Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Abstract

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branchedchain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and acetohydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km r ). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ⌬ilvA ⌬panB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.Corynebacterium glutamicum is used for the production of a number of amino acids, including valine. Acetohydroxy acid synthase (AHAS) (EC 4.1.3.18), also called acetolactate synthase, is the key enzyme of the pathways for biosynthesis of branched-chain amino acids (isoleucine, valine, and leucine) ( Fig. 1) and therefore is a potential target for metabolic engineering. The enzyme catalyzes decarboxylation of pyruvate and its condensation either with another molecule of pyruvate to produce acetolactate (a precursor of valine and leucine) or with 2-ketobutyrate to produce acetohydroxybutyrate (a precursor of isoleucine). The reactions play a key role in determining the relative fluxes to different end products (2). Three AHAS isoenzymes, differing in their genetic determinations, biochemical properties, and regulation of both biosynthesis and activity, were described for Escherichia coli and Salmonella enterica serovar Typhimurium (26). The isoenzymes AHAS I, II, and III are encoded by the genes ilvBN, ilvGM, and ilvIH, respectively. Expression of these genes is subject to different multivalent repression by branched-chain amino acids. Expression of the ilvGMEDA operon is controlled by transcriptional attenuation mediated by all three amino acids, whereas the ilvBN operon is controlled by attenuation mediated only by valine and leucine. Expression of ilvIH is regulated by Lrp (leucine-responsive protein) (29). The large subunits (IlvB, IlvG, and IlvI) are responsib...

Acetohydroxyacid Synthase, a Novel Target for Improvement of l -Lysine Production by Corynebacterium glutamicum

“…Since the AHAS reaction is one of the bottlenecks for efficient L-valine production (3,5,14), we therefore originally intended to use the ⌬C-T ilvN AHAS for improvement of L-valine production strains (3,5). Having tested the kinetic properties and having found the nearly fourfold-reduced V max,P values of the mutated AHAS, we realized that chromosomal introduction of the AHAS ⌬C-T ilvN into L-valine producer strains most probably does not lead to improved L-valine production.…”

“…Together with the acetohydroxy acid isomeroreductase gene ilvC, the two genes form the ilvBNC operon (6,10). On the one hand, AHAS is subject to feedback inhibition by the three BCAAs (10,14). On the other hand, expression of the ilvBNC operon is controlled by an attenuation mechanism, leading to an about twofold-higher expression in response to the shortage of BCAAs (32).…”

Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

“…The AHAS from C. glutamicum WT is feedback inhibited to ca. 50% by L-valine, L-leucine, or L-isoleucine; however, there is no cumulative effect for all three BCAAs and AHAS inhibition remains at 50 to 60% inhibition in the presence of all three BCAAs (14,17,29). Due to the structural similarities of the BCAAs with their corresponding 2-keto acids, it was conceivable that the latter also have an effect on AHAS activity.…”

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

“…There have only been a few reports of attempts to engineer increased valine content in bacteria. These studies in bacteria either increased the pathway flux to valine (Radmacher et al, 2002;Blombach et al, 2007), utilized mutated valine-tolerant RSUs to circumvent end-product inhibition (Elisakova et al, 2005), or combined both strategies (Park et al, 2007).…”