초록
<P><B>Background</B></P><P><SMALL>L</SMALL>-Glutamate is an important precursor in the <SMALL>L</SMALL>-arginine (<SMALL>L</SMALL>-Arg) biosynthetic pathway. Various methods, including polyoxyethylene sorbitan monopalmitate (Tween 40) addition and <I>dtsR1</I> disruption, have been widely used to induce <SMALL>L</SMALL>-glutamate overproduction in <I>Corynebacterium glutamicum</I>. In this study, a novel strategy for <SMALL>L</SMALL>-Arg overproduction through Tween 40 trigger and Δ<I>dtsR1</I> mutant were proposed in <I>Corynebacterium crenatum</I>.</P><P><B>Results</B></P><P><I>Corynebacterium crenatum</I> mutant (CCM01) was selected as a host strain, whose <I>argR</I> was lethal via mutagenesis screening, the <I>proB</I> gene was knocked out, and <I>argB</I> was replaced by <I>argB</I> M4 (E19R, H26E, D311R, and D312R) to release <SMALL>L</SMALL>-Arg feedback resistance. After Tween 40 trigger in the logarithmic period, <SMALL>L</SMALL>-Arg production increased from 15.22 to 17.73 g/L in CCM01 strain. When <I>NCgl1221</I> and <I>dtsR1</I> disruption (CCM03), <SMALL>L</SMALL>-Arg production drastically increased to 27.45 g/L and then further to 29.97 g/L after Tween 40 trigger. Moreover, the specific activity of α-oxoglutarate dehydrogenase complex (ODHC) decreased, whereas the regeneration of NADP<SUP>+</SUP>/NADPH significantly increased after <I>dtsR1</I> disruption and Tween 40 trigger. Results of real-time PCR showed that the transcriptional levels of <I>odhA</I>, <I>sucB</I>, and <I>lpdA</I> (encoding three subunits of the ODHC complex) were downregulated after Tween 40 trigger or <I>dtsR1</I> disruption. By contrast, <I>zwf</I> transcription (encoding glucose-6-phosphate dehydrogenase) showed no significant difference among CCM01, CCM02 (Δ<I>NCgl1221</I>), and CCM03 (Δ<I>NCgl1221</I>Δ<I>dtsR1</I>) strains without Tween 40 trigger but evidently increased by 5.50 folds after Tween 40 trigger.</P><P><B>Conclusion</B></P><P>A novel strategy for <SMALL>L</SMALL>-Arg overproduction by <I>dtsR1</I> disruption and Tween 40 trigger in <I>C. crenatum</I> was reported. Tween 40 addition exhibited a bifunctional mechanism for <SMALL>L</SMALL>-Arg overproduction, including reduced ODHC activity and enhanced NADPH pools accumulation by downregulated <I>dtsR1</I> expression and upregulated <I>zwf</I> expression, respectively.</P>