Strain improvement of the industrially important isolate A. niger using EtBr for higher yield of the product

BACKGROUND

Strain improvement is the science and technology of modifying and enhancing microbial strains to increase their metabolic capabilities.

Genetic engineering has made significant progress and is now widely used in various industrial microbiological fields. When Hopwood et al. initially described the generation of hybrid antibiotics through genetic engineering, there was a high expectation that fusing genes from several antibiotic pathways would produce a large number of novel antibiotics.

Even though numerous novel antibiotics have been produced and discovered on a laboratory scale for so long, their low yield prevented their industrial manufacture.

The regulatory proteins that govern the expression of secondary metabolites can be divided into two types, positive control and negative control, much like the operons that control the gene expression for carbohydrate metabolic pathways.

The generation of the pigmented antibiotics undecyl prodigiosin and actinorhodin in a liquid-grown culture of Streptomyces coeli colour is dependent on the presence of the pathway-specific regulatory genes red-D and actII-ORF4, illustrating examples of positive control. In contrast, in negative control, the genes encoding secondary metabolite production must be activated by the elimination of repressor proteins.

For strain enhancement, traditional UV or chemical mutagenetic programmes may favour mutants with increased production of pro-regulatory proteins or the inactivation of repressor proteins. There are no examples of using genetic engineering to change regulatory genes in strains used in industrial manufacturing.

Microorganism strain enhancement is a crucial strategy for raising bioprocess productivity.

In general, there are three key methods for raising a microorganism’s productivity:

Selection of high-yielding mutants

In this method, a population of microorganisms is screened for variations that outperform the parental strain in terms of product production. By plating cultures on media containing a selection agent that is poisonous to the original strain but not to the mutant, high-yielding mutants are most frequently selected.

Colonies from the plates can then be grown in liquid culture, and mutants with high yield can subsequently be isolated by selecting for growth on minimal media.

Genetic engineering

This strategy entails changing a microorganism’s genetic makeup to increase productivity. Inserting genes from other organisms that encode enzymes that catalyse the creation of the desired product is one typical tactic.

Metabolic engineering

This strategy entails changing a microorganism’s metabolic pathways to shift its resources toward producing the desired product. Knocking out genes that are not necessary for the synthesis of a product and replacing them with genes from other organisms that encode enzymes that catalyse the production of the desired product is one typical tactic.

REQUIREMENTS

S. no.
1	Soil sample
2	Potato dextrose agar
3	Petri dish
4	Test tube
5	Ethidium bromide (EtBr)
6	Phosphate buffer saline
7	Centrifuge
8	Sterile distilled water
9	Inoculation loop
10	Spatula

PROCEDURE

1. The soil sample collected from garden.³
2. 1gm soil weigh, dissolve in 10 ml of sterile distilled water, mix well and 1 ml of sample to petri dish pour molten PDA on the petri dish, swirled to make uniform layer, incubate at 30˚c for 3-5 days.
3. Take a loopful of Aspergillus niger spores and add to 10 ml of sterile distilled water, prepare spore suspension by using serial dilution method.
4. Add 2 ml spore suspension to the 6-8 test tube.
5. Add Ethidium bromide (EtBr) 2-10 mg/ml to each test tubes, kept one test tube as positive control (without EtBr), and incubate it at room temperature for 60 min.¹
6. Centrifuge the cells at 5000 rpm for 10 min, and pellet were washed with PBS buffer.
7. The washed spores spread on the PDA plates and incubate for 48-72 hours at 30˚c.
8. For selection of mutants, the grown colonies again strike on the medium containing 2-deoxy, D-glucose as a selective marker, and incubate for 48 hours at 30˚c.³
9. Select colonies that turned yellow brown colour zone around as EtBr mutant.

CONCLUSION

The A. niger spores were treated with 2-10 mg/ml EtBr for 1 hours. The mutated colonies show yellow brown colour zones in the presence of the selective marker.

REFERENCES

1. Kamalambigeswari R, Sangilimuthu Alagar, Narender Sivvaswamy, “Strain Improvement through Mutation to Enhance Pectinase Yield from Aspergillus niger and Molecular Characterization of Polygalactouronase Gene”, Journal of Pharmaceutical science and research, Vol. 10 (5), 2018.
2. Sadia Javed, Muhammad Asgher, Munir A. Sheikh and Haq Nawaz, “Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid-State Fermentation”, Food Biotechnology, 2010.
3. Babitha Bhaskaran, Sushmitha Shankar and Nagaraj Sonu, “Genetic modification of Aspergillus niger by induced mutagenesis for lipase enzyme production”, International Journal of Life Science, Issue 11, January 2018.

Also read:

• Determination of Growth curve of yeast and compute growth rate & growth yield
• Determination of TDP & TDT of E.coli for designing of a sterilizer
• Screening for citric acid producing organisms
• Effect of inhibitors on enzyme activity
• Methods for purification of enzymes
• Isolation of O & H Antigen from Salmonella typhi

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