Isolation of Mutants That Do Not Produce Feedback Inhibitors or Repressors

Isolation of Mutants

Introduction

Mutants that do not produce certain feedback inhibitors or repressors are valuable for producing intermediates of unbranched pathways, as well as intermediates and end products of branched pathways.

Learning Outcomes

• Understand biochemical approaches to isolate mutants lacking feedback inhibition or repression.

General Principles

• These mutants lack inhibitors or repressors, lifting pathway control. The control factors, essential for growth, must be in the medium at concentrations allowing growth without triggering normal control reactions.

Auxotrophs vs. Prototrophs

• Auxotroph: An organism unable to produce a specific compound needed for growth or requiring more nutrients than normal.
• Prototroph: An organism deriving nutrients from inorganic sources or not requiring specific nutritional substances for growth.

Unbranched Pathway

In an unbranched pathway normally controlled by feedback inhibition or repression of the first enzyme by the end product $E$, an organism auxotrophic for $E$ (due to the inability to convert $C$ to $D$) will accumulate $C$ if $E$ is supplied at a level sufficient for growth but insufficient for inhibition or repression.

Branched Pathway

In a branched pathway controlled by concerted inhibition of the first enzyme by $E$ and $G$, a mutant auxotrophic for $E$ (inability to convert $C$ to $D$) will accumulate $C$ if $E$ is supplied for growth but not inhibition. This occurs because the control of the end product $G$ on the conversion of $C$ to $F$ remains.

Double Auxotrophs

• Requires feeding of both $E$ and $G$ at limiting concentrations.
• A double mutant with a deletion for $G$ production between $F$ and $G$ will accumulate $F$.

Accumulation of End Products in Branched Pathways

• Accumulation of an end product (e.g., $E$) in a branched pathway normally controlled by the concerted effects of $E$ and $I$. If the mutant is auxotrophic for $I$ and $G$ (inability to convert $C$ to $F$), supplying $G$ and $I$ in quantities sufficient for growth without causing inhibition will lead to $E$ accumulation.

Isolation of High-Producing Strains

• Auxotrophic mutants can lead to high-producing strains if the auxotrophic mutation is at the correct site.
• Recovering auxotrophs is simpler than directly recovering high producers.
• The best approach is to select relevant auxotrophs and then screen them for productivity.

Detecting Productive Strains

Over-layering Colonies

• Productive strains among auxotrophs are detected by over-layering mutant colonies with agar suspensions of bacteria auxotrophic for the required product.
• High-producing mutants are identified by the growth of the overlay around the producer.

Methods for Recovery of Auxotrophic Mutants

Common Methods

1. Enrichment culture
2. Techniques for visual identification of mutants

Enrichment Culture

• Expose the population in minimal medium to an antimicrobial agent that affects only dividing cells, killing prototrophs and allowing auxotrophs to survive.
• Penicillin is a common inhibitory agent used in enrichment techniques (Davis, 1949).

Davis’ Technique

1. Culture mutated spores in liquid complete medium.
2. Harvest by centrifugation, wash, and resuspend in minimal medium plus penicillin.
3. Only growing prototrophic cells are susceptible to penicillin; auxotrophs survive.
4. Harvest by centrifugation, wash to remove penicillin and lysed cell products, and resuspend in complete medium to allow auxotroph growth.
5. Purify on solidified medium.

• The nature of isolated auxotrophs can be determined by the design of the complete medium. Adding one supplement to the minimal medium allows isolation of mutants auxotrophic for that additive.

Spore Germination Technique

• Culture mutated spores in minimal medium; only prototrophs germinate.
• Treat the spore suspension with a compound that kills germinated prototrophic spores, leaving ungerminated auxotrophic spores unharmed.
• Isolate auxotrophic spores by washing and culturing on supplemented medium.

Filtration Enrichment Method

1. Inoculation: Add mutated spores (auxotrophs + prototrophs) to liquid minimal medium.
2. Incubation: Shake for a few hours to allow prototrophic spores to germinate while auxotrophic spores remain dormant.
3. Filtration: Pass the suspension through a suitable filter (e.g., sintered glass).
4. Retention of Prototrophs: Germinated prototrophic spores are retained by the filter.
5. Collection of Auxotrophs: Auxotrophic spores pass through and are enriched in the filtrate.
6. Isolation of Auxotrophic Mutants: Use the enriched filtrate for further analysis or experiments.

Visual Identification of Auxotrophs

• Alternating exposure of suspected colonies to supplemented and minimal media.
• Colonies that grow on supplemented media but not on minimal media are auxotrophic.
• Replica plating is used to achieve alternating exposure.
• Allow survivors of mutation treatment to develop colonies on petri dishes of supplemented medium, then transfer a portion of each colony to minimal medium.

Sandwich Technique

1. Seed survivors of mutation treatment in a layer of minimal agar in a petri dish.
2. Incubate for 1 or 2 days and mark developed colonies on the base of the plate.
3. Pour a layer of supplemented agar over the surface.
4. Colonies appearing after a further incubation period are auxotrophic because they were unable to grow on the minimal medium.

Corynebacterium glutamicum

• $C$. glutamicum is a biotin-requiring organism that produces glutamic acid under biotin-limited conditions.
• Mutants used for other amino acid production must be supplied with optimal biotin levels for growth.
• Biotin-limited conditions will result in these mutants producing glutamate instead of the desired amino acid.

Auxotrophic Mutants of C. glutamicum for Lysine Production

• Auxotrophic mutants have been used for lysine production.

Control of Aspartate Family of Amino Acids

• Aspartokinase (1st enzyme): Controlled by concerted feedback inhibition of lysine and threonine.
• Homoserine dehydrogenase: Subject to feedback inhibition by threonine and repression by methionine.
• The first enzyme in the route from aspartate semialdehyde to lysine is not subject to feedback control.
• The control system in $C$. glutamicum is relatively simple.

Homoserine Auxotrophs

• A homoserine auxotroph of $C$. glutamicum, isolated by penicillin selection and replica plating, produces lysine in a medium containing low homoserine or threonine plus methionine.
• The mutant lacked homoserine dehydrogenase, allowing aspartic semialdehyde to be converted solely to lysine. The resulting lack of threonine removed the concerted feedback inhibition of aspartokinase.

Arginine Production in C. glutamicum

• The major control is feedback inhibition of the second enzyme, acetylglutamic acid phosphorylating enzyme, though the first enzyme may also be regulated.
• A citrulline-requiring auxotroph accumulates ornithine from glucose in the presence of limiting arginine and excess biotin.
• The mutant lacked the enzyme converting ornithine to citrulline, ceasing arginine synthesis and removing pathway control.

Purine Nucleotide Production

Control Sites

1. Phosphoribosyl pyrophosphate (PRPP) amidinotransferase: Feedback inhibited by AMP, slightly by GMP.
2. PRPP amidinotransferase synthesis: Repressed by cooperative action of AMP and GMP, along with other enzymes to IMP, coded by the pur operon in B. subtilis.
3. IMP dehydrogenase: Feedback inhibited and repressed by GMP.
4. Adenylosuccinate synthase: Repressed by AMP but not significantly inhibited.

Mutants and Inosine Excretion

• Mutants auxotrophic for AMP or doubly auxotrophic for AMP and GMP excrete inosine at levels up to $15 g dm^{-3}$.
• AMP auxotrophs (lacking adenylosuccinate synthase) require small adenosine quantities but accumulate inosine due to removed inhibition and cooperative repression of PRPP amidinotransferase.
• AMP and GMP double auxotrophs produce inosine due to removed controls normally imposed by the two end products, requiring small concentrations of adenosine and guanosine.