5. Microbial Strain Improvement

Question 1

Characteristics of ideal mutants

Answer 1

• increased ability in producing metabolites
• increased tolerance to inhibitors
• grow in a more economical media
• resistance to bacteriophage

Question 2

Advantages of N streptococci mutants

Answer 2

• overproduce b-galactosidase
• super nisin producing mutants
• phage resistance

Question 3

Four ways to obtain mutants

Answer 3

1. selection/continuous screening
2. classic mutation
3. genetic engineering
4. gene cloning

Question 4

mechanism of continuous screening

Answer 4

DUE TO SPONTANEOUS MUTATION
- change in base pair that occur randomly
- error during replication
- spontaneous alteration of base

Question 5

characteristics of continuous screening occurence

Answer 5

low rate (10^-10 to 10^-6)

Question 6

are silent mutations selected in continuous screening?

Answer 6

Not when phenotypic features are used as indicators

Question 7

Types of amino acid hyperproducer (continuous screening)

Answer 7

1. wild type
2. auxotroph mutants
3. regulatory mutants

Question 8

Which wild type mutants are selected in for amino acid hyperproducer?

Answer 8

Those capable of accumulating amino acids in a specially formulated growth medium

Question 9

Microbes for WT AA HP

Answer 9

Arthrobacter
Brevibacterium
Corynebacterium
Microbacterium

(produce L-glu, require biotin for growth)

Question 10

Example of WT AA HP mechanism

Answer 10

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

Question 11

what are auxotroph mutants

Answer 11

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

Question 12

Example of auxotroph mutants mechanism

Answer 12

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

Question 13

How to make auxotroph mutants?

Answer 13

ANTIBIOTIC ENRICHMENT
(penicillin, nalidixic acid, D-cycloserine, 5-fluoroacyl)

1. WT will die in penicillin containing medium, leaving the ungrowing auxotroph
2. auxotroph rescued with addition of required nutrients (growth factors)

Question 14

What are regulatory mutants?

Answer 14

Mutants incapable of sensing feedback inhibition as its regulatory is “ruined”

• false signal is sent so WT die off
• those resisting feedback regulation survive

Question 15

How to obtain regulatory mutants (lycine producer)?

Answer 15

(case in point: brevibacterium)

1. brevibacterium grown in the presence of lysine toxic analog AEC
2. AEC acts similarly to Thr, gives “false signal” to Asp kinase and shuts down lysine synthesis, WT die
3. AK is no longer sensitive (regulatory site influenced), so mutants capable of living in the presence of AEC no longer sensitive to feedback inhibition
4. Mutants grow and synthesize Lys in the presence of Lys

Question 16

What are deregulatory mutants?

Answer 16

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

Question 17

examples of commercial isolates (selection and enrichment) for deregulatory mutants

Answer 17

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

Question 18

Mutagens in classical mutation

Answer 18

UV, base analogs, alkylating agents, intercalating agents

(chemical agents are carcinogens, hazardous)

Question 19

Examples of classical mutation

Answer 19

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

Question 20

Methods of gene transfer

Answer 20

1. transposon mutation
2. conjugation
3. trandsuction
4. transformation

Question 21

2 applications of genetic engineering

Answer 21

1. gene transfer
2. protoplast fusion

Question 22

What is transposon

Answer 22

JUMPING GENE
- segment of DNA can insert itself to many loci of genome
- has gene encoding for transposition
- has marker gene (R)

Question 23

Example of transposon mutation

Answer 23

B. subtilis
- 5x amylase compared to WT
- insert Tn 5 (Kan R)

Question 24

How does conjugation occur?

Answer 24

Plasmid transfer through sex pili

Can be done in E. coli, Streptomyces, Streptococcus

Question 25

Examples of conjugation

Answer 25

• Transfer of ethythromycin plasmid
• from S. faecalis
• to L. casei, L. acidophilus, L. reuteri, L. salivarus

Question 26

What is transduction

Answer 26

LYSOGENY
Introduction of foreign gene
by temperate phage
to host chromosomal DNA

LYTIC
Phage inserted in host chromosome
escape host
brings host DNA

Question 27

examples of transduction

Answer 27

in cheese starter

group N Streptococci (S.lactis,S.cremoris,S.lactis subsp. diacetylactis)

Question 28

What is transformation?

Answer 28

Introduction of foreign DNA to “competent cells”