The long history of application to the dairy industry has established Lactococcus lactis (L. lactis), the lactic acid bacterium, as one of the most extensively characterized low GC organisms. The relatively simple metabolism of L. lactis has also made it an attractive target for metabolic engineering for the production of non-food related chemicals. Moreover, the status of being the first genetically modified organism to deliver immunoproteins alive to human has brought L. lactis considerable fame in biomedical research. Beside the exceptional industrial relevance of L. lactis, it is also an important subject for basic research in cellular metabolism because L. lactis exhibits an interesting metabolic shift. Under anaerobic conditions, on fast fermentable sugars, L. lactis produces lactate as the primary product, known as homolactic fermentation but on slowly fermentable sugars, significant amounts of formate, acetate and ethanol are formed, known as mixed-acid fermentation. This shift is termed the mixedacid shift. This type of shift between a low-yield and a high-yield metabolism has drawn a lot of research focus and has similarly been observed in other bacteria, yeast and even tumor cells. Efforts have been put to find out the mechanism regulating the mixed-acid shift as well as to answer questions such as why L. lactis prefers such a switch. Until now, some pieces of evidence have been reported and several factors and models have been proposed as the keys to regulating the shift, including the expression level of certain genes in glycolysis and fermentation pathways, the levels of the cofactors NADH, NAD+, ATP and ADP, the balance between catabolism and anabolism, etc. In this project, we studied the mixed-acid fermentation of L. lactis by (i) examining the roles of the enzymes in the mixed-acid fermentation pathway under different growth conditions; (ii) testing the predicted effect of the cofactors NADH, NAD+ on the mixed-acid shift proposed in previous studies; (iii) looking into the connection between amino acid metabolism and the mixed-acid shift; and (iv) contrasting the difference regarding the mixed-acid shift between two widely studied laboratory strains of L. lactis, MG1363 that shifts significantly and IL1403 that does not shift. We have measured the promoter activities of several mixed-acid genes which suggested that the regulatory elements governing the transcriptional regulation of the mixed-acid genes in MG1363 and IL1403 were different. This led us to performing experimental control analysis of the role of pyruvate formate-lyase (PFL) in MG1363 and IL1403. The expression of PFL in MG1363 appeared to be optimized for growth rate when growing on maltose whereas overexpressing PFL in IL1403 was probably detrimental. The two homologous acetate kinases in MG1363 were also chacterized with respect to the transcription and enzyme activities. The isozymes were found to have complementary physiological roles that became important in acetate-producing or acetate-assimilating conditions respectively. The proposed roles of NADH and NAD+ on the mixed-acid shift were tested by perturbation via introducing activities of 2,3-butanediol dehydrogenase and supplying extracellular acetoin as an oxidizing agent. The additional NAD+- regenerating activities allowed a faster growth of MG1363 on maltose by shifting ethanol production into acetate production and also stimulated formate and acetate production in IL1403. Dependance of the mixed-acid fermentation of MG1363 on amino acid availability was observed and the impact of individual amino acids could differ significantly. Meanwhile, a computational method for combining metabolic flux analysis and elementary mode analysis was developed and applied to analyse a case of amino acid metabolism of L. lactis.
Chan, S. H. J. (2014). Elucidating Flux Regulation of the Fermentation Modes of Lactococcus lactis: A Mutlilevel Study. Kgs. Lyngby: Technical University of Denmark. https://doi.org/10.11581/DTU:00000016