초록
<P><B>Abstract</B></P><P>Despite the fact that the area of glycolysis in <I>Lactococcus lactis</I> has been intensively studied, only a limited number of studies have been focused on the regulation of uptake of glucose itself. Using the tool of the glucostat fed-batch mode of culture, it was demonstrated in our earlier work that the concentration of glucose regulates its uptake rate and that the control of the glycolytic flux resides to a large extent in processes outside the pathway itself, like glucose transport and the ATP consuming reactions, while allosteric properties of key enzymes like phosphofructokinase (PFK) have a significant influence on the control. Extending our work, we report here the results of fermentations with engineered <I>L. lactis</I> strains with altered PFK activity in which the <I>pfk</I>A gene from <I>Aspergillus niger</I>, and its truncated version <I>pfk</I>13 that encodes a shorter PFK1 fragment were cloned. The results in this study suggest that, under the optimum for the microorganism applied microaerobic conditions, the glycolytic capacity of <I>L. lactis</I> was significantly increased in engineered strains with increased PFK activity. The transformant strain in which the truncated <I>pfk</I>13 gene of <I>A. niger</I> was expressed performed more efficiently as it was able to grow successfully in glucostat cultures with 277mM glucose – while the optimum glucose concentration for the parental strain was 55mM. The present work demonstrates the direct effect of PFK activity on the glycolytic flux in <I>L. lactis</I> since a twofold increase in specific PFK activity (from 7.1 to 14.5U/OD<SUB>600</SUB>) resulted in a proportional increase of the maximum specific rates of glucose uptake (from 0.8 to 1.7μMs<SUP>−1</SUP>gCDW<SUP>−1</SUP>) and lactate formation (from 15 to 22.8g lactate (gCDW)<SUP>−1</SUP>h<SUP>−1</SUP>).</P>