STRAIN IMPROVEMENT

YOGA DWI JATMIKO, PH.D

TEKNIK DASAR KULTUR SEL DAN JARINGAN

25 NOVEMBER 2021
• The diversity of micro-organisms may exploited still by searching for
strains from the natural environment able to produce products of
commercial value
• The first stage in the screening for microorganism of potential
industrial application is their isolation
• Isolation involves obtaining either pure or mixed cultures followed by
their assessment to determine which carry out the desired reaction
or produce the desired product.
Culture Dependant Microbial Community Analysis
• Isolation
• The separation of individual organisms from the mixed
community
• Enrichment Cultures
• Select for desired organisms through manipulation of
medium and incubation conditions
• Inocula
• The sample from which microorganisms will be isolated
 STRAIN IMPROVEMENT
Introduction

vStrain- A Strain is a group of species with one/ more characteristics that distinguish it from other sub
groups of the same species of the strain .
- each strain is indentified by a name, number or letter.
Example:- E.coli Strain K12

The Science and technology of manipulating and improving microbial strains, in order to enhance their
metabolic capacities for biotechnological applications, are referred to as strain improvement.

vStrain Improvement-The Science and Technology of manipulating and improving microbial strains in
order to enhance their metabolic capacities is known as Strain Improvement
 Criteria to select an industrially important microorganism
q The nutritional characteristics of the organism:- It is frequently required that a process be carried out using a very
cheap medium or a pre-determined one, e.g. the use of methanol as an energy source. These requirements may be met
by the suitable design of the isolation medium.

q The optimum temperature of the organism:- The use of an organism having an optimum temperature above 40°
considerably reduces the cooling costs of a large-scale fermentation and, therefore, the use of such a temperature in the
isolation procedure may be beneficial.

q The reaction of the organism with the equipment to be employed and the suitability of the organism to the type of
process to be used.

q The stability of the organism and its amenability to genetic manipulation.

q The productivity of the organism, measured in its ability to convert substrate into product and to give a high
yield of product per unit time.

q The ease of product recovery from the culture.

Slide Time: 10:40)

ndustrial that that is strain that is develop in that is five main steps involved, one
Purpose of Strain Improvement

v Increase the productivities

v Regulating the activity of the enzymes
v Increasing the permeability
v To change un used co-Metabolites
v Introducing new genetic properties into the organism by Recombinant DNA technology /
Genetic engineering.
Obtaining improved strains

• Select from existing populations

• Mutation using chemicals or radiation

• “Classical” Genetics: conjugation, Transposon, transduction, etc.

• Genetic Engineering….strain construction, plasmid vectors,

temperature sensitive promoters, gene shuffling using cassettes
etc.
Sources of Potential Industrial
Microorganisms
1. Culture collections.
Public e.g. NCCLS
Private i.e. within industry

2. Existing processes often yield hyper-producing strains due to self

mutation…these may appear different on plates.

3. The natural environment – Biodiscovery.

Biodiscovery
To “strike it rich” try environments
that:
• Have high biodiversity
• Are extreme
• Are unexplored
• Encourage the dominance of suitable
organisms
Biodiscovery: DNA Route

Collect isolates or go the “DNA

route”:
• Make total community DNA extracts
– can screen at this level or:
• Put fragments (random or selected)
into a suitable host.
• Screen these recombinant
organisms.
• Artificial chromosomes (BACs and
YACs) can carry whole pathways.
Isolation and Screening of Industrial Strain
Isolation from the environment is by:
• Collecting samples of free living microorganism from anthropogenic or natural
habitats.
• These isolates are then screened for desirable traits.
• Or by sampling from specific sites:
• Most with desired characteristics are found among the natural microflora
• After sampling of the organism the next step is of enrichment.
Screening
• Subsequent isolation as pure cultures on solid growth media involves choosing or
developing the appropriate selective media and growth conditions.
• Next step to enrichment and isolation is Screening.
• The pure cultures must be screened for the desired property; production of a
specific enzyme, inhibitory compound, etc.
• Selected isolates must also be screened for other important features, such as
stability and, where necessary, non-toxicity.
Enrichment and Isolation
• Enrichment culture is a technique resulting in an
increase in the number of a given organism relative to
the numbers of other types in the original inoculum.
• Enrichment Cultures
• Can prove the presence of an organism in a habitat
• Cannot prove an organism does not inhabit an
environment
• The ability to isolate an organism from an environment
says nothing about its ecological significance
Enrichment
• Enrichment in batch or continuous system on a defined growth media and
cultivation conditions are performed to encourage the growth of the organism
with desired trait.
• This will increase the quantity of the desired organism prior to isolation and
screening.
• Ex :
• Tetrathionate broth à used for Salmonella Typhi
• Selenite F broth à used for isolation of Shigella
Some Enrichment Culture Methods
Culture Preservation
• Preservation of cultures by freezing, drying, or a combination of the two
processes is highly influenced by resistance of the culture to the damage caused
by rapid freezing, the dehydrating effects of slow freezing, or damage caused
during recovery.
• To minimize damage, agents have been used that protect against ice formation
by causing the formation of glasses upon cooling.
• Methods to protect against the negative effects of dehydration include
adaptation to lower effective water activity by pre-incubation in high osmotic
pressure solutions.
• Damage caused by thawing after freezing can be minimized by rapid melting and
by the composition of the medium used for growth after preservation.
Culture Preservation
• There are various preservation methods .
• Storage at reduced temperature à agar, liquid nitrogen
• Storage in dehydrated form à dried culture, lyophilization

• To date, preservation in liquid nitrogen is still the most successful long-term

method.
Preservation in Distilled Water
• This method (Castellani method, 50 years ago) was extensively tested on 594
fungal strains:
• 62% of the strains growing and maintaining their original morphology.
• In another study, 76% of yeasts, filamentous fungi, and actinomycetes survived
storage in distilled water for 10 years.
• The pathogen Sporothrix schencki concluded that even though long-term survival
was good when this procedure was used, there was a noted loss in virulence.
• Castellani technique should be considered as one of the options for practical
storage of fungal isolates.
Preservation under Oil
• One of the earlier preservation methods was the use of mineral oil to prolong the
utility of stock cultures.
• Mineral oil has been found to prevent evaporation from the culture and decrease
the metabolic rate of the culture by limiting the supply of oxygen.
• This method is more suitable than lyophilization for the preservation of non-
sporulating strains.
Lyophilization
• One of the best methods for long-term culture preservation of many
microorganisms is freeze-drying (lyophilization).
• The commonly used cryoprotective agents are skim milk (15% [wt/vol]
for cultures grown on agar slants and 20% for pelleted broth cultures)
or sucrose (12% [wt/vol] final concentration).
• It should be noted that some plasmid--containing bacteria are
successfully preserved by this method.
• Storage over Silica Gel
• Neurospora has successfully been preserved over silica gel.
• Preservation on Paper
• Drying the spores on some inert substrates can preserve spore-
forming fungi, actinomycetes, and unicellular bacteria.
• Fruiting bodies of the myxobacteria, containing myxospores, may be
preserved on pieces of sterile filter paper and stored at room
temperature or at 6°C for 5 to 15 years.
• Preservation on Beads
• The method involving preservation on beads (glass, porcelain) ,
developed by Lederberg, is successful for many bacteria.
Liquid Drying
• To avoid the damage that freezing can cause, a liquid—drying preservation
process is applied.
• It has effectively preserved organisms such as anaerobes that are damaged by or
fail to survive freezing.
• This procedure was preferred over lyophilization for the maintenance of the
biodegradation capacity of six gram--negative bacteria capable of degrading
toluene.
Cryopreservation
• Microorganisms may be preserved at - 5 to - 20°C for 1, to 2 years by freezing
broth cultures or cell suspensions in suitable vials.
• Deep freezing of microorganisms requires a cryoprotectant such as glycerol or
dimethyl sulfoxide (DMSO) when stored at -70°C or in the liquid nitrogen at -156
to -196°C.
• Broth cultures taken in the mid--logarithmic to late logarithmic growth phase are
mixed with an equal volume of 10 to 20% (vol/vol) glycerol or 5 to 10% (vol/vol)
DMSO.
• Alternatively, a 10% glycerol-sterile broth suspension of growth from agar slants
may be prepared.
Preservation in Liquid Nitrogen
• Storage in liquid nitrogen is clearly the preferred method for preservation of
culture viability.
Protocol for Cryopreservation with Cryoprotectants by a Two-stage
Freezing Process, and Revival of Culture
• After centrifugation the supernatant is removed and the pellet, consisting of
microbial cells, is dissolved in an ice-cold solution containing polyvinyl
ethanol (10% [wt/vol]) and glycerol (10% [wt/vol]) in a 1:1 ratio.
• Due to the presence of polyvinyl ethanol, a viscous thick cell suspension is
obtained, which is kept for about 30 minutes in an ice bath for equilibration.
• During equilibration, an aliquot of 0.5 to 1.0 ml of the cell suspension is
dispensed into each plastic cryovial or glass ampoule.
• They are tightly closed, clamped onto labeled aluminum canes, and placed
at -30°C for about 1 h or for a few minutes in the gas phase of liquid nitrogen
to achieve a freezing rate of about 1°C/min.
• The canes are then placed into canisters, racks, or drawers and frozen rapidly
at -80°C or in liquid nitrogen.
Protocol for Cryopreservation with Cryoprotectants by a Two-stage
Freezing Process, and Revival of Culture

• For revival of cultures, the frozen ampoules are removed from the liquid
nitrogen.
• For thawing, they are immediately immersed to the neck in a water bath at
37°C for a few seconds.
• The thawed cell contents of the ampoule or vial are immediately
transferred to membranes to form a thick layer.
• The resulting bacterial membranes with immobilized cells are used as a biological
component of a biosensor for activity measurements.
e discuss about the method of industrial strain development
Methods of Strain Improvement
Ø Mutant Selection
Ø Recombination
Ø Recombinant DNA Technology

MUTANT SELECTION

v A MUTATION is a Sudden and Heritable change in the traits of an organism.

v Application of Mutagens to Induce mutation is called MUTAGENESIS.
v Agents capable of inducing mutations are called MUTAGENS Physical –
Particulate and Non-Particulate
Chemical – Base analog, Deamine & Alkylating agents, Acridine Dyes.
v Mutation occurring without any specific treatment are called “Spontaneous Mutation.”

v Mutation are resulting due to a treatment with certain agents are known as
“Induced Mutation.”

v Many Mutations bring about marked changes in the Biochemical Characters of practical interest these are
called Major Mutations – these can be used in Strain Improvement

Ex: Streptomyces griseus-Streptomycin-Mannosidostreptomycin Ex:

Streptomyces aurofaciens(S-604) –
Produce 6-demethyl tetracycline in place of Tetracycline

v In contrast, most improvements in biochemical production have been due to the Stepwise accumulation of
so called Minor genes.
Ex: Pencillium chrysogenum – Strain E15-1 was obtained which yield 55% more penicillin than original strain
Isolation of Mutant

Isolation of Auxotrophic Mutants:- it has a defect in one of its biosynthetic pathways, so it require a
specific Bio-molecule for normal growth & development.
Ex: Phe‾ mutant of C.glutamicus – require Phe for growth so, it accumulates Tyrosine.
Analogue – Resistant Mutant:- it have feed back insensitive enzymes of the biosynthetic pathway.
Feed - back inhibition- Tyr‾ mutant of C. glutamicus were selected for resistance to 50mg/L of p-
flurophenylalanine (analogue of phenylalanine).
Revertants from non producing mutants:-
Mutant mutates back to its original phenotype, its called
Reversion and mutant is called Revertant.
Ex: Reversion mutant of Streptomyces viridifaciens showed over 6-fold
increase in chlortetracycline production over the original strain.
RECOMBINATION

v Defined as formation of new gene combinations among those present in

different strains.
v Recombination used for both genetic analysis as well as strain
improvement
v To generate new products

v Recombination may be based on:-

- Cross over
- Transformation
- Conjugation
- Transduction
- protoplast fusion – The fusion between non producing strains of two species
( Streptomyces griseus and Streptomyces tenjimariensis) has yielded a strain
that produces indolizomycin, a new Indolizine antibiotic.
RECOMBINATION DNA TECHNOLOGY
rDNA Technology or Genetic Engineering involves the isolation and cloning of genes of interest,
production of the necessary gene constructs using appropriate enzymes and then transfer and expression of
these genes into an suitable host organism.

This technique has been used to achieve 2 broad objectives:

- Production of Recombinant proteins
- Metabolic Engineering
1. Recombinant proteins:-These are the proteins produced by the transferred gene / transgene ; they
themselves are of commercial value.
Ex: Insulin, Interferons etc..are produced in Bacteria

2.Metabolic Engineering :- When metabolic activities of an organism are modified by introducing into it
transgenes, which affect enzymatic, transport and /or regulatory function of its cells its known as Metabolic
Engineering
Examples – Over production of the amino acid Isoluecine in C. glutamicum& Ethanol by E.coli
General strategies for enhanced production of recombinant proteins in
Pichia pastoris. Strategies are divided into strain engineering wise and
Z. Yang, Z. Zhang
process engineering wise.
Fig. 1. General strategies for enhance
combinant proteins in P. pastoris. Strate
strain engineering wise and process
Considerations were indicated for ea
combined strategy could be used for p
poses.
 Upstream Processes

Microorganism
Fermentation raw materials
Initial isolation Sources of carbon, nitrogen, phosphorus and
sulphur, minor elements, trace elements,
growth factors, water, etc. (availability, cost,
Strain improvement stability, and pretreatment and sterilization
requirements)

Production strain
Constraints: nutritional requirements, metabolic Media development
controls, shear sensitivity, temperature optima,
morphology, O2 and CO2 effects and requirements,
Propagation Maintenance
genetic stability, metabolic by-products, viscosity
medium medium
effects

Starter culture Production

propagation medium

+/–
Oxygen
pH control
Supported or suspended growth.
Antifoam
Fermenter type, stirring mechanism, size,
Cooling/heating
geometry, mode of operation,
Fermentation instrumentation and automation

Downstream Processes in situ DSP

Cell separation
ex situ DSP centrifugation
Influenced by product
concentration and stability. or filtration
Other considerations are Biomass waste:
yield at each step, process if product is Harvested cells Spent medium
costs and purity requirements extracellular

Intracellular Extracellular
or product
periplasmic product

Concentration
Cell disruption step
Primary
recovery

Cell Centrifugation
debris or ultrafiltration

Cell-free Inclusion Medium

extract bodies concentrate

Dialysis, precipitation, partition, Product

chromatographic steps, ultrafiltration, distillation, etc. purification

Crystallization, drying, lyophilization, Finishing

sterile filtration, packaging, etc. processes

Effluent Finished product

Fig. 7.1 An outline of upstream and downstream processing operations.

 Enzyme Biocatalysis 21

Construction of host/vector system Upstream

Cultivation for gene expression Midstream

Intracellular enzyme Extracellular enzyme

Cell harvest Extracellular medium harvest

Cell lysis

Cell debris removal Concentration of medium

Affinity chromatography Downstream

Precipitation of
total protein Chromatography purification

Final product formulation

Figure 3 General bioprocess scheme for enzyme production.

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