Strain Improvement and Development

Strain Improvement Strategies

• A strain is a specific variant or subtype of a microorganism (e.g., virus, bacterium, or fungus). Microbial strains are differentiated by their genetic makeup using metagenomic methods.
• Industrial strains are preferable for product recovery because they synthesize one main component.
• Strain development is crucial for efficient biotechnological processes at an industrial scale, using microbial strains that produce a high concentration of the desired product. Improving the biosynthetic capabilities of microbes to produce a higher quantity of a desired product is called strain improvement.
• Isolating microorganisms from natural habitats is the first step in developing producer strains.
• Screening methods are employed to isolate microorganisms of interest from mixed populations using highly selective procedures.
• Strain improvement involves enhancing the industrial strain's ability to tolerate the culture environment and produce a high amount of the desired metabolite.
• The rate of production is controlled by the organism's genome. Increasing production rates involve inducing necessary changes in the genome. This is also known as genetic improvement
• Proper strains used in industry are considered safe (GRAS).
• Targets for strain improvement include rapid growth, genetic stability, non-toxicity to humans, large cell size for easier removal from culture fluids, and the ability to utilize cheaper substrates.
• Eliminating compounds that interfere with downstream processing and increasing overall productivity are also key targets. Optimizing carbon and nitrogen sources, decreasing cultivation costs, and lowering requirements for oxygen are also parts of strain improvement.
• Additional enzymes and compounds that inhibit contaminant microorganisms are also targeted for production.

Methods of Strain Improvement

• Mutation and mutant selection
  • A mutation is a sudden, inheritable change in an organism's traits.
  • Spontaneous mutations occur without specific treatment.
  • Induced mutations result from treatment with certain agents (mutagens).
  • Mutagenesis is the application of mutagens to induce mutations.
  • Mutagens are classified as physical, chemical, and biological. Subcategories under each of these classifications exist, like radiation, base analogs, etc.
• Recombination
  • Transduction: Transfer of bacterial DNA between cells by a bacteriophage. Two types: general and specialized transduction.
  • Transformation: Uptake of foreign genetic material (naked DNA) by bacteria. Competent cells are able to take up genetic material.
  • Conjugation: Transfer of genetic material between bacterial cells by direct contact or a bridge-like connection. Types include F+ x F-, Hfr x F-, and F' x F- conjugation.
• Protoplast fusion: Dividing cells are caused to lose their cell walls, creating protoplasts. Fusing mixed protoplast populations is enhanced via polyethylene glycol (PEG).
• Parasexual recombination: A rare form of sexual reproduction in certain fungi where nuclei from different strains fuse, resulting in the formation of new genes. This is useful in fungi that lack sexual reproduction cycles.

Genetic Engineering

• Genetic engineering (GE) is the process of using recombinant DNA (rDNA) technology to alter an organism's genetic makeup. It is a three basic step process:
  • Isolate DNA fragments from a donor organism.
  • Insert the isolated DNA fragment into a vector genome.
  • Grow the recombinant vector in an appropriate host organism.

Improvement of Microbial Processes by Genetic Engineering

• Primary metabolites: New processes for producing amino acids and vitamins have been developed using recombinant DNA technology.
• Secondary metabolites: Studies show antibiotic biosynthesis genes are often clustered and entire pathways can be cloned.
• Microbial enzymes: Genes encoding microbial enzymes are cloned and expressed at levels much higher than natural levels. Examples include lipases for detergents and amylase for production purposes.
• Polymers, fuels, foods, and beverages: Recombinant manipulation of Xanthomonas campestris increased xanthan production. Dehydrogenase and decarboxylase genes from bacteria are inserted into novel organisms, modifying product production for biofuel or food production. Bioconversions using genetically modified organisms, such as Candida pasteurianum are used for converting glycerol to other compounds.

Practical Considerations for Strain Improvement

• Selecting organisms that tolerate high concentrations of chemicals in the environment, selecting auxotrophic mutants (those that require added nutrients for growth), exposing organisms to the mutagen when in their haploid stage, selecting mutants with morphology or medium selectivity, or acclimatization to increase the concentration of a toxic pollutant in a gradual selection method.

Strain development is crucial for efficient biotechnological processes at an industrial scale. Improving the biosynthetic capabilities of microbes produces a higher quantity of a desired product, called strain improvement. Screening methods are employed to isolate microorganisms of interest from mixed populations using selective procedures.