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Recent biotechnological approaches used to enhance the yield and diversity of amino acids, vitamins, antibiotics and biofuels

Advancements in science have been related to a great extent to biotechnology. As the days pass and new discoveries are made, biotechnology has played a big role in it. The following are the four scientific fields and the effects that biotechnology has had on them.

Amino Acids

If Amino acids can be produced by fermentation or microbial methods, they can be used in industries related to feed, cosmetics, food, chemicals and pharmaceuticals, which can be seen by the fact that, there has been a five to seven percent growth in this business every year. Thus biotechnology aims to produce them using these methods. Amino acids are being produced by the use of Escherichia coli microbe and the Corynebacterium glutamicum microbe for the last fifty years. Mutation and selection were the two initial methods being used to produce strains of amino acids. Now that biotechnology has advanced, new methods have been developed to, not only increase production but also to produce more different types of amino acids. This has been done by the scientists, by designing functions of transportation, metabolic pathways and mechanism to regulate them. The metabolic activities that take place within the micro organism are controlled by the target genes. With advancements in bio- technology these genes can be identified and then targeted, to get them to perform the metabolic activities. This has been possible because of the use of methods such as metabolic flux analysis Genome sequencing, genomics and proteomics. This has been a big advancement, both quantitatively as well as qualitatively. Moreover, we have been able to get this far because of the exact knowledge that we have about the networks and metabolic pathways (1) (2).

In order to produce Lysine, a lot of time has been dedicated to work on E. coli and C. Glutamicum. In previous works done on C. Glutamicum, the strains that were produced were not very stable when compared to the wild strains. Now, in order to produce more lysine's, mutant alleles to relating 3 target genes, called pyc, lysC and hom for the purpose of homoserine dehydrogenase, aspartokinase, and pyruvate carboxylase repectively have been put into the wild strains. When this was done, it was seen that not only did lysine production increase, but at the same time, this new strain also retained the vitality as well as the robustness of the wild strain. Likewise, answers to the metabolic pieces that were missing in the pathway of lysine biosynthesis have also been found by annotation of genome sequencing and whole genome sequencing. By carrying out a study of genomes and comparing them, certain mutations have been found. These mutations are very helpful for improving the synthesis of Lysine. By comparing and analysing the metabolically produced strain and the wild type strain, it has been made possible to produce a much better strain, which can be used for lysine production. The wild strain was not able to make use of galactose, starch or lactose in order to grow. Studies being carried on by scientists on E coli have shown that mutated C. Glutamicum can now utilise the galactose, lactose and the starch. However, enhancing the production and diversity of Amino acids is a continuing process. In respect of lysine, the main producer is still C. Glutamicum.(3)(4)

Lysine in plants is also a major area of study. One such plant is corn. Corn has low lysine content. But, with advancements in bio-technology, corn has been genetically engineered. In other words, corn with higher lysine content has been produced. Opaque2 is one such type of genetically engineered corn. Likewise, amount of Amino acids in plants has also increased with advancements ion bio-technology. (5)

Vitamins

The amount of vitamins that people have in the form of food from animal as well as plant sources is not enough. For this reason, production of vitamins has become very essential. With, an increasing number of people suffering from vitamin deficiencies, production of vitamins have become even more important. Moreover, production of chemicals causes environmental pollution and affects the health of the common man. In the current era, bioconversion and fermentation are being studied by scientists the most, to carry on biotechnological production. The biological ways and means being used for this purpose also carry a lot of importance. It is not easy to come up with such strains which can produce vitamins fast and in large quantity. This is mainly because, before doing so one has to be able to understand how, metabolic regulation and pathway elucidation takes place. Vitamin synthesis is intricate and is also restricted to the world of prokaryotic. However, there is a solution to this as well. Fermentation has been improved due to use of methods such as r-DNA. Likewise, this technology has also helped in producing compounds of chiral vitamins by immobilising bioconversions. Another important point is, removing the vitamins from the fermentation mixture. (6)(7)

Euglena gracilis Z is a prokaryote which produces antioxidant vitamins, such as vitamin C& E and b-caretone all at the same time. The studies that have been done on this prokaryote all show that when the culture of two steps was used it caused an increase in the content of vitamins, and as a result vitamin yield were much higher. The two steps used are as follows: first the species is grown photoheterophically. In the second step, the species is put into photoautotrophic conditions. The resultant increase in the vitamin content was because; the different conditions of the lights secondary metabolites to be produced in excess (8). Likewise, to produce Vitamin B12, in industries microbes such as Propionibacterium, Bacillus, Pseudomonas and Methanobacterium have been used. Similarly, by using the latest tools and technology vitamin yield was also increased in Propionibacterium freudenreichii during research. By using various types of vectors, ten diversified genes which belonged to the gene families of cob hem, and cbi were expressed in the species P. Freudenreichii. This caused Vitamin B12 production, to increase. The vitamin level was seen to be different with different types of genes. For instance, when the cobU and cobs genes were used, it was seen that vitamin B12 production increased by a very small amount. On the other hand, a 1.50 times increase in production was seen when the cobA, cbiLF and cbiEGH genes were used. In the same way, a 2.20 fold increase in production of VitaminB12 was seen when multigene recombinant, such as the exogenous hemA gene and endogenous hemB and cobA were used. Such an increase was not witnessed when the genes used were non-recombinant. Producing this same vitamin chemically requires nearly seventy steps. Thus, if we consider this point of view; biotechnology has achieved a big breakthrough. (9)

Some studies have also been done on vitamin bio synthesis in plant. Vitamin C& E and Provitamin A synthesis pathways have been studied in detail. The scientific community has two basic agendas as far as these studies are concerned. The first is to increase the vitamin content in edible plants, so that they can give higher nutritional value to their consumers and the second is to be able to extract the vitamins which are useful to us from the tissues if plants. Here to, metabolic engineering will play a big role in helping to achieve the above purpose. (10)

Antibiotics

One of the biggest and most glaring examples of the exceptional use of biotechnology is the industrial production of antibiotics. The antibiotics industry is one of the biggest commercial and industrial markets in existence today. These antibiotics are one the biggest contributors of therapeutics, in comparison to any other drugs. It has become extremely important to constantly come up with new antibiotics, to counter the different and new diseases that are emerging and also to encounter the re-emerging diseases as we. Moreover, pathogens have started to develop immunity to certain antibiotics; thus it is important to come up with new ones. With population increasing the health dropping, new technologies such as pathways, combinatorial chemistry, directed evolution of genes and biochemistry hold the key to our future problems. (11)

When the first two pathogens, Mycoplasma genitalium and Haemophilus influenza came up in 1995, frantic efforts were started to find out bacterial targets. The agenda behind this was characterization of bacterial genes. The reason was that, the world wanted to produce antibiotics with precise targets. In the present world sequencing of genomes of more than five hundred bacterial pathogens has been done, and drugs which target specific diseases have been produced. (12) By performing the analysis of genomes, scientists have been able to identify those genes which produce antibiotics in different types of organisms. Clusters which have encodes of putative products have been found, in the actinomycetes species. Those gene clusters which were involved in the synthesis of natural- products have identified through the scanning of genomes by High throughput. By using this particular method, other breakthroughs were also made. Biosynthetic pathways have identified in actinimycetes species which encode antibiotics such as the enediyne antitumor antibiotic. The application as well as the scope of the diversity natural-products will increase by the usage of such techniques.

Cephamycins, penicillins and cephalosporins have been biochemically as well as genetically characterized for their biosynthetic pathways. Other than the isopenicillin N epimerase (cefD) gene all other genes which are important for the production of b-lactam antibiotic have been cloned. When production of the fermentation broths is being done, genes such as the cefE gene are amplified. The reason for doing so is that, their amplification causes a reduction in the deacetoxycephalosporin level and an increase in the production of cephalosporin C. When the cefE gene is expressed as well as transferred to P. Chrysogenum from S. Clavuligens it causes an acid known as (adipyl 7-ADCA) i.e. adipyl-7-aminodeacetoxycephalosporanic acid to be produced. By using this method new antibiotics can be produced, which would not only have P. Chrysogenum biosynthetic capacity but would also be much cheaper to produce and thus would be available in the market at a lower price. The antibacterial activity of Adipyl 7-ADCA is extremely effective against both gram negative as well as positive bacteria. (14)

Another breakthrough made by scientist was the separation an antibacterial element from the bacteria called Myxobacteria. This antibacterial element is very effective in fighting against those pathogens which are responsible for Tuberculosis. These antibacterial elements are called ripostatin, myxopronin and corallopronin. These antbacterial elements work by causing the RNA polymerase enzyme to be inhibited. The way that these substances work on the RNA polymerase is amazing and has not been witnessed before now in any other drug. Thus it can be said that, these substances can also act against those bacteria which have developed immunity to normal day antibiotics. However, they are not considered as antibiotics up till now as they have not been registered as such. Moreover, the research relating to these finding has not been published as well. In other terms, they show the same work that biotechnology has shown regarding the increment in antibiotic diversity and their yield.(15)

Biofuels

There are many reasons why research is being done on bio fuels. Some of these reasons are high oil prices, problem in oil supply, pollution by use of fossil fuels, etc. Even though many people in the scientific world are of the view that fossil fuels can be replaced by bio fuels but in order for that to happen certain things must be viable and fall into place. Firstly, bio fuels should be economically suitable, they should give clean energy and lastly their production should be possible without reducing the supply of food. In today's world two types of bio fuels are being used. The first is produced from soya bean and is called biodiesel and the second which is produced from corn is known as ethanol. Since microbes have the ability to convert the renewable biomass found in plants, into energy it has been suggested that now biomass should be used to produce bio fuel rather than food grains. Biomass is now being turned into valuable products and fuels, because of the fact that there has been a lot of advancement in the past few years in the field of process chemistry, genetics and bioengineering. This whole method is called a "bio-refinery". Currently bioethanol is the only bio fuel being manufactured on an industrial scale. More study and research needs to be conducted before biomethane, biodiesel, biohydrogen and biobutanol can be produced industrially.

Producing bio fuel from animal fat, cooking oil or crops is not a feasible option. One of the best producers of bio fuels are micro algae. They use sunlight like plants, but unlike them are more efficient producers. In fact some microalgae can out produce oil crops. Microalgae can produce bio fuel in the following ways: biohydrogen being produced by way of photobiological process. Similarly biodiesel can be produced from microalgae oil and lastly algal biomass being digested an aerobically to produce methane. Besides producing bio fuels, these micro algae can also produce substances like hydrocarbons, lipids and complex oils. This also leads to the idea that natural carbon sources such as sugars can be used for the growing of microorganisms, which are heterotrophic for producing biodiesel. But, since the sources of these organic carbons are plants, this process would have a very low efficiency. Moreover, in order to do so, microalgae biomass, which are rich in oils will have to be produced on a massive scale. It is not possible to produce this biomass, in this way and this is the main reason for not turning this process into a commercial activity. (19)

Another way in which compounds which are biodegradable can be used to produce energy is by use if MFC's i.e. Microbial fuel cells. By using the anode as an acceptor of electron, these microbes can help in generating electrical outputs. Besides carbohydrates, these Microbial fuel cells can also act on waste water, which consists of many different complex substances. The Microbial fuel cells produce electricity by converting bioconvertible substrates. This happen when the microbes start accepting insoluble electrons which is the MFC anode, instead of accepting nitrates and oxygen which are the natural acceptors of electrons. Electron shuttles as well as membranes can be used for this kind of transfers. Reduction happens when the electrons flow via a resistor to the cathode. In contrast to anaerobic digestion, electrical currents are produced by MFC's along with gasses such as CO2. The advantages that MFC's give us in relation to bio energy production are that, firstly they can do their work at low temperatures, secondly they can produce electricity by directly converting the substrate energy and lastly they do not need any kind of energy input or gas treatment. This shows us that if we use them, we can have a diversified range of energy producing fuels at our disposal. (20) Therefore, it is very clear that the research on the biotechnology is going at a neck break speed. Although much of the research is not yet commercial, but the prospects that the research is showing are certainly very bright.

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