86) Use of e.Coli for the biotechnological production

E. coli is frequently used as a model organism in microbiology studies. Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Many lab strains lose their ability to form biofilms. These features protect wild type strains from antibodies and other chemical attacks, but require a large expenditure of energy and material resources.

Because of its long history of laboratory culture and ease of manipulation, E. coli also plays an important role in modern biological engineering and industrial microbiology. The work of Stanley Norman Cohen and Herbert Boyer in E. coli, using plasmids and restriction enzymes to create recombinant DNA, became a foundation of biotechnology.

E. coli is a very versatile host for the production of heterologous proteins, and various protein expression systems have been developed which allow the production of recombinant proteins in E. coli. Researchers can introduce genes into the microbes using plasmids which permit high level expression of protein, and such protein may be mass-produced in industrial fermentation processes. One of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin.

Many proteins previously thought difficult or impossible to be expressed in E. coli in folded form have also been successfully expressed in E. coli. For example, proteins with multiple disulphide bonds may be produced in the periplasmic space or in the cytoplasm of mutants rendered sufficiently oxidizing to allow disulphide-bonds to form,while proteins requiring post-translational modification such as glycosylation for stability or function have been expressed using the N-linked glycosylation system of Campylobacter jejuni engineered into E. coli.

Modified E. coli cells have been used in vaccine development, bioremediation, production of biofuels and production of immobilised enzymes.

87) Microbes in milk and dairy products

The fermentation is usually performed by lactic acid bacteria which ferment the lactose in milk and convert it to lactic acid leading to precipitation of the proteins.

There is a tremendous variety of fermented dairy products in many regions in the world. The properties of each product depend on the local strains used for thefermentation.

Many lactic acid bacteria have also been investigated for medicinal health benefits in the past few decades but so far the results are inconclusive.

Fermented milk or dairy products have been part of human diet since ancient times. Various fermented products are made by different strains. Lactic acidfermentation is performed most often by lactic acid bacteria. Due to their abundance in nature, including mucosal surfaces of the human body, and their use in fermented foods they are labeled as GRAS (generally recognized as safe). The main genera that belong to the lactic acid bacteria group are: Lactobacillus,Leuconostoc, Lactococcus, Pediococcus and Streptococcus. These bacteriaferment the carbohydrates in milk, the major one being lactose, to lactic acid and some other products. The acid precipitates the proteins in the milk and that is why fermented products are usually of thicker consistency than milk. The high acidity and low pH hinders the growth of other bacteria, including pathogens. Some lactic acid bacteria can produce agents with antimicrobial properties. Since milk is rich in many nutrients such as protein, calcium, phosphorus, and B vitamins dairy products are an excellent food.

Some of the most popular and widespread cultured dairy products are yogurt and cheese.

Records of yogurt preparation as food date back to centuries BCE. Classic yogurt is the result of the fermentation of two main bacterial species: Lactobacillus bulgaricus and Streptococcus thermophilus. Sometimes other lactic acid bacteriaare added as well. Yogurt is most often made of cow's milk although milk from sheep, goat, water buffalo, camels and yaks is used as well depending on the region of cultivation.

To make yogurt, the milk is first heated to 80ºC or boiled to kill any pathogenicbacteria and to denature the milk proteins to prevent the formation of curds. After it is cooled down to about 45ºC, the starter culture of the two species is mixed well with the milk and incubated at the same temperature for a few hours. In many countries, the traditional food is yogurt without any sweeteners which could be consumed plain or used to prepare a variety of dishes usually with vegetables (Figure 0). Yogurt has been traditionally consumed in Eastern cultures as a cold drink after mixing with water (e.g., lassi, ayran, doogh). After the industrialization of yogurt production in the twentieth century, yogurt with added sweetener and fruit or fruit jam has become popular in the Western world.

Cheese is another popular and ancient dairy product. It consists of milk proteins and fat together with lactic acid bacteria. It has longer shelf life than uncultured milk. Currently there are a few hundred varieties of cheese produced all over the world. Making cheese is similar to yogurt but after acidification usually with lactic acid bacteria (Lactococci, Lactobacilli, Streptococci), the solids are separated from the whey by coagulation with rennet and processed further to yield the final product. Depending on the type of cheese, the solids could go straight to packaging or other bacteria or mold could be added (e.g., Penicillium mold for blue cheese) for additional fermentation.

Other fermented and widely consumed cultured dairy products include kefir (lactic acid bacteria and yeasts are used for the fermentation), sour cream (fermented cream), cultured buttermilk (fermented cow's milk withStreptococcus lactis or Lactobacillus bulgaricus only).

Lactic acid bacteria have been researched for medicinal health benefits. In the early twentieth century, the Nobel laureate in medicine, Elie Metchnikoff, believed that the longevity of peasants in Bulgaria and the Russian steppes was due to their high consumption of milk-fermented products. He hypothesized that the lactic acidbacteria would inhabit the gut after consumption, create and acidic environment as they grow and multiply, and hence prevent the growth of proteolytic. After it was discovered that Lactobacillus bulgaricus can not live in the human gut, the idea was abandoned. Years later, strains of Lactobacillus acidophilus were found to thrive in the gut after implantation and the research started again. The term "probiotics" was introduced and defined as live microorganisms that provide beneficial effects for their host when administered in adequate concentration. Most of the researched species were isolated from different fermented dairy products. The research has been focused on curing or preventing a number of diseases like diarrhea, intestinal inflammations, urogenital infections, allergies, etc. Some species have been prepared and sold as nutritious supplements. However, so far there has not been enough evidence to establish a definite cause and effect relationship about any of the marketed products.

89)Yeasts,their application Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described (estimated to be 1% of all fungal species). Yeasts are unicellular, although some species with yeast forms may become multicellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae, as seen in most molds.  Most yeasts reproduce asexually by mitosis, and many do so by an asymmetric division process called budding.

By fermentation, the yeast species Saccharomyces cerevisiae converts carbohydrates to carbon dioxide and alcohols – for thousands of years the carbon dioxide has been used in baking and the alcohol in alcoholic beverages.  

Commercial Applications

Yeast has long been considered to be the organism of choice for the production of alcoholic beverages, bread, and a large variety of industrial products. This is based on the ease with which the metabolism of yeast can be manipulated using genetic techniques, the speed with which it can be grown to high cell yields (biomass), the ease with which this biomass can be separated from products and the knowledge that it is generally recognized as safe (GRAS).

The budding yeast S. cerevisiae and other yeast species have long been used to ferment the sugars of rice, wheat, barley, and corn to produce alcoholic beverages such as beer and wine. Yeast produce wine by fermenting sugars from grape juice (must) into ethanol. Although wine fermentation can be initiated by naturally occurring yeast present in the vineyards, many wineries choose to add a pure yeast culture to dominate and control the fermentation. The bubbles in champagne and sparkling wines are produced by a secondary fermentation, typically in the bottle, which traps the carbon dioxide. Carbon dioxide produced in wine production is released as a by-product. One yeast cell can ferment approximately its own weight in glucose per hour.

Saccharomyces cerevisiae or baker’s yeast has long been used as a leavening agent in baking. Baker’s yeast ferment sugars present in dough, producing carbon dioxide and ethanol. The carbon dioxide becomes trapped in small bubbles in the dough, which causes the dough to rise. Sourdough bread is an exception, as it is not produced using baker's yeast, but is instead made with a combination of wild yeast and bacteria.

In addition to these traditional uses yeast has also been used for many other commercial applications. Vegans often use yeast as a cheese substitute and it is often used as a topping for products such as popcorn. It is being used in the petrochemical industry where it has been engineered to produce biofuels such as ethanol, and farnesene, a diesel and jet fuel precursor. It is also used in the production of lubricants and detergents. Yeast is used in the food industry for the production of food additives including colorants, antioxidants, and flavor enhancers.

Application to Human Disease and Research

Several approaches have been used to learn more about human genes once a connection between a human and yeast gene is made. In one approach, after a human disease-associated gene is discovered the sequence is compared to the sequences of all genes in the yeast genome to identify the most similar yeast gene(s). To study whether the genes are functionally related, the human gene is then expressed in a yeast stain where the yeast gene has first been inactivated by mutation. This allows researchers to determine whether or not the human gene is able to rescue viability, growth, or more specific defects associated with loss of the yeast gene, a method referred to as functional complementation. If the pathways and/or processes that a yeast gene is involved in are conserved, much can be learned about the function of the human gene based on what is already known about the related yeast gene. Once functional complementation has been established, researchers can use this system to further characterize the function of the related human gene product. Less directed approaches that often utilize high-throughput (HTP) techniques to randomly screen thousands of human genes at one time to identify gene or genes with complementing activity. Such approaches have successfully been used to identify conserved cell cycle regulators (CDC2), genes involved in cancer, and genes involved in neurodegenerative diseases.

There are many scenarios where studies can provide valuable information to researchers about the cellular pathways and/or processes a human gene is involved in when a related yeast gene is not present.

Yeast is becoming the organism of choice in studies aimed at the identification of drug targets and the mode of action of various drugs. Chemogenomics or chemical-genomics refers to the screens that use a combination of chemicals and genomics to probe drug targets and potentially identify novel drugs. Two main approaches have been used in these chemical-genomic studies. In the first, a genome-wide collection of diploid strains is constructed where one of the two identical copies of a gene is deleted, thereby lowering the levels of a particular gene product. Target genes and genes involved in the target pathway become more sensitive to the compound and are preferentially identified in this kind of screen. In a second approach, nonessential genes are systematically deleted and the collection screened with a drug to look for genes which buffer the drug target pathway. This approach is expected to identify genes required for growth in the presence of the compound. Additional approaches using overexpression screens have been used to identify genes involved in drug resistance including the potential drug target. Comparing the expression profile of yeast cells deleted for a gene to those of wild type yeast cells treated with a particular drug can also be an effective way to identify genes which may tell the researchers something about how the drug works in cells.