A TERM PAPER

ON

THE ROLE OF GENETIC ENGINEERING IN FERMENTATION

TECHNOLOGY

BY

NWIYI, IKECHUKWU U.

M.TECH/SNAS/2013/4207

DEPARTMENT OF MICROBIOLOGY

FEDERAL UNIVERSITY OF TECHNOLGY, MINNA

ADVANCED MICROBIOLIAL GENETICS (MCB 624)

COURSE LECTURER

DR. M.E. ABALAKA

FEBRUARY, 2015

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TABLE OF CONTENT

Title page … … … … … … … 1

Table of Content … … … … … … … 2

SUMMARY … … … … … … … 3

1.0 Introduction … … … … … … … 4

2.0 Fermentation … … … … … … … 5

2.1 Fermentation Industries … … … … … … … 7

2.2 The Range Of Fermentation Processes … … … … … 8

2.3 Fermentation Using Whole Living Cell … … … … … 9

2.3.1 Comparative Advantages of Fermentations Using Whole Cells and Isolated

Enzymes … … … … … … … … 9

2.4 The Process of Enzyme Technology … … … … … 11

3.0 The Relationship of Genetics To Fermentation… … … … … 12

3.1 Genetic Engineering and Fermentation Industry … … … … 15

3.2 Fermentation Efficiency … … … … … … 16

3.3 Protein Engineering: Applications In Food Industry … … … … 17

3.4 Environmental Assessment of Increased Fermentation Efficiency… … 18

3.5 The Genetic Engineering Applications: Improving Brewing, Wine-Making and Baking

Yeasts … … … … … … … … 19

3.6 Genetic Engineering for Improved Xylose Fermentation By Yeasts … … 20

Conclusion … … … … … … … … 22

References … … … … … … … … 24

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SUMMARY

The application of modern biotechnology to food production presents new opportunities and

challenges for human health and development. Recombinant gene technology, the most well-

known modern biotechnology, enables plants, animals and microorganisms to be genetically

modified (GM) with novel traits beyond what is possible through traditional breeding and

selection technologies. It is recognized that techniques such as cloning, tissue culture and

marker-assisted breeding are often regarded as modern biotechnologies, in addition to genetic

modification (WHO, 2005). 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 biotechnology’s 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 (Peacock, 2010). Many traditional fermentations rely

upon the hydrolytic actions of indigenous or deliberately added enzymes. These enzymes derive

from the metabolic activities of acceptable microbes or extracts of plant materials or animal

glands. In recent and scientifically informed times these activities have been identified and

frequently isolated to provide enhanced potency and controlled action resulting in a substantial

industrial production of commercial enzymes (Godfrey, 1997). 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 (OTA, 1984; Stanbury, et al., 2003). This review paper

examines the role of genetic engineering in fermentation technology with particular reference to

the food industry.

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1.0 INTRODUCTION

Biotechnology dates from the dawn of civilization, where the earliest farmers selected edible

plants to grow as crops and saved some of the seeds for the next season, and domesticated cattle,

pigs, sheep, and goats. Over the years, farmers bred both the plants and animals they liked and

learned how to best produce them with irrigation and weed control for plants and growing grain

and forages for the animals. Early civilizations around the globe also used yeast to make alcohol

and bread—yeast being a living microorganism (a fungus)—long before its role in fermentation

was understood (Peacock, 2010). Biotechnology involves the use in industry of living organisms

or their components (such as enzymes). It includes the introduction of genetically engineered

micro-organisms into a variety of industrial processes (OTA, 1984). The European Federation of

Biotechnology defined “Biotechnology is the integration of natural sciences and engineering

sciences in order to achieve the application of organisms, cells, parts thereof and molecular

analogues for products and services” (Buchholz, et al., 2005). According to the Codex

Alimentarius Commission (CAC 2001a), modern biotechnology is defined as the application of

(i.) in vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and

direct injection of nucleic acid into cells or organelles, or (ii.) fusion of cells beyond the

taxonomic family, that overcome natural physiological reproductive or recombination barriers,

and that are not techniques used in traditional breeding and selection (WHO, 2005). Peacock,

2010 states that biotechnology is the manipulation of living organisms for purposes other than

their original intent. It is nearly as old as civilization itself. It began with food; agriculture in its

most basic sense is biotechnology.

The field of biotechnology has been divided using colours namely; red, green, white and blue.

Red biotechnology relates to medicine, and green biotechnology relates to food, white

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biotechnology, also called industrial biotechnology, uses natural processes such as fermentation

and enzymes to create products formerly made with chemicals. Bioplastics made with vegetable

oil and starches instead of petroleum are examples of white biotechnology. Blue biotechnology

encompasses all aspects of marine biology and genomics (Peacock, 2010).

Biotechnology persists to generate new tools and techniques based on molecular and genomic

approaches to food, nutrition, health and well-being. Biotechnology offers the potential for

greater efficiency for producers and processors, as well as additional benefits for consumers. In

foods, biotechnology may allow better and more efficient use of raw materials and by-products,

through improved and novel enzymes and microbes optimised for fermentation. Biotechnology

could also improve food safety and ensure traceability across the food chain (GOS, 2012).

One of the primary goals of biotechnology is to feed the world’s 6 billion people, but there is

substantial disagreement over the best way to accomplish this (Peacock, 2010). In the agro-food

industry, biotechnology continues to generate new tools and techniques based on molecular and

genomic approaches to food, nutrition, health and well-being. Biotechnology offers the potential

for greater efficiency for producers and processors, as well as additional benefits for consumers.

In foods, biotechnology may allow better and more efficient use of raw materials and by-

products, through improved and novel enzymes and microbes optimised for fermentation.

Biotechnology could also improve food safety and ensure traceability across the food chain. For

example, state-of-the-art polymerase chain reaction based tests will radically cut the time it takes

to find and identify pathogenic bacteria in food, while increasing the sensitivity with which

pathogens can be detected (GOS, 2012).

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Historically, biotechnology can be divided into three groups;

Fermentation processes are mostly concerned with improvement of new phyla of

microorganisms and their innovation in the course of their cultivation.

Use of biotechnological processes for elimination of toxic and other wastes and for

transformation of these wastes to non-toxic compounds that can be further utilized.

Techniques of genetic and cellular manipulation followed by cultivation of animal cells,

plant cells and microorganisms.

2.0 FERMENTATION

Zymotechnology is the science of fermentation, and it is often considered the forerunner of

modern biotechnology (Peacock, 2010). Fermentation is one of the oldest methods of food

processing. The history of fermented foods has early records in Southeast Asia, where China is

regarded as the cradle of mold-fermented foods, and in Africa where the Egyptians developed

the concept of the combined brewery-bakery (Nout, et al., 1986; McNeil and Harvey, 2008).

Fermentation has been used for preserving food for hundreds of years and virtually every culture

has, as part of its diet, a variety of fermented milk, meat, vegetable, fruit, or cereal products.

Microorganisms, including bacteria, yeasts, and mold, produce a wide range of metabolic end

products that function as preservatives, texturizers, stabilizers, and flavoring and coloring agents

(Harlander, 1992). Microbial fermentation is essential to production of wine, beer, bologna,

buttermilk, cheeses, kefir, olives, salami, sauerkraut, and many more. The metabolic end

products produced by the microorganisms flavor fermented foods. For example, mold-ripened

cheeses owe their distinctive flavors to the mixture of aldehydes, ketones, and short-chain fatty

acids produced by the fungi (Glazer and Nikaido, 2007).

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2.1 FERMENTATION INDUSTRIES

The food processing, pharmaceutical, and chemical industries make up the three major users of

fermentation taking advantage of advances in molecular genetics (OTA, 1984). Nearly all the

products of biotechnology are manufactured by microorganisms; fermentation is an

indispensable element of biotechnology’s support system. These industries have benefited

immensely from these new discoveries. Industrial fermentation processes begin with suitable

microorganisms and specified conditions; e.g., careful adjustment of nutrient concentration. The

products are of many types: alcohol, glycerol, and carbon dioxide from yeast fermentation of

various sugars; butyl alcohol, acetone, lactic acid, monosodium glutamate, and acetic acid from

various bacteria; citric acid, gluconic acid, and small amounts of antibiotics, vitamin B12, and

riboflavin (vitamin B2) from mold fermentation (OTA, 1984; Sahlin, 1999; Dequin, 2001;

Encyclopædia Britannica, 2014).

Two processes now exist both of which benefit from genetic engineering;

Fermentation technology, living organisms serve as miniature factories, converting raw

materials into end products.

Enzyme technology, biological catalysts extracted from living organisms are used to make

the products.

The food industry was the first to exploit microorganisms to produce alcoholic beverages and

fermented foods. In the early 20th century, the chemical industry began to use the technology to

produce organic solvents like ethanol, and enzymes like amylase, used at the time to treat

textiles. Following the end of World War II, the pharmaceutical industry, began applying

fermentation to the production of vitamins and new antibiotics (OTA, 1984).

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2.2 THE RANGE OF FERMENTATION PROCESSESS

The modern fermentation industry, which is largely a product of the Twentieth century, is

dominated by aerobic cultivations intended to make a range of higher value products than simple

ethanol (McNeil and Harvey, 2008). Stanbury et al., 2003 have identified that they are five major

groups of commercially important fermentations;

i.) Those that produce microbial cells (for biomass) as the product.

ii.) Those that produce microbial enzymes.

iii.) Those that produce microbial metabolites.

iv.) Those that produce recombinant products.

v.) Those that modify a compound which is added to the fermentation – the

transformation process.

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 (OTA, 1984; Stanbury, et al., 2003).

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

products of genes. But proteins (as enzymes) can also be used in thousands of additional

conversions to produce practically any organic chemical and many inorganic ones as well (OTA,

1984; McNeil and Harvey, 2008).

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2.3 FERMENTATION USING WHOLE LIVING CELL

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 (OTA, 1984;

Stanbury, et al., 2003).

2.3.1 COMPARATIVE ADVANTAGES OF FERMENTATIONS USING WHOLE

CELLS AND ISOLATED ENZYMES

The field which studies the use of enzymes or whole cells as biocatalysts for industrial synthetic

chemistry is known as Biocatalysis (Johannes, et al., 2006). They have been used for hundreds of

years in the production of alcohol via fermentation, and cheese via enzymatic breakdown of milk

proteins. Microbial cells play a leading role in ‘chemo-enzymatic synthesis’ because of their

great diversity. Several microbes with unique catalytic abilities have been found through

intensive screening and put to practical use (Ishige, et al., 2005; Buchholz, et al., 2005).

Over the past few decades, major advances in our understanding of the protein structure–function

relationship have increased the range of available biocatalytic applications. In particular, new

developments in protein design tools such as rational design and directed evolution have enabled

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scientists to rapidly tailor the properties of biocatalysts for particular chemical processes

(Johannes, et al., 2006).

Johannes, et al., 2006 and Ishige, et al., 2005 give the following as advantages of whole cell

fermentation over isolated enzymes;

1.) Compared with isolated enzymes, whole cell catalysts can be much more readily and

inexpensively prepared. Because enzymes in cells are protected from the external

environment, they are generally more stable in the long-term than free enzymes.

2.) The whole-cell biocatalysis approach is typically used when a specific biotransformation

requires multiple enzymes or when it is difficult to isolate the enzyme.

3.) A whole-cell system has an advantage over isolated enzymes in that it is not necessary to

recycle the cofactors (non-protein components involved in enzyme catalysis).

4.) In addition, it can carry out selective synthesis using cheap and abundant raw materials

such as corn starches.

5.) Microbial cells have been most commonly used for industrial purposes because of their

diversity and ease of handling. Rapid advances in the life sciences have greatly increased

the availability of whole cell catalysts. The recombinant DNA technique is a good

example.

6.) It has enabled the overproduction of a desired enzyme in various heterologous hosts

(microorganism, plant, and animal cells), and it allows the use of cells as ‘bags stuffed

with catalysts’. This technique has even made it possible to modify metabolic pathways

in the host cells, such as cofactor regeneration systems, biosynthesis of complex

metabolites, etc.

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2.4 THE PROCESS OF ENZYME TECHNOLOGY

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 (OTA, 1984;

Bornscheuer, et al., 2012).

Enzyme technology – a sub-field of biotechnology – new processes have been and are being

developed to manufacture both bulk and high added- value products utilizing enzymes as

biocatalysts, in order to meet needs such as food (e.g., bread, cheese, beer, vinegar), fine

chemicals (e.g., amino acids, vitamins), and pharmaceuticals. Enzymes are also used to provide

services, as in washing and environmental processes, or for analytical and diagnostic purposes

(Buchholz, et al., 2005).

The technology for producing and using commercially important enzyme products combines the

disciplines of microbiology, genetics, biochemistry and engineering, which have developed and

matured through time both singly and in an interactive manner. Demands for new enzymes arise

from the development of new processes or from the unsatisfactory performance of known

enzymes in established processes. The revolution in gene technology over the last two decades

has had a big impact on enzyme industry. Genetic engineering techniques have enabled enzyme

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manufactures to produce sufficient quantities of almost any enzyme no matter what the source,

while protein engineering allows the properties of the enzymes to be adjusted prior to production

(Hatti-Kaul, 2009).

Production of new microbial enzyme starts with screening of microorganisms for desirable

activity using appropriate selection procedures. The level of enzyme activity produced by an

organism from a natural environment is often low and needs to be elevated for industrial

production. Increased in enzyme levels is often achieved by mutation of the organism. An

alternative strategy that has gained favour is production of the enzyme in a recombinant

organism of choice whose growth conditions are well optimized and whose GRAS status is

established. Random or site directed mutagenesis with the purpose of engineering the activity

and stability properties of an enzyme prior to its production is becoming a common practice. The

microorganisms used for enzyme production are grown in fermenters using an optimized growth

medium. Both solid state and submerged fermentation are applied commercially; however the

latter is preferred in many countries because of a better handle on aseptic conditions and process

control. The enzymes produced by the microorganism may be intracellular or secreted into the

extracellular medium (Sahlin, 1999; Buchholz et al., 2005; Hatti-Kaul, 2009).

3.0 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-

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organisms can be made to produce substances beyond their natural capacities (OTA, 1984;

Dequin, 2001; Lennox, et al., 2003).

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-flavours (e.g. H2S for wine

strains), fast dough fermentation, osmo-tolerance, rehydration tolerance, organic acid resistance

(baker’s strains), flocculation and carbohydrate utilization (brewer’s strains) (Dequin, 2001;

Hatti-Kaul, 2009).

Current industrial approach to fermentation technologies therefore considers two problems:

First, whether a biological process can produce a particular product

Second, which micro-organism has the greatest potential for production and how the

desired characteristic can be engineered for producing more of it.

The rapid development of ‘white biotechnology’ involves a continuous search for more efficient

organisms to provide the fermentation of the various products. This development comprises both

the identification and selection of the most suitable host organisms and the genetic modification

of these organisms (DME, 2006). 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.

1.) Genetic engineering can increase an organism’s productive capability (a change that can

make a process economically competitive) but it can also be used to construct strains with

characteristics other than higher productivity.

2.) Properties such as objectionable color, odor, or slime can be removed.

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3.) The formation of spores that could lead to airborne spread of the microorganism can be

suppressed.

4.) The formation of harmful byproducts can be eliminated or reduced.

5.) 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 (OTA, 1984; Ishige, et al., 2005; Johannes, et al.,

2006; Bornscheue, et al., 2012).

In recent times, the genetic characterization of micro-organisms has advanced at a rapid pace

with exponential growth in the collection of genome sequence information, high-throughput

analysis of expressed products i.e., transcripts and proteins and the application of bioinformatics

which allows high throughput comparative genomic approaches that provide insights for further

functional studies. Genome sequence information, coupled with the support of highly advanced

molecular techniques, have allowed scientists to establish mechanisms of various host-defensive

pathogen counter-defensive strategies and have provided industry with tools for developing

strategies to design healthy and safe food by optimizing the effect of probiotic bacteria, the

design of starter culture bacteria and functional properties for use in food processing.

Characterization of the genomes of lactic acid probiotics has, for example, shed light on the

interaction of pathogens with lactic acid bacteria (de Vos, 2001). Nucleotide sequences of the

genomes of many important food microbes have recently become available. Saccharomyces

cerevisiae was the first food microbe for which a complete genome sequence was characterized

(Goffeau, et al., 1996). This was followed by genome sequencing of the related yeast,

15

Kluyveromyces lactis (Bolotin-Fukuhara et al., 2000) as well as filamentous fungi which are

major enzyme producers and have significant applications in the food-processing industry (FAO,

2010).

3.1 GENETIC ENGINEERING AND FERMENTATION INDUSTRY

Genetic engineering has been defined as the artificial manipulation, modification, and

recombination of DNA or other nucleic acid molecules in order to modify an organism or

population of organisms (Britannica encyclopedia 2014). It refers to any process in which an

organism’s genome is intentionally altered. Genetic engineering does not encompass traditional

breeding techniques because it requires manipulation of an organism’s genes through cloning or

transformation via the addition of foreign DNA (Peacock, 2010).

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. For example, a DNA

fragment may be isolated from one organism, spliced to other DNA fragments, and put into a

bacterium or another organism. This process is called cloning because many identical copies can

be made of the original DNA fragment. In another example of genetic engineering, a stretch of

DNA, often an entire gene, may be isolated and its nucleotide sequence determined, or its

nucleotide sequence may be altered by in vitro mutagenic methods. These and related activities

in genetic engineering have two basic objectives: to learn more about the ways nature works and

to make use of this knowledge for practical purposes (OTA, 1984; Schleif, 1993).

Regardless of the industry, the same three criteria must be met before genetic technologies can

become commercially feasible. They include the need for:

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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 then shown 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 (OTA, 1984; Schleif, 1993).

3.2 FERMENTATION EFFICIENCY

The development that has taken place over the past decade in this area is unprecedented with

respect to the efficiency increases it has provided for the industrial processes based on

fermentation. Increases in efficiency/yield of 5-10% are common, and this exceeds the parallel

efficiency increases of any competing technologies by far. Genetic modification in this way

provides a technology leap favouring the use of fermentation processes radically and implying a

continued efficiency improvement of any fermentation operation, and thereby also an

environmental efficiency increase. Moreover, the fermentation efficiency increase leads to

increased cost efficiency of enzyme production, and this strongly contributes to the continuous

gain of new fields of enzyme application from conventional processes using chemical auxiliaries

(DME, 2006).

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3.3 PROTEIN ENGINEERING: APPLICATIONS IN FOOD INDUSTRY

An important application area of protein engineering regarding food industry is the wheat gluten

proteins. Their heterologous expression and protein engineering has been studied using a variety

of expression systems, such as E.coli, yeasts or cultured insect cells. Wild type and mutant wheat

gluten proteins were produced to compare them to each other for protein structure-function

studies. Generally, E.coli expression systems were suggested as suitable systems for many

applications, because of their availability, rapid and easy use, as well as high expression levels

(Tamas & Shewry, 2006). Food industry makes use of a variety of food-processing enzymes,

such as amylases and lipases, the properties of which are improved using recombinant DNA

technology and protein engineering. The deletion of native genes encoding extracellular

proteases, for example, increased enzyme production yields of microbial hosts. In fungi, for

example, the production of toxic secondary metabolites has been reduced to improve their

productivity as enzyme-producing hosts (Olempska-Beer et al., 2006; Johannes, et al., 2006).

In food industry, amylases are used for liquefaction and saccharification of starch, as well as in

adjustment of flour and bread softness and volume in baking (Kirk et al., 2002). Recently, the

production of “functional foods” is becoming increasingly important for food industry.

Particularly, the production of industrial products and functional foods from cheap and

renewable raw agricultural materials is desirable. Conversion of starch to bioethanol or to

functional ingredients like fructose, wine, glucose and trehalose, for example, has been studied.

Such a conversion requires microbial fermentation in the presence of biocatalysts such as

amylases to liquefy and saccharify starch. To improve the industrially important properties of

amylases, such as high activity, high thermo- and pH-stability, high productivity, etc.;

recombinant enzyme technology, protein engineering and enzyme immobilization have been

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used. In a recent review article, rice was given as a typical example for biocatalytical production

of useful industrial products and functional foods from cheap agricultural raw materials and

transgenic plants (Akoh et al., 2008; Turanli-Yildiz, et al., 2012).

Another major group of enzymes utilized by food industries is constituted by lipases. They are

used in many applications of food industry such as for the stability and conditioning of dough (as

an in situ emulsifier), and in cheese flavour applications (Kirk et al., 2002). As lipases are

commonly used in food industrial applications, having toxicologically safe lipases is an

important requirement of food industry. The commercial lipase isoform mixtures prepared from

Candida rugosa meet this requirement. Obtaining pure and different C. rugosa lipase isoforms is

possible by means of computer modelling of lipase isoforms, and protein engineering methods

such as lid swapping and DNA shuffling (Akoh et al., 2008; Ishige, et al., 2005; Turanli-Yildiz,

et al., 2012).

3.4 ENVIRONMENTAL ASSESSMENT OF INCREASED FERMENTATION

EFFICIENCY

The environmental aspects of the achieved increase in fermentation efficiency are quite

unambiguous. The efficiency increase leads to resource savings on both raw materials, energy,

water and other auxiliaries involved in the fermentation. Consequently, environmental impacts

from the production of such resources are reduced accordingly. An increase of 5-10% per years

corresponds to a halving of resource consumption and its related environmental impact over

around 10-20 years provided that the efficiency increase stay at the same level as seen over the

latest years (the realism of this has not been evaluated). The potential environmental trade-off is

the use of more genetic modification that underlies the efficiency increase (DME, 2006).

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3.5 THE GENETIC ENGINEERING APPLICATIONS: IMPROVING BREWING,

WINE-MAKING AND BAKING YEASTS

Yeasts are one of the most important groups of microorganisms in food industry and alcohol

production. Saccharomyces ceravisiae, is a species of budding yeasts. Throughout history, yeast

has proven to be very useful to mankind due to its essential role in the production of beer, bread,

and wine. Recently, it has proved useful for the production of enzymes, pigments, antioxidants,

and for various other medical applications (Hesham, 2010). 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 (baker’s strains),

flocculation and carbohydrate utilization (brewer’s strains) (Dequin, 2001).

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 decade, 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. Dequin, 2001 states that

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.

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Classical genetic approaches were first applied to wine yeast strains in the middle of the 1980s,

in response to increasing demand for new characteristics resulting from the development of pure

culture strains. Strains with new properties, e.g. with mitochondrial markers, flocculation

properties or expressing the killer toxin, have been generated by mutagenesis, hybridization or

cytoduction (Barre et al. 1993; Dequin, 2001).

3.6 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 (Jeffries and Shi, 1999).

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 was 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.

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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 (Jeffries and Shi, 1999; Siddique, 2013).

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CONCLUSION

Biotechnology continues to generate new tools and techniques based on molecular and genomic

approaches to food, nutrition, health and well-being. Biotechnology offers the potential for

greater efficiency for producers and processors, as well as additional benefits for consumers. In

foods, biotechnology may allow better and more efficient use of raw materials and by-products,

through improved and novel enzymes and microbes optimised for fermentation. It could also

improve food safety and ensure traceability across the food chain (GOS, 2012).

Fermentation has contributed much to the well-being and wealth of human populations over

millennia; it will continue to do so to an even greater extent in the future. The demand for

fermentation skills is likely to increase in the immediate future (McNeil and Harvey, 2008). The

fermentation industry today is very much in a state of flux, with rapid changes in location,

product spectrum, and scale of processes occurring. To a large extent this has been brought about

by macroeconomic forces compelling the relocation of large scale bioprocesses outside high

labour cost regions, but also by the successful deciphering of the human genome with its myriad

of new therapeutic targets, and the significant advances in the construction of advanced

fermentation expression systems for making novel proteins and antibodies (Stanbury, et al.,

2003; McNeil and Harvey, 2008).

The wide range of economic activities that are built around fermentation provide an illustration

of the potential of the new bio-economy. The new bio-economy will be a crucial part of the

future of agriculture, both by providing new technologies to improve varieties as well as

expanding the range of products that are produced by agriculture. It will lead to a transition to a

more sustainable economic system with more reliance on renewable resources and reuse. The

23

new bio-economy will require moving outside of our comfort zone and may require us to take

calculated risks, but within a technological framework, we can control and mitigate these risks.

The evolution of the new biotechnology, like the fermentation-based technology, will encounter

resistance, but its potential is so vast that it is likely to prevail (Ziberman and Kim, 2011).

24

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