초록
<P><B>Abstract</B></P> <P>Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. <I>Methylobacterium extorquens</I> AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in <I>M. extorquens</I> AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in <I>M. extorquens</I> AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA–derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. <SUP>13</SUP>C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and <I>fumC</I> overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of <I>M. extorquens</I> AM1. This work was the first attempt to regulate <I>M. extorquens</I> AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> First establishment of high throughput screening method for <I>M. extorquens</I> AM1. </LI> <LI> First SATRE strategy to control carbon flux in <I>M. extorquens</I> AM1. </LI> <LI> Up to 0.055mol acetyl-CoA / mol methanol in fed-batch fermentation. </LI> </UL> </P>