ISOLATION AND SCREENING OF INDUSTRIAL MICROORGANISMS

The success of an industrial fermentation process chiefly depends on the strain of

microorganism used. An ideal producer or economically important strain should have the

following characteristics:

 The microorganism that will be suitable for industrial use must produce the substance

of interest.

 The organism must be available in pure culture, genetically stable and must grow in

large –scale culture

 It must be possible to maintain cultures of the organism for a long period of time in

the laboratory and in the industrial plant.

 The culture should preferably produce spores or some other reproductive cells form

so that the organism can be easily inoculated into large fermentor

 Industrial organism should grow rapidly and produce the desired products in a

relatively short period of time. Rapid growth and product formation are desirable for

several reasons: (i) Expensive large scale equipment will not be tied up so long if the

products are produced quickly. (ii) If the organisms grow rapidly, contamination of

the fermentor will be less likely to occur (iii) If the organism grow rapidly, it will be

much easier to control the various environmental factors of the fermentor.

 Industrial microorganisms should not be harmful to humans or economically

important animals or plants.

 It should be possible to remove the microbial cells from the culture medium

relatively easily.

 Industrial microorganism should be amenable to genetic manipulation

Isolation of Microorganisms

The first step in developing a producer strain is the isolation of concerned microorganisms

from their natural habitats. Alternatively, microorganisms can be obtained as pure cultures

from organization, which maintain culture collections. The microorganisms of industrial

importance are, generally, bacteria, actinomycetes, fungi and algae. These organisms occur

virtually everywhere, e.g., in air, water, soil, surfaces of plants and animals and plants and

animals tissues. But most common sources of industrial microorganisms are soils, and lake

and river mud.

Often the ecological habitat from which a desired microorganism is more likely to be

isolated will depend on the characteristics of the product desired from it, and of process

development. For example, if the objective is to isolate a source of enzymes, which can

withstand high temperatures, the obvious place to look will be hot water springs. A variety

of complex isolation procedures have been developed, but no single method can reveal all

the microorganisms present in a sample. Many different microorganisms can be isolated by

using specialized enrichment techniques, e.g., soil treatment (UV irradiation, air drying or

heating, filtration or continuous percolation, washings from root systems, treatment with

detergents or alcohols, pre-inoculation with toxic agents), selective inhibitors (anti-

metabolites, antibiotics, etc.), nutritional (specific C and N sources), variations in pH,

temperature, aeration, etc. The enrichment techniques are designed for selective

multiplication of only some of the microorganisms present in a sample. These approaches

however take a long time (20-40 days), and require considerable labor and money.
 INDUCTION OF MUTATION IN MICRO-ORGANISM AND PLANTS FOR THE
PURPOSE OF OVER PRODUCTION
Generally wild strains are not suitable for industrial fermentation because of low yield of the

metabolite. In such cases, genetic improvement of strain becomes essential for

overproduction of a particular metabolite.

Genetic approaches for augmenting production of metabolites by microorganisms

Mutation

Mutant generation of the existing wild strains is the most practiced strategy for enhancing

the yield of primary and secondary metabolites. For overproduction of primary metabolites,

feedback inhibition by the end product of a particular pathway is suppressed by generation

of auxotrophs i.e. mutation to cause accumulation of metabolite of interest.

Mutants resistant to anti-metabolites through modification of enzyme structure at allosteric

site or modification of operator or regulator gene to express the enzyme constitutively is

another method employed for the over production of primary metabolites. Overproduction of

secondary metabolites is regulated by the structural genes directly participating in their

biosynthesis. Regulatory genes, antibiotic resistance gene and genes involved in primary

metabolism also affect the biosynthesis of secondary metabolites. Mutant generation has

improved the yield of certain antibiotics by 15-400 times in comparison to wild strains.

Greater insight of the biochemical pathways, fluxes of intermediates inside and outside of

cells, enzymes’ role, are being now studied and applied to in metabolic engineering.

Recombinant DNA technology

Recombinant DNA technology is used for modification of biochemical pathways which

ultimately overproduce a particular metabolite and better utilization of media components.

The bioinformatics tools now being used in metabolic engineering for closer understanding

of the gene networks in biochemical pathways determine the hotspots that could be modified

to increase the metabolite yield. Metabolic engineering involves directed improvement of

cellular properties through the modification of specific biochemical reactions or the

introduction of new genes using recombinant DNA technology with the goal to increase

process productivity, such as the production of antibiotics, biosynthetic precursors or

polymers. Examples of metabolic engineering include the microbial production of indigo

(Genencor) and propylene glycol (DuPont).

Mutation and its Application in Crop Improvement

Globally, the current human population is increasing day by day and expected to reach 9

billion by 2050 and that will lead to food scarcity on earth. To overcome this increasing

demand for food and proper nourishment, an improvement in food production is urgently

needed. The envisaged in food production is daunting because of limited available arable

land, depleting water resource and varying climatic condition. The difficulties are also

compounded by urbanization, salinization, biotic stress, drought and desertification that

result in a reduction of arable land. Moreover, changing climatic conditions and subsequent

variations also limit food production. There are different mechanisms for harnessing the

heritable variations encoded in the genetic makeup of existing crop plants so as to use them

in the crop improvement programs. The incorporation of desired traits from non-adapted

landraces/crop wild resources can speed up crop improvement. Putative parental material can

also be induced to mutate so as to obtain new genes that control desired traits for new crop

variety development. Among the different strategies to enhance crop improvement programs,

induced mutagenesis has contributed immensely by creating mutant varieties with improved
and desirable genetic changes in agronomically important traits of the crop plants.

Mutagenesis has become more efficient in combination with advanced molecular biology

techniques and in vitro culture methods that result in enhancement of crop

improvement/breeding program particularly under the global climate change. Such induced

mutagenesis also helps in the mining of new gene alleles that do not occur in the germplasm.

Numerous induced mutagenesis methods are available for plants. Over the last century,

physical mutagens (e.g. fast neutron, UV, X-ray and gamma radiations), chemical mutagens

[e.g. N-methyl-N-nitrosourea (MNU), sodium azide, hydrogen fluoride (HF), methyl

methanesulfonate (MMS) and ethyl methanesulfonate (EMS)] and biological mutagens

(Agrobacterium and transposon-based chromosomal integration) have been widely explored.

The EMS-induced mutation is a highly effective method and, therefore, commonly used in

crop breeding to develop improved crop varieties. In addition, induced mutagenesis is being

applied to improve medicinal plants due to their high demands.

Role of Mutation Breeding in Crop Improvement and Some Highlight of Mutant

Varieties i) Genetic Enhancement of Rice: The impact of induced rice mutants in

applied research is best exemplified by the development of improved rice varieties through

mutation breeding. The first rice varieties KT 20-74 and SH 30-21, developed through

induced mutation, were released in China in 1957 and the first variety Yenhsing-1,

developed by a cross- breeding programme with a mutant. Soon afterwards, the semi dwarf

mutant Reimei was released in Japan which have significantly increased yield because of

their lodging resistance. Calrose 76 and Basmati 370, semi dwarf varieties of rice with short

and stiff straw have revolutionized the rice production in USA and Pakistan respectively. In

Pakistan, a new variety ‘Kashmir Basmati’ which matures early and has cold tolerance, and
retains the aroma and cooking quality of the parent, was derived from induced mutation in

Basmati 370. Several high yielding rice mutants were released in India under the series PNR

and some of these were early in maturity and had short height. Among these, two early

ripening and aromatic mutation-derived rice varieties, PNR- 381 and PNR- 102, are popular

for cultivation in Haryana and Utter Pradesh. A Rice mutant, Zhefu 802 was cultivated in

China in a span of ten years. In Thailand, gamma ray irradiations expedite the release of an

aromatic indica variety of rice RD6 in 1977. In Australia, nine rice mutant varieties Amaroo,

Bogan, Echua, Harra, Labong, Jarrah, Langi, Millin and Namaga have been developed. The

induction of thermo sensitive genic male sterile (TGMS) mutant in Japonica rice mutant

PL-12, which is controlled by a single recessive gene has an immense contribution in

designing the strategies for the production of hybrid rice varieties.

ii) Developing Draught and Salinity Tolerance in Wheat Crop: Sharbati Sonora, a

semi dwarf and non-lodging mutant variety has made a significant contribution to wheat

production in India. Sharbati Sonora produced from red grained Mexican variety Sonara 60

by gamma irradiation at the Indian Agriculture Research Institute, New Delhi, India. A high

yielding mutant Stadler, developed in Missouri, USA had resistance to leaf rust and loose

smut, better lodging resistance and early maturity. In Italy, Durum wheat cultivation area

was significantly expanded due to the cold tolerant mutant varieties.

iii) Enhancing Lodging Resistance in Barley Crop: Mutation breeding has been very

successfully used in breeding barley, the introduction of Diamant and Golden Promise, a

gamma-ray induced semi-dwarf mutant revolutionized brewing industry in Europe. A large

number of barley cultivars were developed from crosses involving Diamant in Europe. Since

decades these high yielding mutants have been used as the parents of many leading barely
varieties released in Europe. Centenario, high yielding, high protein content, early maturity

and resistance to yellow rust, was released in 2006 contributes significantly to the food

security of the country. Luther, gamma ray induced mutant had 20% increased yield, higher

tillering and lodging resistance and Pennrad, had winter hardiness, better lodging resistance

and early ripening.

iv) Developing Early Maturing Varieties of Peanut: Several peanut mutants (Yueyou

No. 5, Yueyou No. 22, Yueyou No. 33, Yueyou 551, Yueyou 187) induced with gamma

radiation were released in China as high yielding varieties under the series Yueyou, some

(Changua No. 4, Lainog, Yueyou 551-38 and Yueyou 551) of those were early in maturity

with improved yield. A Mutant peanut variety TG 26 developed at Bhabha Atomic Research

Centre, Bombay. It is a semi-dwarf plant, early maturity, compact pod setting, greater pod

bearing, higher harvest index and field tolerance to major diseases.

v) High Yielding and Wilt Disease Resistant Chickpea Mutants: A series of High

Yielding and Wilt Disease Resistant Chickpea Mutants such as Pusa – 408 (Ajay), Pusa –

413 (Atul), Pusa – 417 (Girnar), and Pusa – 547, developed at I.A.R.I., New Delhi, are based

on the direct use of induced micro-mutants in a legume crop in the world. Mutant variety

Pusa – 547, released in 2006 has thin testa, attractive bold seeds, better cooking quality and

high yield performance under late sown conditions of North-Western region of India.
 STRAIN SELECTION/DEVELOPMENT AND ENHANCEMENT

STRAIN SELECTION/DEVELOPMENTS

The Science and technology of manipulating and improving strains, in order to enhance their

metabolic capacities for biotechnological applications, are referred to as strain improvement.

Several options are open to an organization pursing industrial biochemistry to help maximize

its profit in the face of its competitor’s race for the same market. The organization may

undertake more aggressive marketing tactics, including more active packaging while leaving

its technical procedures unchanged. It may use its human resources more efficiently and

hence reduce cost or it may adopt a more efficient extraction system for obtaining the

material from the fermentation broth. The operations in the fermentor may also be improved

in term of a better medium, better environmental conditions, or better engineering control of

the fermentor process.

To appreciate the basis of strain improvement it is important to remember that the ability of

an organism to make any particular product is predicated on its capability for the secretion of

a particular set of enzymes. The production of the enzyme itself depends ultimately on the

genetic make-up of the organisms. Improvement of strain can therefore be put down in

simple terms as follows:

(i) selecting suitable producing strains from a natural population with the varying

genetic configurations

(ii) manipulation of the existing genetic apparatus in a particular organism

(iii) regulating the activity of the enzymes

(iv) in the case of metabolites secreted extra-cellularly, increasing the permeability

The targets of strain improvement are listed below:

(i) Rapid growth

(ii) Genetic stability

(iii)Non-toxicity to humans

(iv) Large cell size, for easy removal from the culture fluid

(v) Ability to use cheaper substrates

(vi) Elimination of the production of compounds that may interfere with downstream

processing

(vii) Increase productivity

(viii) To improve the use of carbon and nitrogen sources

(ix) Reduction of cultivation cost (lower price in nutrition and lower requirement

for oxygen)

(x) Production of additional enzymes and compounds to inhibit contaminant

microorganisms.

The activities of micro-organisms can be done by enhanced by optimizing environmental

conditions (physical parameters e.g. temperature and agitation, chemical parameters e.g. pH

and O2 concentration and biological parameters e.g. enzymes) and nutrition of

microorganisms (carbon, nitrogen and mineral sources). Microbial activities can also be

enhanced by methods involving foreign DNA (recombination; this entails genetic

engineering, transduction, conjugation, transformation and protoplast fusion) and methods

that do not involve foreign DNA (mutagenesis).

 GENE DOSAGE AND ITS APPLICATION IN INDUSTRIAL PROCESSES

Gene dosage refers to the number of copies of a gene present in an organism's genome, or

complete "library" of genetic information. Many organisms, including humans, store genetic

information on paired chromosomes. Each member of a pair of chromosomes contributes a

"single dose" of the genes contained on that chromosome. Sex chromosomes, however, tend

to differ between males and females; human males have a single X chromosome and a single

Y chromosome while human females have a pair of X chromosomes. Various regulatory

processes known as "dosage compensation" are in place to ensure that gene dosage remains

at proper levels in both males and females despite the genetic imbalance caused by different

genes.

Generally, more copies of a gene or higher gene dosage will result in increased expression of

the proteins for which the genes code. To a significant extent, however, the genes on male

and female sex chromosomes are expressed at comparable levels despite the difference in

gene dosage. If this were not the case, females with their two X chromosomes could over-

express certain genes, or males with their single X and single Y chromosomes could under-

express certain genes. Either of these alternatives could cause severe mutations or death, so it

is important that the genes are expressed at comparable levels in spite of the difference in

gene dosage.

A set of regulatory mechanisms and processes known as dosage compensation are

responsible for maintaining the expression of genes at appropriate levels. Different

organisms have different means of regulating the expression of their genes, and some even

make use of multiple methods of dosage compensation. Gene expression in human females

is regulated through X-inactivation, through which one of the female's two X chromosomes
becomes an inactive "Barr body." The result of X-inactivation is that males and females each

only have a single X-chromosome that is actually expressing its genetic information and

contributing to gene dosage.

In some organisms, such as the fruit fly or Drosophila melanogaster, the expression of genes

on the male X chromosome is doubled to match the gene dosage of the female's two X

chromosomes. The roundworm, or Caenorhabditis elegans, presents an interesting case, as it

exists most commonly as a hermaphrodite with two X chromosomes, though some have only

a single sex chromosome, X, and are classified as male. Dosage compensation in

Caenorhabditis elegans results in the partial repression of the expression of genes on both of

the X chromosomes in hermaphrodites.

Dosage compensation is a genetic regulatory mechanism which operates to equalize the

phenotypic expression of characteristics determined by genes on the X chromosome so that

they are equally expressed in the human XY male and the XX female. Dosage compensation

also occurs in other organisms like the fruit fly Drosophila melanogaster and the round

worm Caenorhabditis elegans.

Species can have different mechanisms of dosage compensation. In human females (XX),

one chromosome is inactivated (see X-inactivation), resulting in a heterochromatic and

largely genetically inactive Barr body. Drosophila males (XY) double the expression of

genes along the X chromosome. In C. elegans hermaphrodites (XX), both X chromosomes

are partially repressed. Any of these mechanisms results in balancing the relative gene

expression between males and females (or, in the case of C. elegans, hermaphrodites and

males).
In plants (which lack dimorphic sex chromosomes), dosage compensation can occur when

aberrant meiotic events or mutations result in either aneuploidy (the chromosome state in

which there is a loss or a gain of single chromosomes, and the chromosome number is not an

exact multiple of the basic number in the genome) or polyploidy (the chromosome state in

which each type of chromosome is represented more than twice).

Genes on the affected chromosome may be up-regulated or down-regulated to compensate

for the change in the normal number of chromosomes present.

In Humans: Women with two normal X chromosomes have the same blood levels of X-

chromosome protein products, such as factor VIII, as normal men, who of course have only

one X chromosome. An exception to this phenomenon of dosage compensation is the level

of steroid sulfatase in blood, which is increased in women compared with men. Not

surprisingly it has been shown that the locus for steroid sulfatase (deficiency of which causes

a skin disorder known as ichthyosis) is in the pseudoautosomal region.

Assignment

1. Write detailed note on how microbial activities can also be enhanced using the

following techniques:

a) genetic engineering

b) transduction

c) conjugation

d) transformation

e) protoplast fusion

f) mutagenesis

2. Give detailed description on the application of dene dosage in industrial processes.