Strain Improvement Flashcards
Metabolism
Sum of catabolic (breakdown, energy yielding) and anabolic (synthesis, energy requiring) activities
What is the difference between primary and secondary metabolism?
Primary = during balanced growth of organism (nutrient excess) - tropophase
Secondary = absence of growth (nutrient limitation) - idiophase
What are the three aims of strain improvement?
- Improve yield of a target product
- Reduce the amount of a contaminant/by-product
- Introduce a new metabolic capacity`
How can mutations occur in the genome?
Spontaneous
Radiation
Chemicals (alkylating agents, intercalators)
Biological (transformations, transposons, genetic crossing)
What are forward, back and second site mutations?
Forward = mutation that inactivates a gene
Back = True reversal at original site of mutation
Second site = Second aa change overcomes effect of the first mutation
Describe how metabolic engineering can increase the yield of Isoleucine production.
Threonine converted to Isoleucine by 5 enzymes.
High levels of Isoleucine (more than 1000) means Threonine Deaminase (E1) is allosterically inhibited so Threonine can’t bind.
Through metabolic engineering, the allosteric site on threonine deaminase can be removed meaning the enzyme isn’t inhibited even at high levels of Isoleucine
Describe strain improvement through gene shuffling.
Rounds of recombination between previously selected strains of same or closely-related species.
Complex traits can be altered
Generate strain libraries available for screening for a desired trait.
Describe evolutionary engineering for anaerobic use of arabinose by S. cerevisiae.
Wild type cannot use 5 carbon sugars (such as xylose and arabinose) and instead needs 6cs (glucose).
Xylose and Arabinose are sugars redundant in plant cell mass.
1. Metabolic engineering of Sc by introducing necessary genes from Lactobacillus plantarum.
2. Evolutionary engineering for selection (serial transfer of cultures of a period of thousands of hours)
What are cloned genes useful for?
- Determining sequence and sequence of regulatory regions
- Introducing extra copies of a gene
- Deleting a gene (e.g. by double cross-over) and replacing with selectable marker
Describe genetic transformation in Bacteria
Gram negative e.g. E.coli: - DNA introduced into competent cells (use Ca2+ and heat shock in strains deficient in restriction/modification)
Gram positive e.g. B. subtilis - Natural competence of protoplasts (Ca2+/polyethylene glycol), electroporation
Describe genetic transformation in fungi.
More complicated in that genes need to be introduced into the nucleus.
Usually involves protoplasts (strip cell wall in one part of cell, membrane in that part bulges) + DNA + Ca2+ + polyethylene glycol.
May involve electroporation, metal ions, agrobacterium, shooting
What is the difference between processing aids and ingredients?
Processing aids do not remain in food, may be residuals from earlier stage (most enzymes)
e.g. alpha-amylases added during baking
Ingredients are food components added for specific purpose and remain functional in final product - need to be labelled
e.g. invertase from S. cerevisiae hydrolyses sucrose to mix of glucose and fructose (sweeter without calorific change)
Which products must be labelled as GM?
Products made from GM source
Products with intentional use of GM ingredients at any level
Which products don’t need to be labelled as GM?
Products from GM technology (e.g. enzymes)
Products from animals fed on GM feed
Accidental presence of GM material (0.9% for approved GM varieties or 0.5% for unapproved GM varieties)
