Fermentation Tech

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Role of Genetic Engineering in

Fermentation TechnologyFERMENTATION BIOTECHNOLOGY

Submitted by : Saima Siddique

Submitted to : Sir Tahir Baig

Session : UG-3

ATTA-UR-REHMAN SCHOOL OF APPLIED BIOSCIENCES

ASSIGNMENT


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INTRODUCTION

Biotechnology is the application of scientific and engineering principles to the processing of different

materials with biological agents to provide goods and services. Essentially, biotechnology harnesses the

catalytic power of biological systems, whether by direct use of enzymes or through the use of the

intricate biochemistry of whole cells and micro-organisms. Biotechnology involves the use in industry of

living organisms or their components i.e. enzyme. It includes the introduction of genetically engineered

micro-organisms into a variety of industrial processes. Because nearly all the products of biotechnology

are manufactured by micro-organisms, fermentation is an indispensable element of biotechnologys

support system. Defined in this way, biotechnology encompasses everything from the technology of

bread-making to that implicated in the manufacture of human insulin from a bacterium induced to take

up a non-bacterial gene and produce the protein coded by that gene.

The potential of biotechnology for increasing agricultural productivity is high, in terms of both increasing

the yields of cultivated plants and of obtaining foodstuffs with higher nutritional value. Numerous

foodstuffs are manufactured using fermentation, and enzymes are now widely used as processing aids in

food manufacturing. Acetone, citric acid, ethanol, and other chemicals are, or have been, produced

industrially by fermentation.

FERMENTATION

For centuries natural fermentation processes have been used to produce products including cheese,

yoghurt and beer. On the other hand, since the birth of genetic engineering (1970s), genes have been

spliced between different types of micro-organims in order to enable the production of products using

transgenic E.colibacterium and other micro-organisms. At present, transgenic E.colibacterium are used

in production of almost all manner of things including chymosin (as required in the making of cheese), as

well as human growth hormones, synthetic insulin and first generation bioplastics and biofuels.

Over the years, the scope and efficiency of the fermentation process has been gradually improved and

refined. Two processes now exist, both of which will benefit from genetic engineering.

In fermentation technology, living organisms provide miniature factories, converting rawmaterials into end products.

In enzyme technology, biological catalysts take from living organisms are utilized to make theproducts.

FERMENTATION INDUSTRIES

The food processing, chemical, and pharmaceutical industries are the three major users of fermentation

today.


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The food industry was the first one to exploit micro-organisms to produce alcoholic beveragesand fermented foods.

In the early 20th century, the chemical industry began to use the technology to produceorganic solvents like ethanol, and enzymes like amylase, used at the time to treat textiles.

About thirty years later after the World War II, the pharmaceutical industry followed thechemical industrys lead, applying fermentation to the production of vitamins and new

antibiotics.

Today, approximately 200 companies in the United States and over 500 worldwide use fermentation

technologies to produce a wide variety of products. Most use them as part of production processes,

usually in food processing. But others manufacture either proteins, which can be considered primary

products, or a host of secondary products, which these proteins help produce. For genes can make

enzymes, which are proteins; and the enzymes can help make alcohol, methane, antibiotics, and many

other substances.

Fermentation technologies are so useful for producing proteins partly because these are the direct

products of genes. But proteins in the form of enzymes can also be used in thousands of additional

conversions to produce practically any organic chemical and many inorganic ones as well.

FERMENTATION USING WHOLE LIVING CELLS

Originally, fermentation used some of the most primitive forms of plant life as cell factories e.g.

Bacteria were used to make yogurt and antibiotics Yeasts to ferment wine The filamentous fungi or molds to produce organic acids

More recently, fermentation technology has begun to use cells derived from higher plants and animals

under growth conditions known as cell or tissue culture. In all cases, large quantities of cells with

uniform characteristics are grown under defined, controlled conditions.

COMPARATIVE ADVANTAGES OF FERMENTATIONS USING WHOLE CELLS AND ISOLATED ENZYMES

At present, it is still uncertain whether the use of whole cells or isolated enzymes will be more useful in

the long run. There are advantages and disadvantages to each. With isolated enzymes, genetic

manipulation can readily increase the supply of enzymes, while with whole organisms, a wide variety ofmanipulations is possible in constructing more productive strains.

THE PROCESS OF ENZYME TECHNOLOGY

Although live yeast had been used for several thousand years in the production of fermented foods and

beverages, it was not until 1878 that the active agents of the fermentation process were given the name


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enzymes (from the Greek, meaning in yeast). The inanimate nature of enzymes was demonstrated

less than two decades later when it was shown that extracts from yeast cells could effect the conversion

of glucose to ethanol. Finally, their actual chemical nature was established in 1926 with the purification

and crystallization of the enzyme urease.

Fermentation carried out by live cells provided the conceptual basis for designing fermentation

processes based on isolated enzymes. A single enzyme situated within a living cell is needed to convert a

raw material into a product. A lactose-fermenting organism, e.g., can be used to convert the sugar

lactose, which is found in milk, to glucose (and galactose). But if the actual enzyme responsible for the

conversion is identified, it can be extracted from the cell and used in place of a living cell. The purified

enzyme carries out the same conversion as the cell, breaking down the raw material in the absence of

any viable micro-organism. An enzyme that acts inside a cell to convert a raw material to a product can

also do this outside of the cell.

THE RELATIONSHIP OF GENETICS TO FERMENTATION

Applied genetics is intimately tied to fermentation technology, since finding a suitable species of micro-

organism is usually the first step in developing a fermentation technique. Until recently, geneticists have

had to search for an organism that already produced the needed product. However, through genetic

manipulation a totally new capability can be engineered; micro-organisms can be made to produce

substances beyond their natural capacities.

The most striking successes have been in the pharmaceutical industry, where human genes have been

transferred to bacteria to produce insulin, growth hormone, interferon, thymosin a-1, and somatostatin,

In general, once a species is found, conventional methods have been used to induce mutations that can

produce even more of the desired compound. The geneticist searches from among hundreds of mutants

for the one micro-organism that produces most efficiently.

The current industrial approach to fermentation technologies therefore considers two problems:

First, whether a biological process car produce a particular product Second, which micro-organism has the greatest potential for production and how the desired

characteristic can be engineered for it.

Finding the desired micro-organism and improving its capability is so fundamental to the fermentation

industry that geneticists have become important members of fermentation research teams.

Genetic engineering can increase an organisms productive capability (a change that can make aprocess economically competitive) but it can also be used to construct strains with

characteristics other than higher productivity.

Properties such as objectionable color, odor, or slime can be removed. The formation of spores that could lead to airborne spread of the microorganism can be

suppressed.


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The formation of harmful byproducts can be eliminated or reduced. Other properties, such as resistance to bacterial viruses and increased genetic stability, can be

given to micro-organisms that lack them.

Applying recent genetic engineering techniques to the production of industrially valuable enzymes may

also prove useful in the future.

GENETIC ENGINEERING AND FERMENTATION INDUSTRY

Genetic engineering is not in itself an industry, but a technology used at the laboratory level. It allows

the researcher to alter the hereditary apparatus of a living cell so that the cell can produce more or

different chemicals, or perform completely new functions. The altered cell, or more appropriately the

population of altered identical cells is, in turn, used in industrial production. It is within this framework

that the impacts of applied genetics in the various industries is examined.

Regardless of the industry, the same three criteria must be met before genetic technologies can becomecommercially feasible. They include the need for:

1. a useful biochemical product;

2. a useful biological fermentation approach to commercial production; and

3. a useful genetic approach to increase the efficiency of production.

The three criteria interrelate and can be met in any order; the demonstration of usefulness can begin

with any of the three. Insulin, e.g., was first found to have value in therapy; fermentation was thenshown to be useful in its production; and, now genetic engineering promises to make the fermentation

process economically competitive. In contrast, the value of thymosin a-1, has not yet been proved,

although the usefulness of genetic engineering and fermentation in its production have been

demonstrated.

STRAIN IMPROVEMENT FOR BIOCATALYSIS PROCESSES AND FERMENTATION BY GENETIC

ENGINEERING TECHNOLOGY

In the past, enzyme engineering required the prior knowledge of the 3-D structure of the enzyme before

any rational design of mutation could be carried out. Twenty years ago, the first complete gene cluster

encoding the actinorhodin biosynthetic pathway was cloned and characterized. Afterwards, the gene

clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade,

breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites

to completion. Now, the development of high-throughput DNA sequencing and DNA microarray

techniques enables researchers to identify the regulatory mechanisms for the overproduction of

secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the

rational application of metabolic pathway engineering.


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The use of whole cells or partially purified enzymes as catalysts has been increased significantly for

chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR

technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes

with

Increased stability to a wide range of pHs, temperatures and solvents Increased substrate specificity, reaction rate and enantioselectivity .

Using genetic engineering techniques, the objectives for enzyme engineering have been mostly

achieved.

THE POTENTIAL OF GENETIC ENGINEERING FOR IMPROVING BREWING, WINE-MAKING AND BAKING

YEASTS

Yeasts have been used to produce food and beverages since the Neolithic age. Their use in fermentation

was recognized in 18361838; and Louis Pasteur demonstrated their unequivocal role in the conversion

of sugar to ethanol and carbon dioxide in 1861.

Originally, fermentation was spontaneous. The first pure yeast culture was obtained by Emil Christian

Hansen from the Carlsberg Brewery in 1883. A pure culture of wine yeast was subsequently obtained by

Mller-Thurgau from Geisenheim (Germany) in 1890. The genetic improvement of industrial strains

traditionally relied on classical genetic techniques (mutagenesis, hybridization, protoplast fusion,

cytoduction), followed by selection for broad traits such as fermentation capacity, ethanol tolerance,

absence of off-flavors (e.g. H2S for wine strains), fast dough fermentation, osmotolerance, rehydration

tolerance, organic acid resistance (bakers strains), flocculation and carbohydrate utilization (brewers

strains).

One major advantage of gene technology over classical genetic techniques is that just one characteristic

can be precisely modified, without affecting other desirable properties. In addition, molecular biology

approaches have introduced a new dimension. The expression of heterologous genes has substantially

increased the possibilities. In the past 20 years, impressive progress has been made in the development

of molecular techniques for Saccharomyces cerevisiae. These advances have been successfully applied

to industrial strains in the past decade, allowing the development of a new generation of specialized

industrial yeast strains. The principal targets for strain development fall into two broad categories:

(1) improvement of fermentation performance and simplification of the process and

(2) improvement of product quality, e.g. organoleptic and hygienic characteristics.

GENETIC ENGINEERING FOR IMPROVED XYLOSE FERMENTATION BY YEASTS

A few yeasts are known to ferment xylose directly to ethanol. However, the rates and yields need to be

improved for commercialization. Xylose utilization is repressed by glucose which is usually present in

lignocellulosic hydrolysates, so glucose regulation should be altered in order to maximize xylose

conversion. Xylose utilization also requires low amounts of oxygen for optimal production. Respiration

can reduce ethanol yields, so the role of oxygen must be better understood and respiration must be

reduced in order to improve ethanol production. This is being made possible by using genetic

engineering.


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Xylose is the second most abundant carbohydrate in the lignocellulosic biomass hydrolysate. The

fermentation of xylose is essential for the bioconversion of lignocelluloses to fuels and chemicals. A few

yeasts are known to ferment xylose directly to ethanol. However, the rates and yields need to be

improved for commercialization. Wild-type strains of Saccharomyces cerevisiae are unable to utilize

xylose. A modified genome shuffling method were developed to improve xylose fermentation by S.

cerevisiae. Recombinant yeast strains were constructed by recursive DNA shuffling with the

recombination of entire genome of P. stipitis with that of S. cerevisiae. After two rounds of genome

shuffling and screening, one potential recombinant yeast strain ScF2 was obtained. It was able to utilize

high concentration of xylose and produced ethanol. The recombinant yeast ScF2 produced ethanol more

rapidly than the naturally occurring xylose-fermenting yeast.

REFERENCES

1. http://www.explainingthefuture.com/genetics.html2. http://archive.unu.edu/unupress/unupbooks/uu09ue/uu09ue17.htm3. http://www.biotechnologyforbiofuels.com/content/pdf/1754-6834-5-46.pdf4. http://www.princeton.edu/~ota/disk3/1981/8115/811505.PDF5. OTA (Office of Technology Assessment). Commercial Biotechnology: An International

Analysis. OTA-BA-218. January. Washington, D.C.: Government Printing Office. 1984

6. Strain improvement for fermentation and biocatalysis processes by genetic engineeringtechnology: Shu-Jen Chiang , Society for Industrial Microbiology.

(http://link.springer.com/content/pdf/10.1007%2Fs10295-004-0131-z)

7. The potential of genetic engineering for improving brewing, wine-making and bakingyeasts:S.Dequin, : 19 April 2001 / Accepted: 20 April 2001 / Published online: 15 June 2001

Springer-Verlag 2001

(http://bio.ijs.si/~upetrovic/png/GM_yeast_wine_making_brewing_baking_AEM_56_577.pdf

)

8. Ethanol fermentation from biomass resources: current state and prospects: Yan Lin . ShuzoTanaka (http://awe.mol.uj.edu.pl/~allel/s6/pliki/mbPrz_seminaria/ethanol.pdf)

9. Genetic engineering for improved xylose fermentation by yeasts: Jeffries TW, Shi NQ. USDA,Forest Service, Institute for Microbial and Biochemical Technology, Madison, WI 53705, USA.

(http://www.ncbi.nlm.nih.gov/pubmed/10533434)
http://www.ncbi.nlm.nih.gov/pubmed?term=Jeffries%20TW%5BAuthor%5D&cauthor=true&cauthor_uid=10533434http://www.ncbi.nlm.nih.gov/pubmed?term=Shi%20NQ%5BAuthor%5D&cauthor=true&cauthor_uid=10533434http://www.ncbi.nlm.nih.gov/pubmed?term=Shi%20NQ%5BAuthor%5D&cauthor=true&cauthor_uid=10533434http://www.ncbi.nlm.nih.gov/pubmed?term=Jeffries%20TW%5BAuthor%5D&cauthor=true&cauthor_uid=10533434

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