초록
<P><B>Background</B></P><P>Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out <I>Citrobacter werkmanii</I> ∆<I>dhaD</I> was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply.</P><P><B>Results</B></P><P>By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by <I>ldhA</I>, and ethanol dehydrogenase coded by <I>adhE</I>) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in <I>C. werkmanii</I> ∆<I>dhaD</I> resulted in the strain <I>C. werkmanii</I> ∆<I>dhaD</I>∆<I>ldhA</I>∆<I>adhE</I>::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism.</P><P><B>Conclusions</B></P><P>In this study, a multiple knock-out was created in <I>Citrobacter</I> species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s12934-016-0421-y) contains supplementary material, which is available to authorized users.</P>