Lectrure 3

Question 1

amino acids

Answer 1

All amino acids can be synthesised industrially either by direct fermentation, by enzymes synthesised by micro-organisms, by hydrolysis of proteins or by chemical synthesis (20 aa)

Question 2

what is L-glutamate?

Answer 2

known to provide the umami taste and contributes to various aspects of health.

Question 4

formation of L-glutamate

Answer 4

1) Initially it was produced by hydrolysis of proteins, which was quite expensive as it had to be separated from the rest of the amino acids.
2) Later, it was chemically synthesized, but this was also an expensive process as it had to be separated from its isomer D-Glutamate (tasteless).
3) Then a soil bacterium was discovered that secretes large quantities into the culture medium (Corynebacterium glutamicum) starting the fermentation of wild strains of Actinobacteria, Firmicutes or Proteobacteria for the industrial production of amino acids.

Question 4

Compounds affecting glutamate production

Answer 4

Biotin influences glutamate production, so that adding it to the culture medium increased production, but adding too much decreased it.
The absence of biotin is known to alter the function of the regulatory enzyme OdhI by blocking the Krebs cycle. The regulatory enzyme OdhI controls the 2-oxoglutarate dehydrogenase (ODHC) enzyme complex so that, when dephosphorylated, it is able to inhibit the complex by stopping the Krebs cycle (2-oxoglutarate is converted to L-glutamate). OdhI also facilitates the expression of a membrane transporter for glutamate that allows its accumulation outside the membrane.
Under normal conditions, the Krebs cycle functions in a truncated version, via the glyoxylate cycle, maintaining normal glutamate production. When a high culture density is reached, this pathway is partially inhibited, increasing glutamate production.

Question 4

Regulation of amino acid biosynthesis in E. coli

Answer 4

1) Control of the activity of pre-existing enzymes: This is feedback inhibition as occurs in the synthesis of proline from glutamate (when large amounts of proline are present, it binds to the first enzyme of its synthesis inactivating it).
2) Control of the synthesis of new enzymes:
- Repression: The final product of the pathway (when overexpressed) binds to a regulatory protein or inactive repressor (ArgR) causing a conformational change that allows its binding to the promoter region inhibiting transcription by blocking the binding of RNA polymerase.
- Attenuation: mRNA blocking is controlled, for example, in the tryptophan operon, in which the leader region has the ability to form secondary structures by region pairing –> When there is a lot of Trp it passes quickly through region 1 to region 2 so that regions 3 and 4 form a loop terminating transcription; in contrast, when there is little Trp the ribosome gets stuck in region 1 forming a loop between regions 2 and 3 which has no effect on RNA polymerase.

Question 4

Production of biosynthetic intermediates by auxotrophic mutants: L-Ornithine

Answer 4

L-Ornithine is an intermediate in the synthesis of arginine so, to synthesize it, we must obtain an auxotrophic mutant for arginine that lacks ornithine transcarbamylase activity, which is the first enzyme of the pathway (L-Ornithine + ornithine transcarbamylase = L-Citrulline).

Question 4

how can auxotroph mutants be identified?

Answer 4

To identify the auxotroph, a medium with citrulline is used, but the amount must be well controlled, otherwise ornithine synthesis may be reduced (negative feedback by arginine at higher levels). Arginine should be used as a limiting nutrient, maintaining a normal physiological function without activating the feedback mechanisms.