidate the efflux or secretion of these amino acids into the surrounding medium.
Electroporation, a well-established physical process dealing with living cells (Chang et al., 1992; Neumann et al., 1989; Teissie et al., 2002; 2005; Tsong, 1991; Weaver and Chizmadzhev, 1996), is involved with a rapid structural rearrangement of cell membrane in response to an externally applied electric field resulting in pore formation in the lipid bilayer within a short period of time (Chernomordik et al., 1987; Haest et al., 1997; Teissie and Tsong, 1981). The opening of transient aqueous pores provides a way to transfer ions and water-soluble molecules across the cell membrane (Prausnitz et al., 1995; Sixou and Teissie, 1993; Tekle et al., 1994). In addition, free diffusion is observed even with dextrans and oligonucleotides, molecular weights up to 4kD (Teissie et al., 1999). Electroporation has been studied over the past two decades due to its numerous applications in cellular biology and biotechnology, especially for the purpose of gene transfer and loading of cells with extracellular molecules (Golzio et al., 2004; Faurie et al., 2005; Mir et al., 1999; Neumann et al., 1982; Rols, 2006; Somiari et al., 2000). In addition, the application of this technique in gene therapy, cancer therapy and transdermal drug delivery has given a new approach to treating complicated diseases (Heller et al., 1999; Mir and Orlowski, 1999; Mir et al., 1991; Orlowski and Mir, 1993). Apart from those applications mentioned above, electroporation has been found to be effective in non-thermal food pasteurization (Angersbach et al., 2000; Wouters and Smelt, 1997), selective release of intracellular proteins from recombinant Escherichia coli (Ohshima et al., 1999; 2000; Ohshima and Sato, 2004) and Saccharomyces cerevisiae (Ohshima et al., 1995) and Kluyveromyces lactis (Ganeva and Galutzov, 1999, Ganeva et al., 2001).
There are two types of electroporation that are extensively applied in biosciences i.e., reversible electroporation and irreversible electroporation. Reversible electroporation refers to the process of treating living cells by a moderate strength of electric field in which transient pores are formed on the cell membrane, and thus the membrane is reversibly permeabilized (Faurie et al., 2005; Hapala, 1997; Teissie et al., 1999). However, electropores that are usually observed on the cell membrane within one minute of pulsation can either be resealed within short period of time or remain open for a longer period depending on the voltage and number of pulses applied to the cell suspension (Chang et al., 1992; Faurie et al., 2005; Weaver, 1995). It has been demonstrated that pulsed cells usually recover their original permeability within 30min of incubation at room temperature (RT) (Kinosita and Tsong, 1977; Teissie et al., 1999). This phenomenon has been extensively used in molecular biology and biotechnology, especially for the transformation of bacteria using foreign genes (Golzio et al., 2004; Jaroszeski et al., 1999; Neumann et al., 1982). The genetic transformation of Corynebacteria has been successfully conducted by high voltage electroporation where an occurence of reversible membrane permeabilization is observed (Bonamy et al., 1990; Dunican and Shivnan, 1989; Liebl et al., 1989; Wolf et al., 1989). On the other hand, irreversible electroporation, the application of high intensity electric field causing permanent breakdown of cell membrane, is used to deactivate microorganisms (Angersbach et al., 2000; Schoenbach et al., 2000). Barbosa-Canovas et al. (1999) demonstrated that the application of pulsed electric field is one of the most relevant non-thermal processes for food preservation without altering their organoleptic and nutritional properties. In addition, Ohshima and Sato (2004) carried out an effective bacterial sterilization using high intensity electric pulses in which an induced irreversible disruption of biological membranes occurred, eventually leading to cell death.
However, the mechanisms involved in electroporation still remain unclear. Although it has been suggested that hydrophilic pores are formed in the lipid matrix (Haest et al., 1997; Neumann et al., 1989; Teissie and Tsong, 1981), their existence has never been clearly shown. The molecular processes involved during electroporation are not fully understood due to the complex nature of the cell membrane, although many theoretical studies have been conducted on the formation of pores under the influence of an electric field (Chernomordik et al., 1987). Nevertheless, it is obvious that electric field intensity, number and duration of pulses are the crucial factors for successful electroporation. Hence, the stimulation factors have to be tightly controlled and optimized, especially when working with an uncharacterized strain. Without adjusting those parameters, cells may not return into their normal physiological state, and eventually lose their viability. Although killing of cells by electropulsation is vital in the case of irreversible permeabilization, it is mandatory to maintain cell viability as high as possible while a reversible electropermeabilization is accomplished either for genetic transformation or bioprocessing.
The production of heterologous proteins in bacteria and yeast using recombinant DNA technology is already well-established in the biotech industry. However, the isolation of recombinant proteins from hosts is not straightforward as the foreign proteins are not usually secreted into the surrounding medium. More specifically, E. coli produces foreign protein as an inclusion body that needs to be disrupted in order to obtain the protein of interest. In most cases, cell disruption is performed by ultrasonication or homogenization for the recovery of recombinant proteins. Using these techniques, however, complete destruction of cells makes the purification process of desired protein complicated, and ultimately the process turns into an expensive bioprocess. Moreover, the recombinant proteins are generally contaminated with the other host proteins during their isolation. As these proteins are used for medical purposes or human consumption, they should be free from other host proteins, particularly pyrogen (Hermann, 2003). To resolve this problem, researchers have been trying to develop an alternative strategy for the bioprocess industries.
However, two most important facts concerned with these industrially important bacteria have been demonstrated in the literature. Firstly, C. glutamicum is one of the most important microorganisms in amino acids production, and L-glutamate production is caused by cultivating this bacterium under certain growth conditions, such as biotin limitation, surfactant addition and penicillin addition (Kramer, 1996; 2004). Secondly, the construction of Corynebacteria recombinants, for the enhancement of yield and productivity of amino acids, is successfully carried out by electrotransformation (Bonamy et al., 1990; Dunican and Shivnan, 1989; Liebl et al., 1989), where a reversible permeabilization of cell membrane is observed. Furthermore, electroporation that creates pores on the cell membrane of microorganisms enhances the selective release of proteins and enzymes from cells (Ohshima et al., 1999; 2000; Ohshima and Sato, 2004; Ganeva and Galutzov, 1999, Ganeva et al., 2001). Although different treatments and extensive investigations towards the genetic engineering of Corynebacteria have been conducted in order to increase the yield and productivity of L-glutamate, no research has been carried out on the electropermeabilization of these bacteria for the enhancement of L-glutamic acid production so far. Based on the above literature, it is hypothesised that electroporation may be a potential approach by which an appropriate strength of electric pulse will be applied to the cell suspension or fermentation broth of Corynebacteria in which the production of L-glutamate is secured by the above-mentioned treatments, and hence the secretion ability of L-glutamate could be enhanced through the membrane permeabilization.
This study is focused on the production of L-glutamate by the different strains of Corynebacteria (Brevibacterium lactofermentum, Micrococcus glutamicus and B. flavum) using different treatments, such as biotin limitation, surfactant addition and ethambutol addition. While developing a suitable method for the production of this amino acid in M. glutamicus, the growth studies (OD600nm), glucose consumption and L-glutamate production in presence of a range of biotin concentrations (0-200µg l-1), under the addition of different concentrations of surfactants [Tween 20 (4g l-1), Tween 80 (4g l-1) and Tween 40 (1-4g l-1)] at two different growth points of fermentation (both start and exponential), and in presence of a range of ethambutol concentrations (0-500mg l-1) will be investigated. Furthermore, a very simple and easily accessible method based on centrifugation will be developed in which L-glutamate is separated with a purity of more than 90% from the fermentation broth. In this research, a study on the utilization of pulsed electric field (transient electroporation) during L-glutamate fermentation by M. glutamicus is considered in order to improve the yield of this amino acid, and thereby intensify the bioprocessing.
Beside L-glutamate, the effect of electric pulses on the release of malate dehydrogenase (MDH, cytoplasmic enzyme), glutamate dehydrogenase (GDH, ammonia assimilating enzyme) and total protein will be investigated. In addition, the effect of electroporation factors, such as cellular (growth phase and cell wall rigidity), electrical (field strength, capacitance, number of pulses and pulse gap/resting time in the case of multiple pulsing) and physiochemical (medium conductivity, ionic concentration of electroporation buffer and temperature) on the membrane permeabilization as well as the viability of M. glutamicus will be examined. An attempt will be made in order to assess the permeabilization of electric field treated Corynebacterial cells by Bleomycin (antitumor agent). Furthermore, the effect of hyperosmotic conditions (addition of 0.5-1.5M NaCl into Seed Medium) on the growth of M. glutamicus, L-glutamate production and the activities of MDH, GDH and total protein will be investigated. It will also be examined whether addition of compatible solutes (glycine betaine and proline) has any notable influence on M. glutamicus growth.
The outline of this thesis is as follows-
Chapter 2 contains literature reviews regarding Corynebacteria (taxonomy and cell wall composition, central metabolism, anaplerortic pathways, uptake and ammonia assimilation, and metabolic engineering); industrial production of amino acids by microbial fermentation and the mechanism of L-glutamate efflux under different growth conditions; and the theory of electroporation or electropermeabilization, importance of this approach in biotechnology and factors associated with the successful permeabilization.
Chapter 3 describes the production of L-glutamate in three different strains of Corynebacteria (B. lactofermentum, M. glutamicus and B. flavum) under several growth conditions i.e., biotin limited (1µg l-1), surfactant (Tween 40, 2g l-1) addition and ethambutol (100mg l-1) addition. A range of biotin or Tween 40 or ethambutol concentrations is added in order to determine the optimum amount of agent required for the highest production of L-glutamate. A simple method based on centrifugation is developed for the purification of L-glutamate (90%) from the fermentation broth.
Chapter 4 represents the application of electropermeabilization for the enhancement of L-glutamate secretion produced under biotin limited fermentation of M. glutamicus. The effectiveness of electric pulse for the extraction of cytoplasmic enzymes (MDH and GDH) and total protein of both M. glutamicus and E. coli is also investigated.
Chapter 5 demonstrates the effect of different factors associated with the electroporation on cell viability (both M. glutamicus and E. coli) and membrane permeabilization. Whether the Bleomycin based method is applicable in accessing the membrane permeabilization of M. glutamicus after electroporation is also investigated.
Chapter 6 depicts the osmotic stress associated during amino acid production and demonstrates the effect of hyperosmotic stress on the growth and viability of M. glutamicus, L-glutamate production and cytoplasmic enzymes or protein level.
Chapter 7 concludes the major factors associated with the success of electropermeabilization, and the degree to which the above-mentioned objectives have been met. This chapter also reveals the prerequisites that are required to consider for introducing this approach in intensified bioprocessing, and suggests the directions for future work.
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