For branched chain amino acid synthesis, microorganisms such as Corynebacterium

Question

For branched chain amino acid synthesis, microorganisms such as Corynebacterium glutamicum employ feedback inhibition of the critical enzyme acetohydroxy acid synthase (AHAS) by leucine, isoleucine, and valine (Elisakova et al., Appl Env Micro 2005) (Table 1 and Figure 1). The activity of AHAS is inhibited by any of the three amino acids to 50% of the maximum. However, the inhibitory effect does not exceed 50% even in the presence of all three amino acids. C. glutamicum is used for commercial production of a number of amino acids including valine. AHAS from this organism is comprised of a catalytic and a regulatory subunit encoded by the ilvB and ilvN genes, respectively. Elisakova and collaborators identified mutations in the regulatory subunit of C. glutamicum that conferred resistance to feedback inhibition.

E. Figure 2 shows the amount of L-valine produced in various mutants. Which combination of gene mutations would you include in a strain to boost L-valine production to maximal levels? Explain your rationale using the pathway described in Figure 1.

I know the answer is the ilvA/panB/ilvNM13 one but I just don't know how to explain the answer using the pathway.

Table 1. Gene/enzyme relationships Enzyme Threonine dehydratase Acetohydroxy acid synthase Acetohydroxy acid isomeroreductase Dihydroxvacid dehydratase DHAD Transaminase Gene name Abbreviation TD AHAS ilvB (catalytic subunit), ilvN (small re gulatory subunit ilvc ilvD ilvE AHAIR TA L-threonine TD vate vate AHAS ilvB+ilvN aceto-2-hydroxy 2-acetolactate AHAIR ilvC X2,3-dihydroxyisovalerate 3-methylvalerate DHAD. ilvD 2-keto-3-methylvalerate2-ketoisovalerate panB ivE LLsoleucineLvaline L-valine D-pantothenate -leucine Figure 1. Synthesis of branched chain amino acidS

Explanation / Answer

The main purpose of this research was to create Corynebacterium glutamicum strain which can produce high amount of valine (amino acid).according to the research it was found that the enzyme AHAS (Acetohydroxy acid synthetase) is a key enzyme in the biosynthesis of branched-chain amino acid (valine, leucine, isoleucine).however, this enzyme can be inactivated due to the feedback inhibition by the final product valine, leucine, and isoleucine.

The gene which regulates the feedback response of the AHAS enzyme was found to be. So in order to make Corynebacterium glutamicum strain resistant to feedback inhibition, site-directed mutagenesis was performed on the ilvN gene was performed with the help of plasmid. The mutant strain (ilvNM13) AHAS enzyme did not show sign of feedback inhibition therefore, the valine production was increased.

To further enhance the production of valine in the in (ilvNM13) to deletions were performed in the gene responsible for the enzyme which was related to pathways competing for the valine production. The ilvA gene of the mutant strain (ilvNM13) responsible for the formation of the 2-ketobutyrate intermediate of isoleucine synthesis was inactivated by 368 base pair deletion. The gene panB responsible for converting 2-ketoisovalerate to pantothenate was also inactivated 200 base pair deleted.

Finally, the mutant Corynebacterium glutamicum strain was engineered (ilvA panB ilvNM13) which was resistant to feedback inhibition and was not having two valine production competitive pathways.

Therefore, valine production increased many folds in the engineered mutant strain.

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