Construction Of The Negative Feedback Pathway Of Lactic Acid In Lactobacillus Bulgaricus And Streptococcus Thermophilus

Yoghourt is a kind of widely popular food. In the process of producingyoghourt, Lactobacillus bulgaricus and Streptococcus thermophilus are the commonstarter culture. These lactic acid bacteria would contine produce lactic acid in theprocess of yoghourt transportation, marketing, and store. The phenomenon is calledthe postacidification of yoghourt and will reduce the property of yoghourt. Thisresearch explored the possibility of establishing lactification-negative feedbackpathway using pH inducible promoter, by building mathematic models of lactic acidgeneration and regulation in Lactobacillus bulgaricus and Streptococcusthermophilus, and attempted to regulate lactic acid production based on people’srequirements.In the process of building mathematic models, based on Law of massaction, Michaelis-menten equation, and Hill equation, etc, we build system ofordinary differential equations to describe the change of cellular materials over time.By sensitivity analysis of model parameters, we find out the parameters that couldhave significantly impact on lactic acid production. Then, we establish the mostimportant parameters, and make assignment for them for computer stimulation,combined with biological experiment maneuverability. By discussing the stimulationresults, we could make conclusions as follows.For Lactobacillus bulgaricus, we could establish lactification-negativefeedback pathway by utilizing lacR gene regulated by pH inducible promoter, butthe startup rate of pH-induced promoter need to be in between20h-1and10.5h-1, andthe intensity of lacR ribosome bind site need to be in between564h-1and1128h-1.In the meantime, the normal leaking transportation of pH-induced promoter didn’tshow significant effect on the production of lactic acid.For Streptococcus thermophilus, the model could not find out negativefeedback pathway suitable for establishing pH inducible promoter regulation,because the regulation pathway relative to lactic acid metabolism is morecomplicated, and we are lack of reliable parameters. However, we could firstmeasure the metabolic regulation parameters of HPr(Ser-P) CcpA complex bybiological experiments, and thus improve the established mathematical model forproviding more believable predictions and instruction.According to the results from the model of Lactobacillus bulgaricus, we madea codon optimization for lacR gene and designed the gene rcfB+lacR. Then weconstruct the recombinant plasmid pMG36e-rl with lacR gene regulated by pHinducible promoter rcfB, and establish the foundation for subsequent biological experiments.