Lecture 4 Flashcards
Amino acid production using mutant regulators, immobilized bacteria or enzymes
production of amino acids by fermentation
1- Wild - type strains
2- Auxotrophic muthants
3- Regulatory muthants:
- Unbranched Pathways
- Branched Pathways
Production of aminoacids by regulatory mutants of unbranched pathways:L-Proline
L - proline is obtained from glutamate by fermentation with Serratia marcescens. When there is an excess of proline, it binds to glutamate kinase inhibiting its own synthesis. Therefore, we need a mutant for glutamate kinase that cannot recognize proline. To select the mutant we use toxic structural analogues of proline, so that only mutant bacteria will grow. We also need to deactivate proline oxidase (degrades proline). Being an osmoprotectant, a medium with high salt concentration will increase proline production as a protective mechanism against these culture conditions. The biosynthesis genes can be overexpressed by cloning.
Production of aminoacids by regulatory mutants of unbranched pathways: L-histidine
In the case of histidine we must inhibit histidinase, as well as induce mutagenesis with selection by structural analogues to inhibit the feedback of adenylyl transferase. No increase in production was achieved since the feedback was only partially inhibited; moreover, when total inhibition was achieved, production did not increase either due to the existence of an additional pathway that did not function at a sufficient rate to recover ATP (AICAR enzyme). Therefore, a mutant that inhibits histidinase/adenylyl transferase while overexpressing AICAR is required.
Production of aminoacids by regulatory mutants of branched pathways: Aspartate
Four amino acids (isoleucine, threonine, methionine, lysine) are produced from aspartate, which control both the first enzyme in its pathway and aspartate kinase.
Production of aminoacids by regulatory mutants of branched pathways: Aspartate E.coli
In case of E.coli there are three aspartate kinases so that each amino acid (not isoleucine) inhibits one without totally blocking the synthesis.
Production of aminoacids by regulatory mutants of branched pathways: Aspartate B. flavum
In soil bacteria (Brevibacterium flavum) living in lower nutrient media, regulation is easier because it is controlled by only two amino acids (lysine/threonine).
To find a lysine-only producing strain we would need only a mutant for homoserine dehydrogenase, but it would become auxotrophic for methionine, threonine and isoleucine. Using lysine analogs, a mutant could be found that has inhibited aspartate kinase feedback (only threonine would regulate it). However, these mutants tend to grow worse than the wild-type organism, so we usually resort to protoplast fusion. Among the daughter cells we should look for those with higher production of the metabolite that grow fast. By promoting growth, more fermentations can be carried out in the same time, obtaining more product.
Aminoacid production with enzimes: characteristic
- Allows production in high concentrations
- Requires few reagents
- It has a very high production rate per unit volume, but requires purification of enzymes or very expensive starting reagents.
Aminoacid production with enzimes: Aspartase
Aspartase degrades aspartate to form fumarate, however, in industry the direction of the reaction can be reversed by administering aspartate. We need a continuous production of the enzyme (they end up inactivating) so we can attach the aspartate to a column by passing a solution with fumarate (or add an Escherichia coli that actively produces the enzyme). Since it is a degradation enzyme we must provide aspartate at the beginning to induce the enzyme. Aspartase can also be overexpressed under a constitutive or inducible promoter that can be controlled.
Aminoacid production with enzymes: Aspartate 4- decarboxylase
L-Alanine is obtained from aspartate by aspartate 4-decarboxylase which can be purified from Pseudomonas dacunbae or introduced into Escherichia coli to attach it to the column. Two columns can be coupled to prevent re-degradation of aspartate by its increase.
Aminoacid production with enzymes: hydrolase&racemase
L-lysine is obtained from DL-α-amino-ε-caprolactam using Cryptococcus laurentii hydrolase with 50% efficiency (racemic mixture). Using racemase from Achromobacter obae we can transform the remaining D-isomer into the L-isomer (hydrolase) with100%yield.
