The below mentioned article provides an essay on Microbial Biotechnology.
Traditionally, microorganisms used in industrial fermentation have been selected on the basis of their ability to produce desired substances in quantities that make the process economically feasible. The producing strains have to be continually improved with regard to their performance.
Strain upgradation has been achieved mainly through mutation and selection. With the development of recombinant DNA technology, the scope of selecting microorganisms capable of producing novel products has been opened up.
This was not possible by classical methods of strain improvement. With the help of this new technology, it has been possible to develop microorganisms which can produce human gene products. Thus, the possibilities of generating unique strains of microorganisms capable of synthesizing a great number of useful products have been expanded, and this has added a new dimension to industrial microbiology.
Modern biotechnology has been defined as the “scientific manipulation of living organisms, especially at molecular genetic level to produce useful products.” An important way of such manipulation is importing a gene of choice from a donor to a prospective producer through a vector to produce a transgenic organism. If the imported gene survives and functions properly to produce the gene product, the recipient organism, commonly a bacterium or yeast, can be mass-cultured to obtain the product on commercial basis.
It should be understood that microbial biotechnology in this perspective differs from traditional industrial microbiology mainly in the development of a producing organism. Once this is achieved through application of modern molecular genetic techniques, the cultivation of the producing organism, product recovery etc. are done more or less in the same way as in traditional fermentations.
The important products of microbial biotechnology are shown in Table 11.5. Many of these products are therapeutic proteins coded by human genes. These are high-priced products and the yield need not be necessarily very large. They were previously extracted from animal or human sources.
The new technology makes it possible to obtain the same or even better products from microorganisms or human cell cultures in which the human genes encoding the proteins have been cloned. For example, insulin, a small polypeptide hormone used by diabetics, has been traditionally extracted from pancreas of slaughtered animals. The two human genes encoding the two polypeptide chains have been cloned in E. coli and Saccharomyces and the hormone can now be obtained by fermentation of these transgenic organisms.
Moreover, this new insulin has no side effects, because it is identical to the natural human insulin. The recombinant DNA technology makes it possible to transfer any gene controlling synthesis of a useful protein from a donor to a microbial recipient cutting across all taxonomic barriers enabling the recipient to produce many new novel products which were never produced by microorganisms.
Cloning of eukaryotic genes in prokaryotes is beset with certain inherent problems. Mere transfer of such genes in bacterial hosts does not ensure that the recipient expresses the gene and produces the desired product in the medium. One of the factors that may prevent expression of a eukaryotic gene in a prokaryote host is the presence of introns in the primary transcripts.
The prokaryotic system is unable to splice out the unwanted segments to make an effective messenger, because the mechanism is absent in bacteria. This obstacle can be overcome by employing a c-DNA for cloning instead of the whole eukaryotic gene which includes the introns.
Another difficulty is that bacterial RNA-polymerases often fail to correctly recognises the eukaryotic promoters to initiate transcription of the cloned gene. This problem has been solved by cloning the eukaryotic gene next to a strong bacterial promoter, like that of β-galactosidase. This enables the bacterial polymerase to transcribe the β -galactosidase gene and the eukaryotic gene together to produce a long m- RNA which is translated to give a fusion protein composed of β -galactosidase and the eukaryotic protein.
The eukaryotic protein can be separated by chemical means from the fusion product. An additional advantage is that the eukaryotic protein in the fusion product is protected from destruction by the proteolytic enzymes of bacteria. The β -galactosidase enzyme in E. coli is an inducible one. When a eukaryotic gene is cloned next to the β-galactosidase gene, both can be made functional by the inducer (lactose). This provides an opportunity to control the activity of the cloned gene.
A further complexity often faced with a eukaryotic gene functioning in a prokaryotic host is that the gene-product is not secreted into the growth medium from where it can be isolated and purified. As a result, the synthesised protein accumulates intracellularly as aggregates or inclusion bodies. When this happens, it becomes difficult to recover the eukaryotic protein in desirable form. This problem may be solved by tagging a nucleotide sequence coding for signal peptide which guides the gene product to be transported across the cell membrane.
For avoiding many of these obstacles, attention has been diverted to use simple eukaryotic organisms, specially Saccharomyces cerevisae as the recipient of eukaryotic genes, rather than bacteria, like E. coli. In fact, some of the important products, like insulin, blood-coagulation factor XIII, hepatitis B virus antigen, HIV-1 antigen, α-antitrypsin etc. are now obtained from transgenic S. cerevisae (see Table 11.5).
Somatostatin:
Production of somatostatin, a small human peptide hormone, genetically engineered. E. coli was the first attempt by a group of scientists in 1976. Instead of cloning the human gene, the DNA sequence coding for the 14 amino acid peptide was artificially synthesised in the laboratory. The double-stranded DNA sequence was provided with free single-stranded 5′-ends containing the restriction sites of Eco R1 and Bam HI. A methionine codon was also added to connect the polypeptide to the bacterial β -galactosidase.
The DNA sequence so constructed was inserted into a plasmid with the help of the restriction enzymes, Eco R1 and Bam HI and sealed by DNA ligase. The plasmid contained a β-galactosidase gene with its promoter and operator, as also an ampicillin resistance gene (Ampr). The somatostatin sequence was inserted in frame with the β-galactosidase gene. The recombinant plasmid was next introduced into competent ampicillin sensitive E. coli cells by transformation. The trans-formants containing the somatostatin DNA were selected by plating in ampicillin agar.
The selected trans-formants were grown and β-galactosidase was induced by adding isopropyl thiogalactoside (IPTG). The bacteria produced a fusion protein contain β-galactosidase and somatostatin linked through a methionine residue. The bacteria were harvested and the cell-mass was treated with 70% formic acid an cyanogen bromide (CNBr) to release the cell contents and to cleave somatostatin from the fusion protein.
Cleavage occurred at the carboxyl end of the methionine residue of somatostatin molecule. Somatostatin was isolated from the mixture and purified. In this way, a human polypeptide hormone was produced by transgenic bacteria for the first time.
The procedure followed in somatostatin production is schematically shown in Fig. 11.54:
The first product obtained from a genetically engineered organism produced commercially and given approval for human application in 1982 was insulin. The experimental procedure followed was more or less similar to that adopted for somatostatin, except that the insulin genes for A and B chains of this polypeptide hormone were isolated from human source. The two genes were separately cloned in E. coli, so that some produced the A polypeptide chain and some others the B-chain.
The polypeptides were isolated separately and purified. The purified polypeptides were mixed under conditions which permitted their association and correct folding to produce a functional insulin molecule. This new insulin was called humulin which meant human insulin. Later, yeasts (Saccharomyces cerevisiae) have been used for production of human insulin.
A host of other medically important proteins are currently being produced and many others are expected to appear in coming years. Many of these,, like human growth hormone, tissue plasminogen factor, erythropoietin etc. are among the top-selling therapeutic proteins. Though E. coli and S. cerevisiae are used as producing organisms for human proteins in most cases, transgenic mammalian cell cultures are also used in specialised cases (see Table 11.5).
Although the therapeutic human proteins have been the main products obtained from genetically engineered organisms, other useful materials are also being produced. An important product is antigen for preparation of vaccine. Instead of using live or killed bacteria or viruses as antigens in vaccines, it is much safer to use an antigenic component of the pathogen for inducing immunity.
If the antigenic constituent is a protein, the coding gene can be cloned into a microorganism to obtain the concerned protein by cultivating the transgenic organism. The first vaccine to be prepared in this way and approved for human application in 1986 was the hepatitis B vaccine. The gene encoding a capsid glycoprotein of hepatitis B virus was cloned into yeast through the YEp vector.
The recombinant yeast containing the viral gene could be mass-cultured resulting in production of the hepatitis B glycoprotein. The protein was isolated, purified and used as antigen in the subunit vaccine for protection against infection of hepatitis B. Some other vaccines have been prepared on the same principle. An anti-material vaccine has been developed using a gene encoding a circumsporozoite protein of malarial parasite.
Another vaccine for protecting cattle from foot and mouth disease has also been successfully produced. This disease is caused by an RNA virus. A c-DNA copy of an RNA gene encoding a capsid protein of the RNA virus was prepared by reverse transcriptase and cloned into E. coli. The transgenic bacteria produced the capsid protein which was used as antigen for the food and mouth disease vaccine.
Another attractive proposition has been to develop vaccines in plants, specially fruit-plants. The idea is that the antigenic substance in the fruits would lead to immunization in consumers via the mucous membranes. A gene encoding a specific antigenic protein of a pathogen can be introduced into a plant cell by the already established plant biotechnological system.
Then the recombinant plant cell can be induced to produce a plantlet by the usual culture techniques. Plant-based vaccines have already been developed and are now being tested for their efficacy. Such vaccines will be cheap and will reach a large section of population.
An example of an unusual product obtained from genetically engineered organisms is rennin or chymosin. It is an important enzyme for cheese industry, because of its ability to coagulate milk by partial hydrolysis of kappa-casein. This enzyme has been traditionally extracted from the fourth stomach of newborn calves.
Through modern biotechnology, the same enzyme can now be obtained adequately by cultivating transgenic microorganisms. Initially, the chymosin c-DNA was cloned in E. coli. Although the gene-product was synthesised, the bacteria failed to secrete the enzyme protein in the medium. The secretion by transgenic yeasts was also unsatisfactory for commercial production. Further search for better yield showed that Aspergillus awamori could produce as much as 1 g of the chymosin protein per liter of growth medium.
Modern gene technology has created unlimited scope for getting unusual novel compounds from transgenic microorganisms. For example, possibility of production of artificial protein fibres by transgenic E. coli has been explored by cloning the spider-silk gene. In spite of its fineness, spider-silk has been found to be stronger than man-made artificial fibres.
The cloning of the spider gene coding for the protein of spider-silk in E. coli has produced the possibility of obtaining silk-like fibres by growing bacteria in fermenters. In contrast to the man-made polymer fibres, such protein fibres would be more environment- friendly, because they would be easily biodegraded in nature.
Instead of using natural genes encoding protein fibres, like those of spider and silk-moth, technology has been developed for synthesizing designer genes coding for specially designed fibre proteins. Fibrous proteins generally consist of repeating short sequences of amino acids. The quality and property of the fibre depends largely on the amino acid sequence in the repeating units.
The developments in fibre technology provide opportunity to an experimenter to design a protein having the desirable qualities. The DNA sequence coding for a specially designed protein can then be artificially synthesized and cloned into bacterial hosts. Cultivation of the transgenic bacteria in fermenters would produce the fibre protein.