5. Microbial Strain Improvement Flashcards
Characteristics of ideal mutants
- increased ability in producing metabolites
- increased tolerance to inhibitors
- grow in a more economical media
- resistance to bacteriophage
Advantages of N streptococci mutants
- overproduce b-galactosidase
- super nisin producing mutants
- phage resistance
Four ways to obtain mutants
- selection/continuous screening
- classic mutation
- genetic engineering
- gene cloning
mechanism of continuous screening
DUE TO SPONTANEOUS MUTATION
- change in base pair that occur randomly
- error during replication
- spontaneous alteration of base
characteristics of continuous screening occurence
low rate (10^-10 to 10^-6)
are silent mutations selected in continuous screening?
Not when phenotypic features are used as indicators
Types of amino acid hyperproducer (continuous screening)
- wild type
- auxotroph mutants
- regulatory mutants
Which wild type mutants are selected in for amino acid hyperproducer?
Those capable of accumulating amino acids in a specially formulated growth medium
Microbes for WT AA HP
Arthrobacter
Brevibacterium
Corynebacterium
Microbacterium
(produce L-glu, require biotin for growth)
Example of WT AA HP mechanism
Microbes that produce L-glu and require biotin for growth
- when biotin (high), membrane is strong, L-glu (low)
- when biotin (low), membrane is weak, L-glu (high)
- penicillin added, cell membrane leaky, L-glu synthesized even when biotin high
what are auxotroph mutants
Those that require growth factors (AA) that are not needed by wild type as they have lost the ability to synthesize it (pathway is blocked)
- Mutants will proliferate when medium is supplemented with the growth factor
- Mutants are capable of producing desired amino acids in larger amounts
Example of auxotroph mutants mechanism
Threonine auxotroph: lysine producer
aspartate > b-phosphoaspartate > aspartate semialdehyde (lysine) > homoserine (met) > threonine
THREONINE PATHWAY IS BLOCKED
- lysine inhibits asp kinase 20%
- threoning and lysine inhibits 90%
mutants produce more lysine
How to make auxotroph mutants?
ANTIBIOTIC ENRICHMENT
(penicillin, nalidixic acid, D-cycloserine, 5-fluoroacyl)
- WT will die in penicillin containing medium, leaving the ungrowing auxotroph
- auxotroph rescued with addition of required nutrients (growth factors)
What are regulatory mutants?
Mutants incapable of sensing feedback inhibition as its regulatory is “ruined”
- false signal is sent so WT die off
- those resisting feedback regulation survive
How to obtain regulatory mutants (lycine producer)?
(case in point: brevibacterium)
- brevibacterium grown in the presence of lysine toxic analog AEC
- AEC acts similarly to Thr, gives “false signal” to Asp kinase and shuts down lysine synthesis, WT die
- AK is no longer sensitive (regulatory site influenced), so mutants capable of living in the presence of AEC no longer sensitive to feedback inhibition
- Mutants grow and synthesize Lys in the presence of Lys
What are deregulatory mutants?
Mutants resistant to the analog but may grow in its presence
e.g. AEC (lysine analog) resistant mutant of brevibacterium can produce large amounts of L-lysine
https://sci-hub.se/10.1016/j.procbio.2004.01.037
examples of commercial isolates (selection and enrichment) for deregulatory mutants
B. SUBTILIS
- resistant to valeric acid and actithiazic acid
- produce biotin
BACILLUS SP
- resistant to Trp analog, azaserine, norleucine
- produce Trp (ajinomoto)
CORYNEBACTERIUM
- resistant to deoxyribose and glutamine analog
- produce 5-inosinic acid
Mutagens in classical mutation
UV, base analogs, alkylating agents, intercalating agents
(chemical agents are carcinogens, hazardous)
Examples of classical mutation
CHEESE STARTER
- N-nitroso-N-methylurea mutation of S. cremoris
- increased phage resistance
MILK
- Nitrosoguanidine-induce-mutants of L. bulgaricus and L. casei
- increased acidity and protease activit
SAKE
- UV-induced yeast
- more isobutyl alcohol, less isoamyalcohol
Methods of gene transfer
- transposon mutation
- conjugation
- trandsuction
- transformation
2 applications of genetic engineering
- gene transfer
- protoplast fusion
What is transposon
JUMPING GENE
- segment of DNA can insert itself to many loci of genome
- has gene encoding for transposition
- has marker gene (R)
Example of transposon mutation
B. subtilis
- 5x amylase compared to WT
- insert Tn 5 (Kan R)
How does conjugation occur?
Plasmid transfer through sex pili
Can be done in E. coli, Streptomyces, Streptococcus
