A metabolic model of exopolysaccharide production in Lactobacillus delbrueckii subsp. bulgaricus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

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Exopolysaccharides (EPS) from lactic acid bacteria (LAB) play an important role in enhancing the rheology and texture of fermented dairy foods. Whilst LAB are attractive vehicles for the production of probiotics due to their GRAS (Generally Recognized as Safe) status, the economic production of EPS by LAB remains constrained by their metabolism. Rational metabolic engineering studies aimed at altering the production of EPS and lactic acid require an understanding of the contributions of whole biosynthetic networks, or segments thereof, to the end-products. Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 was confirmed to follow a homofermentative pattern of calabolism, and to export exopolysaccharide (EPS), lactate, and galactose as principal metabolites concurrently with cell growth. The EPS formed was found to diminish after a period of time, probably due to degradation of the polymer. Intracellular glucose resulting from the splitting of lactose taken up into the cell was the principal source of carbon for EPS, lactate and biomass. Kinetic models which were applied to describe the production of EPS, lactate and galactose in batch fermentation suggested, however, that a small percentage of carbon from galactose could have been diverted towards the formation of EPS, lactate, and biomass. Emphasis was placed upon developing a rational screening programme in order to generate a mutant of Lb. delbrueckii subsp. bulgaricus NCFB 2483 with a higher specific yield of EPS production than the parent strain, for the purposes of understanding the flux to sugar-nucleotides associated with raised levels of EPS production. This process resulted in the isolation of a chemically induced mutant of Lb. delbrueckii subsp. bulgaricus NCFB 2483 with a higher specific EPS yield than the parent strain. In addition, it was demonstrated that by changing the environmental conditions viz. by reducing the water activity of the growth medium, carbon flux to the EPS-synthesizing pathways could be raised in batch culture. Detailed information on the enzymatic activities and metabolite levels associated with EPS formation at steady state metabolic conditions was derived from continuous culture studies. The formation of EPS, lactate, and galactose, as well as the distribution of carbon fluxes through the glucose 6-phosphate and glucose-1-phosphate branch points in Lb. delbrueckii subsp. bulgaricus NCFB 2483 was measured at different growth rates in continuous culture. This investigation revealed an enhanced carbon flux to the EPS-synthesizing pathway being associated with higher growth rates, despite a limitation imposed by a "bottleneck" at the glucose-6-phosphate branch-point. Results of similar metabolic flux studies in continuous culture with Lb. delbrueckii subsp. bulgaricus NCFB 2483, grown under conditions of osmotic stress supported this deduction. Comparative metabolic flux investigations between the chemically induced mutant of Lb. delbrueckii subsp. bulgaricus NCFB 2483, previously isolated, and the parent strain, provided further evidence of this constriction of carbon flow. Collectively, these investigations suggested that any improvement in the flux of carbon towards EPS formation in Lb. delbrueckii subap. bulgaricus NCFB 2483 could be effected by the enhancement of the activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase. In all of the metabolic studies undertaken in continuous culture, raised levels of carbon flux toward EPS sugar-nucleotides occurred in conjunction with raised levels of specific lactate production rates. In these instances, raised levels of ATP which were measured could be associated with raised levels of glycolysis and the biosynthesis of certain sugar-nucleotides. These findings implied that the cell dissipated excess cellular energy by the formation of sugar-nucleotides, however it may be that the raised flux toward the sugar nucleotides was a direct response to excess energy available in the cell. Any strategy aimed at enhancing carbon flow to EPS by the diversion of carbon away from glycolysis would need to be counterbalanced by the cell's requirement to generate ATP via this pathway, including its need to maintain its redox balance.
Lactic acid bacteria, Probiotics, Carbon flux