초록
<P><B>Abstract</B></P> <P>Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as <I>Clostridium tetanomorphum</I>. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from <I>C. tetanomorphum</I>, were recombinantly expressed in <I>Escherichia coli</I>. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the <I>in vivo</I> activity. These efforts led to efficient mesaconate production at a titer of 7.81g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96g/L directly from glucose. In summary, we have engineered an efficient system in <I>E. coli</I> for the biosynthesis of mesaconate.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Design of a total biosynthetic pathway to mesaconate. </LI> <LI> Discovery of MutL as the reactivatase of glutamate mutase. </LI> <LI> Engineering of the coenzyme B12 regeneration pathway in <I>E. coli</I> to improve mesaconate production. </LI> <LI> High-level biosynthesis of mesaconte in <I>E. coli</I> from glucose. </LI> </UL> </P>