Industrial Microbiology - Strain Improvement strategies

 STRAIN DEVELOPMENT FOR INDUSTRIAL MICROBIOLOGY

“Strain improvement is defined as improving the production and yielding capacity of a microorganism through the microbiological, biotechnological, or biochemical process”.

Need for strain improvement: Microbes (fungi, bacteria, actinomyces) that live freely in soil or water and produce novel compounds of commercial interest, when isolated from their natural surroundings, are not ideal for industrial use.

Wild strains need to make the product of commercial interest at higher yields to be economically viable.

Microbial strain improvement requires alteration and reprogramming of the DNA (or the genes) in a desired fashion to shift or bypass the regulatory controls and checkpoints.

Features of Improved strains

1. Ability to utilize inexpensive and complex raw materials efficiently.

2. Elimination of the production of compounds that may interfere with downstream processing

3. Increased productivity

4. Provide cellular morphology suitable for rapid growth and product separation.

5. Tolerance to high product concentration.

6. Non-toxic to humans and the environment.

7. Genetic stability of the organism used.

8. Large cell size to excrete the product to facilitate product recovery.

Importance of strain improvement:

It can be done by

Optimizing environmental conditions

·       Modification of physical parameters (temperature, agitation, etc) 

·       Modification of chemical parameters (pH, O2 concentration) 

·       Modification of biological parameters (enzymes )

Optimizing the nutrition of microorganisms

·       Carbon sources

·       Nitrogen sources

·       Mineral sources and other sources

·       Precursor

·       Enzymes

Other includes

I. Method not involving foreign DNA—Mutagenesis

II. Method involving Foreign DNA (recombination)

¯  Transduction

¯  Conjugation

¯  Transformation

¯  Genetic engineering

Proper strain used in Industry Genetically Regarded as Safe (GRAS)

Bacteria

•       – Bacillus subtilis, B. amyloliquefaciens, B. licheniformis

•       – Lactobacillus bulgaricus

•       – Lactococcus lactis

•       – Leuconostoc oenos

Yeasts

•       – Candida utilis

•       – Kluyveromyces marxianus

•       – Kluyveromyces lactis

•       – Saccharomyces cerevisiae

Filamentous fungi

•       – Aspergillus niger

•       – Aspergillus oryzae

•       – Mucor javanicus

•       – Penicillium roqueforti

Mutagenesis is a process of treatment given to microorganisms that will cause an improvement in their genotypic and phenotypic performances.

Types

·       Spontaneous mutation

·       Direct mutation (addition, deletion, substitution, point)

·       Induced mutation.

The process of selecting improved strains follows three basic steps:

  

Mutation:

v  Mutations result from deletion of one or more base pairs, insertion of base pairs, or rearrangement of the chromosome due to breakage and faulty reunion of the DNA.

v  In many cases mutations are harmful, but certain mutations occur that make the organism better adapted to its environment and improve its performance.

Table: Mutagens used for strain improvement

S.No	Mutagen	Site of mutation	Impact
1.	Ionizing radiation X-rays, gamma rays	Single- or double-strand breakage of DNA	Deletions, structural changes
2.	Short wavelengths Ultraviolet rays	Pyrimidine dimerization and cross-links in DNA	Transversion, deletion, frameshift, GC àAT transitions
3.	Base analogues 5-Chlorouracil, 5-bromouracil	Faulty base pairing	ATàGC, GCàAT transition
4.	2-Aminopurine	Errors in DNA replication
5.	Deaminating agents Hydroxylamine (NH2OH)	Deamination of cytosine	GCàAT transition
6.	Nitrous acid (HNO2)	Deamination of A, C, and G	translation, deletion, ATàGC, and/or GCàAT transition
7.	Alkylating agents Mustards, di-(2-chloroethyl) sulfide	Alkylation of C and A	GCàAT transition
8.	Intercalating agents Ethidium bromide, acridine dyes	Intercalation between two base pairs	Frameshift, loss of plasmids, microdeletions
9.	Biological Phage, plasmid, DNA transposons	Base substitution, breakage	Deletion, duplication, insertion

 

Recombination process:

Transduction:

3  It is a method of transfer of bacterial genetic material from one bacterium to another bacterium through bacterial infecting viruses (Bacteriophages).

3  While infecting a bacterium, virus packs some of the host DNA during assembly. These bacterial chromosomes may be integrated to another bacterium during second infection by the viral progeny.

3  Transduction is a commonly used technique in recombination.

3  Transduction was discovered by Zinder and Lederberg in Salmonella.

3  Hershey and Chase used transduction as a tool to confirm that DNA is the genetic material.

Transduction is the transfer of bacterial DNA from one bacterial cell to another by means of a bacteriophage.

              Two types:

Ø  General transduction

Ø  Specialized transduction

Ø  In general transduction, host DNA from any part of the host’s genetic apparatus is integrated into the virus DNA.

o   Occur in both lytic or lysogenic cycle.

o   Used to study linkage information, gene mapping, comparing genomes of two different bacteria, mutagenesis, etc.

o   Ex: E.coli transduction by P1 phage.

Ø  In specialized transduction, which occurs only in some phages, DNA from a specific region of the host DNA is integrated into the viral DNA and replaces some of the virus’ genes.

o   Occur only through the lysogenic cycle.

o   Commonly used for isolation and insertion of genes of choice

o   E.coli transduction by 𝝀 phage

Ø  The method is a well-established research tool in bacteria including actinomycetes but prospects for its use in fungi appear limited.

Applications:

1.      Transduction helps to introduce the genes of interest in animals and plants to express desired characteristics.

2.      Gene therapy

3.      Used in genetics and molecular biology as research tools.

Table: Genetically modified organisms and its products

Method of mutation	Mutated microorganism	Product
Plasmid based Gene cloning	C. glutamicum	amino acid – L lysine, L glutamate
Plasmid based Gene cloning	E.coli cell with Bacilus sphaericus plasmid	L-Alanine
Transductional crosses	Serratia marcescens	L-Threonine.
Metabolic engineering	Lactobacillus plantarum	Sorbitol (sweet)
Genetic engineering of IMP gene	B. subtilis	Nucleosides - Guanosine
Conjugation	carotenoid gene clusters from Erwinia uredovora into E. coli	Carotenoids
Cloning of ace gene	Clostridium acetobutylicum	Solvents – acetone
Metabolic engineering	activating CoA ligase from Pseudomonas putida into P. chrysogenum	increased penicillin production up to 30-fold
Recombinant DNA	Acremonium chrysogenum	Cephalosporin C
Cloning	Streptomyces peucetius	Anthracyclines (Antimtumour)
Recombinant enzymes	A. oryzae	Lipases for leather processing.
Cloning	B. amyloliquefaciens plasmid pUB110 in B. subtilis	α-amylase

Conjugation:

o   Transfer of genetic material from one cell to another cell by direct contact.

o   During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient.

o   The donor bacterium carries a DNA sequence called the fertility factor, or F-factor.

o   Bacterial structure pilus serves to transfer the genetic material (Plasmid).

o   The recipient may have a genetic advantage through the transferred DNA, Ex – antibiotic resistance.

Bacterial Transformation:

Ø  Process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment.

Ø  It was first reported in Streptococcus pneumoniae by Griffith in 1928.

Ø  Transformation is used for genetic manipulation of more than 120 species of at least 35 families,

o   Including the major economic crops, vegetables, ornamental, medicinal, fruit, tree and pasture plants, using Agrobacterium mediated or direct transformation methods.

Application:

Ø  A key step in DNA cloning - preparation of multiple copies of DNA, called DNA cloning.

Ø  Large amounts of specific human proteins, for example, human insulin, which can be used to treat people with Type I diabetes.

Ø  Genetically modify a bacterium or other cell.