초록
<P>Abstract</P><P><I>N</I>-Acetyl-<SMALL>L</SMALL>-glutamate kinase (EC 2.7.2.8) is first committed in the specific <SMALL>L</SMALL>-arginine pathway of <I>Corynebacterium</I> sp. A limited increase of <SMALL>L</SMALL>-arginine production for the <I>arg</I>B overexpression in the engineering <I>C. creantum</I> SYPA-CCB strain indicated that <SMALL>L</SMALL>-arginine feedback inhibition plays an influence on the <SMALL>L</SMALL>-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of <I>I</I><SUB>0.5</SUB><SUP>R</SUP> efficiently. Thereby, the multi-mutated NAGK<SUB>M3</SUB> (including E19R/H26E/H268D) was generated and its <I>I</I><SUB>0.5</SUB><SUP>R</SUP> of <SMALL>L</SMALL>-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust <SMALL>L</SMALL>-arginine producer, the engineered strains SYPA-CCB<SUB>M3</SUB> were constructed. Introducing the <I>arg</I>B<SUB>M3</SUB> gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The <SMALL>L</SMALL>-arginine synthesis was largely enhanced due to the overexpression of the <I>arg</I>B<SUB>M3</SUB>, which is resistant to feedback resistant by <SMALL>L</SMALL>-arginine. Thus <SMALL>L</SMALL>-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the <SMALL>L</SMALL>-arginine metabolic pathway by deregulating the key enzyme of the pathway.</P>