The document discusses strategies for improving microbial strains used in industrial fermentation to enhance product yields. It highlights methods such as genetic modification, random and rationalized selection, and the use of mutagenesis to create strains that are genetically stable, efficient in product formation, and capable of utilizing low-cost substrates. Key objectives include increasing productivity, reducing cultivation costs, and ensuring safety and non-toxicity of the microorganisms.

1. Strain Improvement

3.  Natural isolates usually produce commercially important
products in very low concentrations and therefore every
attempt is made to increase the productivity of the chosen
organism.
 In nature, metabolism is carefully controlled to avoid
wasteful expenditure of energy and the accumulation of
intermediates and enzymes needed for their biosynthesis.
 The potential productivity of the organism is controlled by
its genome and, therefore, the genome must be modified
to increase the potential yield.
 Genetic modification may be achieved by selecting natural
variants, by selecting induced mutants and by selecting
recombinants.
Introduction

4.  Irrespective of the origins of an industrial microorganism, it
should ideally exhibit:
◦ Genetic stability
◦ Efficient production of the target product, whose route of biosynthesis
should preferably be well characterized
◦ Limited or no need for vitamins and additional growth factors
◦ Utilization of a wide range of low-cost and readily available carbon
sources
◦ Amenability to genetic manipulation
◦ Safety, non-pathogenicity and should not produce toxic agents, unless
this is the target product
◦ Ready harvesting from the fermentation
◦ Ready breakage, if the target product is intracellular
◦ Production of limited byproducts to ease subsequent purification
problems.
Attributes of Improved Strain

5.  In many cases strain improvement has been
accomplished using natural methods of genetic
recombination, which bring together genetic
elements from two different genomes into one
unit to form new genotypes.
 An alternative strategy is via mutagenesis. Those
recombinants and mutants are then subjected to
screening and selection to obtain strains whose
characteristics are more specifically suited to the
industrial fermentation process.
 However, such strains are unlikely to survive
Strain Improvement

6.  Several procedures are employed to improve
microbial strains.
 All bring about changes in DNA sequence.
 These changes are achieved by mutation,
genetic recombination, or the modern DNA
splicing techniques of “genetic engineering.”
 Each procedure has distinct advantages.
 In some cases, a combination of one or more
techniques is employed to attain maximum
strain improvement.
Strategies for Strain Improvement

7.  Microbes, generation after generation,
generally inherit identical characteristics as
their parents.
 However, when changes occur in the DNA, they
too are passed on to daughter cells and
inherited in future generations.
 This permanent alteration of one or more
nucleotides at a specific site along the DNA
strand is called a genetic mutation.
Mutation

9.  On the basis of the method of screening and
selection chosen, there are basically two
methods of improving microbial strains
through random mutation:
 (1) random selection and
 (2) rationalized selection.

10.  Random mutagenesis and selection is also referred to as the classic
approach or non-recombinant strain improvement procedure.
 After inducing mutations in the culture, the survivors from the
population are randomly picked and tested for their ability to
produce the metabolite of interest.
 it offers a significant advantage over the genetic engineering route
alone by yielding gains with minimal start-up and sustaining such
gains over years despite a lack of scientific knowledge of the
biosynthetic pathway, physiology, or genetics of the producing
microbe.
 One drawback to the random selection approach is that it relies on
non-targeted, non-specific gene mutations, so many strains need to
be screened to isolate the improved mutant in the mixed
population.
Random Selection

11. mutagenesis of the
population to induce
genetic
variability
random selection and
screening from
the surviving population
of improved strains by
Small scale model
fermentation
assaying of
fermentation broth/agar
for products and scoring
for
improved strains
This process of strain improvement involves
repeated applications of three basic principles:

12. Typical steps in mutation and random strain selection process.

13.  Although the procedure is repetitive and labor
intensive, this empirical approach has a long history of
success and has given dramatic increases in titer
improvement, as best exemplified by the
improvements achieved for penicillin production in
which titers over 50 g/liter are reported—a 4000-fold
improvement over the original parent strain.
 To increase the efficiency of random selection, ways by
which the key steps in the process can drive the
throughput higher without adding labor are typically
sought. In some instances high throughput screens
have been automated with robotics technology.

14.  An alternate approach to random screening requires a basic
understanding of product formation and the fermentation
pathway; this can be acquired through radioisotope feeding
studies and isolation of mutants blocked in various pathways.
 These observations can shed light on the metabolic
checkpoints, and suggest ways to isolate specific mutants. For
example, environmental conditions (pH, temperature,
aeration) can be manipulated, or chemicals can be
incorporated in the culture media to select mutants with
desired traits.
 Rationalized selection for strain improvement does not
generally require a sophisticated understanding of molecular
biology to manipulate environmental or cultural conditions.
Rationalized Selection

15. It can be done by
 Optimizing environmental condition
 Optimizing nutrition of micro-organism
1. Methods not involving foreign DNA-
mutagenesis
Optimization of microbial activity

16.  The best example of the modification of the
permeability of a micro-organism is provided
by the glutamic acid fermentation.
 Kinoshita et al. (l957a) isolated a biotin-
requiring, glutamate-producing organism,
subsequently named Corynebacterium
glutamicum, the permeability of which could be
modified by the level of biotin.
MODIFICATION OF THE
PERMEABILITY

17.  Given figure is a branched pathway
controlled by the concerted
inhibition of the first enzyme in the
pathway by the combined effects of
E and G.
 The mutant illustrated is auxotrophic
for E due to an inability to convert C
to D, resulting in the removal of the
concerted control of the first
enzyme. Provided that E is included
in the medium at a level sufficient to
allow growth but insufficient to
cause inhibition then C will be
accumulated due to the control of
the end product G on the conversion
of C to F.
Auxotrophic mutants

18.  Isolating strains relaxed in regulation can
usually be accomplished by selecting strains
desensitized to feedback inhibition (enzyme
activities) or feedback repression (enzyme
synthesis) involved in the pathway.
 The use of auxotrophic mutant is useless when
the end product controls its own synthesis
independently.
Regulatory mutants

19.  In many microbes, the end products of metabolism, when
accumulated in the microbial cell, inhibit the enzyme
activities of many pathways.
 The end product causes conformational changes by binding
to a specific (allosteric) site on the enzyme, and inhibits
activity.
 The binding is usually noncompetitive.
 Mutation in the structural gene, however, can alter the
enzyme binding site and prevent these inhibitory effects.
 Studying the interaction of various analogs of end products
and their resistance, improved strains can be selected that
lack feedback inhibition and thus overproduce metabolites
of interest.
Mutants Resistant to Feedback
Inhibition

20.  Here intermediates, products of catabolism
(derived from breakdown of compounds
containing carbon, nitrogen, or phosphorus),
or end products regulate the amount of
biosynthetic enzymes synthesized and,
therefore, the amount of final product formed.
 However, mutations at the operator site or
other regulatory sites on the gene relieve such
end-product repression and allow
overproduction of the biosynthetic enzyme.
Mutants Resistant to Repression

21.  Protoplast fusion
◦ Fusing two closely related protoplasts (cells whose walls are
removed by enzyme treatment) is a versatile technique that
combines the entire genetic material from two cells to generate
recombinants with desired traits that cannot be obtained through
a single mutation.
 Transformation
◦ Transformation is the process involving the direct uptake of
purified, exogenously supplied DNA by recipient cells or
protoplasts.
 Conjugation
◦ Conjugation introduces mutational changes in microbes through
unidirectional transfer of genetic material from one strain to the
other; it is mediated by plasmid sex factors.
Genetic recombination

22.  In vitro recombinant DNA technology
◦ By employing restriction endonucleases and
ligases, investigators can cut and splice DNA at
specific sites.
 Isolate the desired gene (DNA fragment) from the
donor cells.
 Isolate the vector (a plasmid or a phage).
 Cleave the vector, align the donor DNA with the
vector, and insert the gene into the vector.
 Introduce the new plasmid into the host cell by
transformation or, if a viral vector is used, by
infection.
 Select the new recombinant strains that express the
desired characteristics.
Cloning and genetic engineering

23.  So far the mutations and the modifications of the strains
discussed have been randomly directed at the level of the
genome of the culture.
 The application of recombinant technology and the use of
synthetic DNA now make it possible to induce specific
mutations in specific genes.
 This procedure of carrying out mutagenesis at a targeted
site in the genome is called site-directed mutagenesis.
◦ It involves the isolation of the DNA of the specific gene and the
determination of the DNA sequence.
◦ It is then possible to construct a modified version of this gene in
which specific bases or a series of bases are changed.
◦ The modified DNA can now be reinserted into the recipient cells and
the mutants selected.

24.  Increase the productivities
 Regulating the activity of the enzymes
 Introducing the new genetic properties into the
organism by recombinant DNA
technology/Genetic engineering.
Purpose

25.  Rapid growth
 Genetic stability
 Non-toxicity to humans
 Ability to use cheaper substrates
 Elimination of production of compounds that
may interfere with downstream processing.
 To improve the use of carbon and nitrogen
sources.
 Reduction of cultivation cost.
 Shorter fermentation time.
IDEAL CHARACTERISTICS OF STRAIN