Advances in Industrial Biotechnology - Essay Example

?Advances in Industrial Biotechnology Biotechnology is defined by the UN Convention on Biological Diversity to be “any technological application thatuses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use” (as quoted byWordIQ.com, 2010). This definition is by far the broadest given for biotechnology. Biotechnology has been employed even in pre-historic times. Traditional food like wine, vinegar, cheese, and bread are just a few of the products of biotechnology. Modern biotechnology products are those that were produced with genetically altered microorganisms or organisms to produce medicine, antibiotics, hormones, proteins, amino acids, and biofuels. Transgenic plants, like Bt corn and herbicide resistant soybeans, were designed to improve agronomic performance, although these are also used in biopharming (pharmaceuticals from plants). Transgenic animals are currently used to express drugs in milk. Biotechnological approaches have been used to enhance the yield and diversity of the important compounds amino acids, vitamins, antibiotics and biofuels. Amino acids Amino acids are the building blocks of proteins. Humans and animals alike can synthesize amino acids, except for eight that have to be supplemented by the diet. Aside from their roles in nutrition, amino acids are also used as food flavour enhancers, for medical uses such as transfusion of proteinaceous food and ammonia detoxification, and in the manufacture of synthetic raw materials in the chemical industry (Okafor, 2007). Globally, glutamic acid, lysine and methionine are produced in the highest amounts, although all amino acids can be and are being synthesized. The flavour enhancer monosodium glutamate is the number one product in terms of tonnage (Demain, 2007). Okafor (2007) reviewed the different methods used to manufacture amino acids. Hydrolysis of high protein products like hair, keratin and feathers is the oldest means of manufacturing amino acids. However, this procedure is highly dependent on the amount of raw material that is available. Amino acids are also chemically synthesized, but the end-products are a mixture of D and L forms which necessitate an additional expensive step to produce the biologically active L-form. Fermentation of carbon and nitrogen materials through the action of different bacterial species is the most economical and feasible to large-scale process. Enzymatic process converts specific substrates to amino acids through specific enzyme catalysis. The most important method for microbiological production of amino acids is through direct fermentation and this is where biotechnological approaches have been utilized the most. The discovery that microorganisms like bacteria, moulds and yeast express amino acids was the stimulus for advancement in this field. Corynebacterium, Brevibacterium, Microbacterium and Arthrobacter are the most common bacterial genera employed for direct fermentation. To induce expression of amino acids, media and culture conditions are modified. However, bacteria will produce the amino acids only to a certain extent because they have this innate control of production to prevent toxic effects. Thus, bacteria have been engineered that do not have the control mechanisms for inhibiting amino acid production. Auxotrophic mutants have increased production of L-glutamic acid (Nakamura, et al., 2007; Asakura, et al., 2007). Regulatory mutants for amino acid production have also been developed. These mutants possess an enzyme that is insensitive to feedback inhibition, and thus continues to overproduce a certain amino acid. For example the bacterium Brevibacterium flavum was engineered to be insensitive to increased lysine concentration which has an inhibitory effect on aspartate kinase activity (Fernandez-Gonzalez, et al., 1996). Aspartate kinase is the only system in the lysine biosynthesis pathway that is sensitive to increased lysine levels. Recombinant DNA technology has greatly enhanced the introduction of genes into current bacterial species in order to improve production efficiency and volume (Okafor, 2007). This approach was largely aided in molecular biology techniques in isolating, cloning and performing functional analysis of important microbial genes. The genes modify the central and terminal pathways of amino acid synthesis, and secretion of the amino acids into the external medium. Other strategies for improving amino acid production with recombinant microorganisms were proposed by Ikeda (2003) (Figure 1). Figure 1. Proposed strategies for improving amino acid production via recombinant DNA technology (Ikeda, 2003). Vitamins Fifty percent of commercial production of vitamins is geared towards the consumption of domestic animals. Vitamins are produced by chemical synthesis, extraction from raw materials, fermentation and bioconversion techniques (Demain, 2007). Of eighteen vitamin products, seven are produced in commercial scale using microorganisms. These vitamins are: vitamin C, vitamin B12, vitamin B13, vitamin F, beta-carotene, riboflavin, linolenic acid, and ergosterol. In terms of annual production value, Vitamin C or ascorbic acid ranks first at US $ 480 million. Vitamin C has been produced traditionally by chemical synthesis (called the Reichstein process). Five steps are required for synthesis of ascorbic acid from glucose, and its conversion to 2-keto-L-gulonic acid, which undergoes reaction with base to form ascorbic acid. Microbial synthesis of vitamin C is now available using genetically modified Erwinia herbicola for efficient conversion of glucose to 2-keto-L-gulonic acid (Anderson, et al., 1985; (Grindley,et al., 1988). Better yields are achieved with Gluconobacter oxydans that has been engineered to express L-sorbose dehydrogenase and L-sorbosone dehydrogenase. The two enzymes convert sorbitol (starting material) directly to 2-keto-L-gulonic acid, thereby bypassing two steps in chemical synthesis of vitamin C (Saito, et al., 1997). Fermentation is currently the major process for the production of riboflavin (vitamin B2). The major microorganisms for fermentation are the moulds Ashbya gossypii and Eremothecium ashbyii, and the bacteria Bacillus subtilis (Demain A, 2007). A. gossypii is a riboflavin overproducer; it makes forty-thousand times more riboflavin than what it needs for growth. Currently, genes are being identified that could further increase the riboflavin yield from A. gossypii (Karos, et al., 2004). The bacterium Cornybacterium ammoniagenes have been engineered with riboflavin biosynthetic genes to express more riboflavin without feedback inhibition (Koizumi, et al., 2000). Other microbial strains are being tested for improved riboflavin production. Industrial production of Vitamin B12 or cyanocobalamin utilizes the strains Propionibacterium shermanii and Pseudomonas denitrificans. These are overproducers, making 100,000 times more vitamin B12 than what is needed for growth. To permit such high production volumes, feedback repression by vitamin B12 should be avoided. Biotin or vitamin H is a product of the action of Serratia marcescens that have been selected for resistances against anti-metabolites of biotin. Presently, research studies are underway to identify genes for high production rates, and for identifying means to avoid feedback inhibition by the desired product. Antibiotics Secondary compounds are usually produced after the growth of the microorganism has slowed down, and do not have any function on the growth of the organism. These compounds function as sex hormones, competitive factors against other organisms, symbiosis agents, cell-to-cell communication, and as ionophores. Antibiotics are the most popular secondary metabolites. The antibiotics are a group of heterogeneous compounds that are biologically active, with different structures and mode of actions, and can attack different microbial activities like nucleic acid synthesis, membrane function (e.g. signalling), germination and electron transport. Since 1940, antibiotics have been used in the fields of medicine, and in basic research. Antibiotic drugs have been very useful in the fight against microbial diseases, but the discovery of new ones dropped after the 1970s (Demain, 2007). Microbes are also the major source of antibiotics. Well-utilized bacteria are strains of Streptomyces griseus which produce more than 40 antibiotics, Bacillus subtilis with more than 60 compounds, and Streptomyces hygroscopicus strains that produce over 200 antibiotics (Demain, 2007). The increased need for more and better antibiotics have also increased the use of recombinant DNA technology to introduce genes that code for antibiotic synthetases into bacteria. These genes encode for modified or hybrid antibiotics (Epp, et al., 1989; Khosla, et al.1996). ?-lactams comprise one of the most important group of antibiotics. In the group are penicillin G, cephamycin C, cephalosporin C and other semi-synthetic cephalosporins and penicillins. Protoplast fusion was utilized in modifying Penicillium chrysogenum, a pencillin producer, to improve its poor sporulation and seed growth characteristics. It was crossed with a low-producing strain that had the desired traits (Kennedy & Turner, 1996). The same organism was also genetically engineered to increase the copies of the penicillin biosynthetic genes and increasing the transcription rates. Penicillin yield were increased by overexpressing phenylacetic acid-activating CoA ligase from Pseudomonas putida (Kennedy & Turner, 1996). Aside from introducing extra copies of biosynthetic genes into the genomes of the penicillin producers, overexpression was also achieved by replacing the normal promoters with another more vigorous promoter. Similar strategies have been employed in other microorganisms to produce other antibiotics. The modification of nutrient requirements of the high producers was also performed to reduce costs for nutrients. A good example is the development of an Acremonium chrysogenum recombinant strain that showed rapid growth in less expensive sulphate nutrition, and produced 40% more cephalosporin than the parental line The progeny included a recombinant which grew rapidly, sporulated, produced cephalosporin C from sulfate and made 40% more antibiotic than the parent. Other improvements are reviewed in Adrio and Demain (2010). It is important to continue to develop new anti-microbials especially with the development of new antibiotic resistant strains of microbes. With more molecular approaches being employed for increasing yields and producing secondary metabolites, (Table 1), it is expected that new and better antibiotics will be produced to combat new super-bugs (Adrio & Demain, 2010). Table 1. Molecular approaches for increasing yields of different secondary metabolites (Table is adapted from Adrio & Demain, 2010). Molecular Approach Secondary Metabolites Produced Protoplast fusion Penicillin G, cephalosporin C, cephamycin C, clavulanic acid, indolizomycin, rifamycins Metabolic engineering Antibiotics (penicillin G, cephalosporin C, cephamycin C, clavulanic acid, semisynthetic cephalosporins), antitumor agents (anthracyclines, glycopeptolides, anthracenones), avermectins, xanthan gum, artemisinin Transposition Daptomycin, tylosin Association analysis Lovastatin Combinatorial biosynthesis Erythromycins, tetracenomycins, tylosin, spiramycins, surfactins Whole genome shuffling Epothilones, spinosad Genome mining Echinosporamicin-type antibiotics, antifungal compounds (ECO-02301) and others Biofuels As the demand and use of fossil fuels escalates, supplies are expected to be depleted. This scenario has triggered the search for other energy sources that are renewable and can replace fossil fuels. Biofuels refer to the wide range of fuels that can be derived from plant or biological biomass. The most popular biofuels are bioethanol and biodiesel. Bioethanol and methanol are produced from the fermentation of carbohydrates and sugars coming from fresh or degraded biomass through the action of microorganisms. Biotechnological approaches are critical in increasing the biomass yield per unit area, in improvement of crop varieties for increased biofuel yields, to increase in land-use efficiency by increased productivity through reduced effects of stresses and cultivation of energy crops in marginal lands, and development of microorganisms for the efficient conversion of biomass to biofuels (EuropaBio, 2008). Thus, a two-pronged approach is recommended: improve the biomass production, and improve the microorganisms in the conversion process. Corn is currently the primary crop used in producing bioethanol. However, with the projected demand on bioethanol, it is not possible for most countries to produce enough corn for fuel. Thus, efforts are exerted to search for alternative crops, specifically those that have high cellulose and hemi-cellulose contents. To degrade and ferment biomass rich in cellulosic matter, it will be necessary to produce recombinant yeasts, recombinant bacteria (Escherichia coli, Klebsiella oxytoca, and Clostridium spp.) (Demain, 2007). Recombinant DNA techniques produced modified E. coli by introducing genes from other organisms, (i.e. genes for alcohol dehydrogenase and pyruvate decarboxylase from Zymomonas mobilis) making it highly efficient in producing ethanol (Lindsay, Bothast, & Ingram, 1995; Ingram, et al., 1987). In the genetically modified strain, the 95% of the fermentation product was ethanol, while the wild-type strain had a product of mixed acids. Clostridium thermocellum, which thrives in warm environments, directly converts cellulose to ethanol (Demain, Newcomb, & Wu, 2005). Other Clostridia strains can also produce acetone, lactate, acetate and butanol, which have potential as biofuels. Aside from cellulosic and lignocellulosic materials that have been proposed to be primary material for the production of renewable biofuels, some feedstocks are rich in fatty acids and are available for recovery. Development of microorganisms with the oil-harvesting trait make fatty-acid-rich feed an attractive alternative to lignocellulosic materials. A study to engineer the microorganisms that can utilize fatty acids as substrate was reported recently (Dellomonaco, et al., 2010). Fermentative pathways were engineered in Escherichia coli so that they can function under aerobic conditions. These pathways enabled the combination of respiratory and fermentation metabolic pathways to proceed, leading to the synthesis of chemicals and biofuels from fatty acid substrates. To illustrate the feasibility of such an approach, the production of target products ethanol and butanol (representing biofuels) and selected biochemicals (propionate, isopropanol, succinate, acetone, and acetate) were measured. Using the engineered bacteria, yields of the target products exceeded those that were produced from sugars, surpassing even the amounts produced from lignocellulosic material. The engineered pathways also showed the feasibility of synthesizing propionate from E. coli, previously, only propionibacteria, can synthesize it. This was the first work that showed success in producing biofuels from fatty acid substrates by recombinant bacteria. Conclusion Biotechnological techniques are the major contributors to the production of compounds that are important in health, nutrition and environmental preservation. 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