The key point of this part is selecting the gene related to anti-acid characteristic. Because the mechanism of acid resistance is complicated, and genes are connected to each other, it is hard to confirm the most significant gene.
Gnomic mutagenesis and high throughout screening are carried out to obtain anti-acid mutant strains with significant higher survival rate under specific acid environment (pH 4.0, 5h). Then analyze the diff-genes data between mutant strain and parent strain on pH 4.0 and pH 7.0 respectively. A dimensionality reduction model is established and five possible acid-resistant genes are obtained. Finally, with experiment verification and pathway analysis, msmK gene is shown to be the key anti-acid gene.
In addition, we also inferred how the component functions by analyzing the main acid tolerance pathways of lactic acid bacteria. The result shows msmK is related to carbohydrate metabolism and sugar transporters. That means it can help provide more energy for bacteria in acid environment to maintain intracellular pHi.
According to previous research, we know that cspD2 can express cold shock protein in lactic acid bacteria. Therefore, we obtained the cspD2 fragment by in vitro synthesis, and constructed the cspD2 overexpression strain. Verify the cold resistance performance of the recombined bacteria with nisin as inducer.
It has been verified that cspD2 can effectively exert anti-freezing effect in the recipient bacteria. CspD2 was ligated to the pNZ8149/msmK plasmid by one-step cloning (seamless ligation), and the recombinant plasmid was introduced into the constructed L.lactis NZ3900/pNZ8149-msmk-cspD2 strain using electroporation. The gfp gene was inserted as a marker gene, and cell viability was characterized by fluorescence intensity. The strain was tested for acid resistance and freezing resistance using a flow cytometer.