Metabolic Regulation Analysis of Recombinant Lactococcus Lactis Based on Gene Expression and Enzyme Activity

Lactic acid bacteria (LAB) are industrially important microorganisms that are widely used in industrial food fermentations for dairy production. However, there is a growing interest in their application in genetic modification and biotechnology processes. Lactococcus lactis is a non-pathogenic bacterium whose genome has been completely sequenced and its metabolic pathways are well studied. These reasons make L. lactis an attractive target for those approaches including creating live vector vaccine. The present study was conducted to evaluate the effect of aerolysin on the metabolic regulation and fermentation characteristics of the recombinant L. lactis. Both L. lactis NZ9000 and Recombinant L. lactis carrying D1 of aerolysin gene (Lac-D1ae) were cultivated in M17 medium supplemented with 0.5% (w/v) glucose incubated at 30°C with an agitation of 150 rpm. Chloramphenichol (7.5 mg/mL) was added to maintain the plasmid. Samples for gene expression and enzyme activity assays were taken during late-exponential growth phase. The alteration of expression of 10 genes (glk, pfk, pyk, ackA, mdh, ldh, pgi, zwf, gnd)responsible for enzymes at the main metabolic pathways i.e. glycolysis, Triarboxylic Acid (TCA) cycle, fermentation and pentose phosphate pathway (PP pathway) were examined by using semi-quantitative RT-PCR and Real-Time PCR. The activity of these enzymes including glucokinase (GLK), phosphofructokinase (PFK), pyruvate kinase (PYK), acetate kinase (ACK), Malate dehydrogenase (MDH), Lactate dehydrogenase (LDH), Phosphoglucose isomerase (PGI), Glucose-6-phosphate dehydrogenase (G6PDH) and 6-Phosphogluconate dehydrogenase (6PGDH) were also measured to understand the metabolic regulation in Lac-D1ae. According to the fermentation results obtained, cell growth, lactate production rate and acetate production rate in the Lac-D1ae were 0.77 g/L, 0.82 mg/L/h and 0.16 mg/L/h, respectively. The values were slightly lower compared to the parental strain (0.8 g/L, 0.84 mg/L/h and 0.18 mg/L/h). Glucose consumption rate also showed a considerable decrease in the recombinant strain (3.48 g/L/h) in comparison with L.lactis NZ9000 (4.27 g/L/h). HPLC results showed the production of lactate (8.20 g/L) and acetate (1.58 g/L) were reduced in Lac-D1ae in contrast with parental strain (8.35 g/L and 1.83 g/L). In conclusion, the fermentation characteristics of the recombinant L. lactis showed that the presence of aerolysin gene has no inhibitory effect on the growth. Furthermore, integrating methods based on gene expression and enzyme activities showed up-regulation of glycolysis and TCA cycle and down-regulation of Pentose Phosphate pathway in the recombinant L. lactis carrying aerolysin gene (Lac-D1ae).

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