초록
<P>Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in <I>Corynebacterium glutamicum</I> that enable improved <SMALL>L</SMALL>-lysine production from glucose. A suitable reporter strain was transformed with a <I>pyc</I> gene library created by error-prone PCR and screened by fluorescence-activated cell sorting (FACS) for cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration. Two pyruvate carboxylase variants, PCx<SUP>T343A,I1012S</SUP> and PCx<SUP>T132A</SUP> were identified allowing 9% and 19% increased lysine titers upon plasmid-based expression. Chromosomal expression of PCx<SUP>T132A</SUP> and PCx<SUP>T343A</SUP> variants led to 6% and 14% higher <SMALL>L</SMALL>-lysine levels. The new PCx variants can be useful also for other microbial strains producing TCA cycle-derived metabolites. Our approach indicates that a biosensor such as pSenLys enables directed evolution of many enzymes involved in converting a carbon source into the target metabolite.</P><BR>[FIG OMISSION]</BR>