Improving the carbon conversion rate in Lactococcus lactis fermentations: Cloning strategies

The TCA cycle is a crucial link between catabolic and anabolic reactions in cells and anaplerotic reactions are responsible for replenishing the citric acid cycle intermediates. Extensive studies on the metabolism of an industrial Aspergillus niger strain have revealed that there are three main metabolic events that replenish TCA cycle intermediates and predispose the cell to product formation: fast uptake of glucose by simple diffusion, unrestricted metabolic flow through glycolysis, and uncoupled NADH re-oxidation resulting in lower levels of ATP and therefore decreased anabolic reactions. Only the activities of certain enzymes characteristic of individual A. niger strains can lead to such intracellular conditions. In this respect, we carried out studies aiming to demonstrate that the productivities and yields of industrial strains of L. lactis can be increased by reinforcing anaplerotic reactions by the transfer of specific A. niger genes into them. The development of a wide variety of cloning systems during the last decade has allowed the improvement of many properties of L. lactis strains that are essential for a large number of industrial dairy and other food fermentations. A central area in research related to L. lactis genetics is the regulation of glycolysis. The mechanisms responsible for regulation of the glycolytic flux and the shift between different fermentation modes are being studied extensively. Most recent research with microaerobic glucostat fed-batch experiments showed that the concentration of glucose influences its specific uptake rate and consequently the glycolytic flux, as well as the fermentation pattern. The highest specific activities of the key glycolytic enzymes PFK, PYK and the LDH were obtained at 55 mM glucose, the area of the highest observed glycolytic flux. Reduction of the glycolytic flux by 55% in the 277 mM glucostat corresponded to an almost identical reduction in PFK activity, indicating a certain controlling influence of this enzyme on the flux, through the glucose effect. Glucostat data also showed that the control of the flux through the glycolytic pathway under the examined conditions, resides to a large extent in processes outside the pathway, like the ATP consuming reactions and glucose transport. A regulation mechanism was proposed governed by the energy state of the cell by which L. lactis can handle the glycolytic flux through the allosteric properties of key enzymes, with PFK having a significant influence on the control. By cloning the pfkA and its truncated version alone and in strategic combinations with aox1 and pkaC genes from A. niger into L. lactis, carbon conversion rates and the yield of lactate and nisin A were improved. L. lactis transformants performed more efficiently under aerobic conditions with high glucose levels in the medium.