How to improve strains without using rDNA technology?

STRAIN IMPROVEMENT BY PARASEXUAL CYCLE & HYBRIDIZATION / PROTOPLAST FUSION –

WHAT IS STRAIN  ?

Strain is a genetic variant or subtype of an organism. (micro-organisms like bacteria, fungi; plants etc).

WHAT IS STRAIN IMPROVEMENT & WHY IT’S NEEDED ?

Strain improvement is the technique that aims at improving the existing strains in terms of their properties like production of certain substances (e.g. Antibiotics, etc) , stability toward environment, production of some new substances etc.

 This is ‘Biotech Era’ .  Biotechnology has got many applications ,from industrial level to common men’s life. 

      As the technology is advancing, there is a need of not only production but improved quality of products. A large no. of applications are based on the bacteria & fungi itself, especially in Fermentation Biotechnology. Hence, there is a need of improving the strains .

There are numerous examples of strain improvement via r DNA Technology e.g. Golden rice (rich in Vitamin A), Herbicide resistant & Insect resistant crops. But there are certain disadvantages in this method –

   1.      Need of sophisticated equipments .

   2.      Expert/Trained human labour.

   3.      Due to above reasons, it’s a costly technique.

Hence, there is a need of ‘alternatives’, especially for a developing country like India where there is problem of trained hands & money.

Following are some of many less costly & effective approaches/methods that can implemented to improve a strain -

1.PARASEXUAL CYCLE :

Parasexual cycle (Parasexuality) , a process peculiar to fungi & single – celled organisms, is a process of transfer of genetic material from one strain to another without involvement of  meiosis or development of sexual characters. (formation of a heterokaryon)

     The process involves :

   1.      Fusion of nuclei of  vegetative Hyphae from different strains. (atleast 2)

   2.      Formation of Diploid clone/ Zygote . In rare case, This diploid clone undergoes abnormal mitosis & hence, abnormal segregation . This may lead to formation of Diploid or Haploid clones in the forthcoming generation.

   3.      The process of recombination during formation of diploid clone may occur by 2 ways-  

                          i.            Mitotic crossing over (producing diploid recombinants)

                        ii.            Haploidization (producing haploid recombinants)

       I.            Mitotic Crossing Over :

In normal mitosis, each chromosome replicates to produce 2 chromatids. These chromosomes align themselves at equator of the cell during Metaphase & get separated by taking away one of 2 chromatids towards each pole of the cell during Anaphase & Telophase. The mitosis ends up with the cleavage of cell into 2 daughter nuclei during Cytokinesis.

   But in Mitotic crossing over, the non-sister chromatids of chromosomes cross-over after replication. After that the Mitosis proceed as usual .As a result , the resultant daughter cells consist of normal chromosomes with some chromosomes with recombination . As during recombination, distal part of chromosomes take place ,hence, will contain & express recessive characters/traits. Thus, as the generations are increased, the expression of recessive character also increases.

    II.              Haploidization –

In haploidization, the chromatids separate & move toward poles of cell in ratio 3:1 (during Anaphase & Telophase).This is due to abnormal Segregation. For example, if there are 2 chromosomes, then from total of 4 chromatids, 3 go to one pole (giving 2n + 1 chromosomes) & one go to another pole. (giving 2n-1 chromosomes). Now the 2n-1 cell contains random genetic information & this increases with further generations.

ORGANISMS UNDERGOING PARASEXUAL CYCLE : A. niger and P. chrysogenum,

as well as the sexual fungus A. Niduzans.

Selection/Isolation of  Recombinant :

The recombinants can be isolated by making parent organisms auxotroph for a certain nutrient & growing them  on a minimal media.  Firstly, a minimal media for 1^st parent is prepared but the nutrient needed for survival of 2^nd parent is added (this nutrient needed by 2^nd parent is not utilized by 1^st parent for growth). Due to this 1^st parent will die out or slow down its growth. These colonies can be easily selected & removed . Now, minimal media for 2^nd parent is prepared & nutrients needed for growth of 1^st parent is added. The previous colonies (that don’t contain 1^st parent) are incubated on it . As a result, the 2^nd parent will die out or slows down its growth . These dead colonies are removed . Now , the colonies left are recombinants (as these have properties of both parents & can grow if minimal media of any one parent is present) .

·         The application of paracycle organisms in industries  have been hindered by many problems like failure of autotrophic marker in case the required product is secondary metabolites, deleterious effect of colour markers used, etc.

APPLICATION OF PARASEXUAL CYCLE IN STRAIN IMPROVEMENT:

 Improvement of Xylanase Production by a    Parasexual   Cross between Aspergillus niger strains:

Introduction:

    1.      Xylanases comprise a group of enzymes able to catalyse the breakdown of xylan-containing substrates. These enzymes have been widely used in process such as paper industry & food industry.

    2.      Filamentous fungi are commonly used to produce xylanases and some improvements have been achieved in enzyme yields either by modifying culture conditions or by isolating mutant strains.

    3.      Aspergillus genera is known for phenonmenon of Parasexual cycle.

  

    4.      Parasexual cycle is being used between 2 strains of Aspergillus to produce recombinants that were strong against toxic glucose analogue - 2-deoxy-D-glucose (2DG) ;overproducing pectinases (Aw99iii2 and Aw96-4) ; less sensitive to carbon catabolite repression than wild type strain  A. niger C28B25.

    5.      A.niger C28B25 ; Aw99iii2 and Aw96-4; diploid (D4) previously retrieved from a parasexual cross between arginine auxotrophs from strains Aw99iii2 and Aw96-4 (strains Aw99ARG and Aw96 4ARG) were assayed for enzymatic activity by dinitrosalicylic acid (DNS) method. This method is based on measurement of O.D. of solution containing reducing sugar , enzyme solution,etc. DNS gives colour to the solution & act as stopping agent. Enzyme activity in a given assay took into account the volume of the assay and expressed as nkat/ml, where one nanokatal represented the amount of enzyme that released 1 nmol of reducing sugar (expressed as xylose equivalents) per second under these assay conditions.

RESULTS :

1)      The D4 mutant gave the highest yield of the enzyme ,even higher than individual parents (Aw99iii2 and Aw96-4 strains) & wild type A.niger.

2)      The D4 was independent of presence of glucose unlike parents. The activity of D4 was higher than Aw99iii2 and Aw96-4  & The wild type strain showed strong repression of Xylanase in presence of glucose.

2. HYBRIDIZATION :

Hybridization is the first method to be considered for improvement of diploid industrial yeast strains. It is the process of combining different varieties of organisms to create a hybrid.  For heterothallic (Heterothallic species have sexes that reside in different individuals.)strains, one can select hybrids by micromanipulating the zygotes formed between meiotic segregants with complementary mating types. For homothallic (Homothallic species have sexes that reside in same individual) strains, the most frequently used of the industrial yeasts , different strategies have to be used. Some approaches use laboratory haploid heterothallic strains with appropriate markers to be crossed with haploid cells from spores of homothallic industrial strains. In these cases, hybrids are easy to detect, and improvement of some industrial yeasts has been described . However, backcrossing is needed to regenerate the industrial strain properties that are lacking in laboratory strains . For two homothallic strains, hybridization can be accomplished by mixing sporulated cultures . Cell fusion can occur between spore germination and diploidization. However, hybrids are obtained with lower frequency, and they are difficult to identify.

Strain improvement by Hybridization :

   1.    A Simple and Reliable Method for Hybridization of Homothallic Wine Strains of Saccharomyces cerevisiae.

A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts.Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains.

Disadvantages of Hybridization :

   1.      The produced hybrids need repeated strain selection via backcrossing. Repeated strain selection is time consuming (in some cases it’s low) & is a labour intensive process.

   2.      There are many bacteria/fungi with hard covers/cell wall that doesn’t allow the parent cells to fuse.

   3.      Due to presence of cell wall fusion efficiency also decreases.

·         Due to all these disadvantages, protoplast fusion was adopted in strain improvement.

Protoplast Fusion in strain improvement :

   1.      The fusion of cells is easily when  cells are without cell wall (protoplasts).

   2.      The protoplasts can be obtained by degradation of cell wall in isotonic solution.

   3.      Nuclei of this protoplast may (& undergo recombination) or may not fuse & the cell regenerates it’s cell   wall.

   4.      A large number of microorganisms have been fused for strain improvement via protoplast fusion. Eg. Members of Bacillus sp. , Streptomyces sp, Corynebacteria, filamentous fungi & yeasts.

                                 

Enzymes used for degradation of cell wall :

       Cellulase and pectinase or macerozyme acts on plant cell wall. Bacterial cell wall are degraded by     the action of lysozyme.Fungal wall degraded by Novozyme -234 which includes glucanase and chitinase. Streptomyces cell wall is degraded by action of lysozyme and achromopeptidase.

       Methods of protoplast fusion :

   1.     SPONTANEOUS FUSION: Protoplast during isolation often fuse spontaneously and this phenomenon      is called spontaneous fiusion .During the enzyme treatment,protoplast from adjoining cells fuse through their plasmodesmata to form multinucleate protoplasts.

   2.     INDUCED FUSION: Sometimes the isolated protoplasts become negatively charged (-10mV to -30mV). This charge resides on outer cell membrane & hence, protoplasts repel each other. These protoplasts are fused by external methods. These methods are :

                                  i.            Mechanical fusion: In this process the isolated protoplast are brought into intimate physical contact mechanically under microscope using micromanipulator or perfusion micropipette.

                               ii.            Chemofusion : Several chemicals has been used to induce protoplast fusion such as sodium nitrate ,polyethylene glycol, Calcium ions(Ca++ ). Chemical fusogens cause the isolated protoplast to adhere each other and leads to tight agglutination followed by fusion of protoplast. Chemofusion is a non specific,inexpensive,can cause massive fusion product, can be cytotoxic and non selecetive and having less fusion frequency.

                             iii.            Electrofusion : In this method, an electric field is applied which cause fusion of protoplasts. In this, 2 capillary microelectrodes are kept close to protoplasts. An electric field of low strength (10Kvm-1) gives rise to dielectrophoretic dipole generation within the protoplast suspension. This leads to pearl chain arrangement of protoplasts. Subsequent application of high strength of electric fields (100 kvm-1) for some microseconds results in electric breakdown of membrane and subsequent fusion. Electrofusion is easy to control having fusion frequency upto 100%, gives reproducibility; less cytotoxic .But equipment is sophisticated and expensive.

Applications of protoplast fusion :

   1.      Easy fusion of protoplasts of fungi where conventional techniques failed.

   2 .      Parasexual Cycle : The organisms with parasexual cycle, whose fusion were not possible , can be fused & recombination can be done –

Protoplasts of P. chrysogenum and P. Cyaneofuluum were fused in presence of  benomyl to give rise to diploid recombinant.

   3.      Production of cephalosporin : Two stains of C. Acremonium were selected, one which is asporulating &slow growing strain & other one which is fast-growing , sporulating with high Cephalosporin synthesis. Protoplasts of these 2 were fused. As a result, a new strain with High production of cephalosporin (40% higher than parents), fast growing & good sporulation was formed. The protoplast fusion resulted in nuclear fusion which is less frequent in conventional methods.

   4.      Production of Penicillin V : Prtoplasts from 2 strains , differing in morphology & products formed ,i.e., Penicillin V (desired product) & OH-V Penicillin (not desired) respectively were fused. Then these were plated on a non-selective medium . Out of 100 colonies ,2 were spotted for high production of Penicillin V & low production of OH-V penicillin, on the basis of their morphological changes from parents colonies. As no selective genetic marker was used, there will be no adverse effect.  

    NOTE -

    RECOMBINATION :

     In recombination, DNA/RNA or part of it get broken down & attached to same or another molecule of DNA/RNA. If recombination occurs in same molecule then it’s Homologous recombination & If it’s between different molecules then it’s Non-homologous Distal end joining.

      Due to recombination, some traits are obtained from both parents organisms (that give DNAs) & some are born new due to new combination of Nucleotides in recombinant DNA.

     Recombination is a process that is involved in almost every strain technique such as Strain improvement via Parasexual cycle organism, hybridization, protoplast fusion,etc.

     Genetic recombination is catalyzed by many different enzymes, called recombinases. RecA, the chief recombinase found in Escherichia coli, is responsible for the repair of DNA double strand breaks (DSBs). In yeast and other eukaryotic organisms there are two recombinases required for repairing DSBs. The RAD51 protein is required for mitotic and meiotic recombination, whereas the DMC1 protein is specific to meiotic recombination.

      Although Recombination is a natural process but there are times when required traits are not produced by natural recombination or some new traits are required to be created or sometimes toxic traits get developed along with desired traits. Hence, Recombinant DNA Technology is being developed. In this, particular part of DNA is cut & joined to particular part of a DNA or some part of DNA is edited by joining some nucleotides. This technique is now widely used & has overtaken natural recombination.