Expression Of Functional Genes In Lignocellulose-dependent Pediococcus Acidilactici DQ2and Knockout Of Target Gene

Pediococcus acidilactici DQ2, isolated by our lab, is thermotolerance and high resistant to the inhibitors derived from lignocellulose and can produce high titer of lactic acid. In order to make this strain more adaptable to the lignocellulose biorefinery engineering process, we modified P. acidilactici DQ2from two aspects. First, we constructed heterologous protein expression system in P. acidilactici DQ2for the first time. The lactic acid bacterial expression vector pMG36e was modified by replacing the promoter P32with PldhL derived from P. acidilactici DQ2, Then expressed two different heterologous genes successfully with the new plasmid pTY36e, green fluorescent protein gene(gfp) from Aequorea victoria and β-glucosidase gene (bglA) from Bacillus polymyxa1.794, the results showed that the β-glucosidase enzyme activity of the recombinant bacteria was4.48U/(g·dry cells) detected in the intracellular fraction. The successful construction of this system layed the foundation for the subsequent genetically engineering of P. acidilactici DQ2. Second, we try to knockout the D-lactic dehydrogenase coding gene ldhD of P. acidilactici DQ2to produce optical pure L-lactic acid. Modified the thermosensitive knockout plasmid pSET4S by replaced the spectinomycin marker with erythromycin and then use the new plasmid pSET4E to construct knockout vector to delete ldhD.Results show that we succeeded in eliminating the production of D-lactic acid, the optical purity of L-lactic acid produced by the mutant strain P. acidilactici ΔldhD112was99.88%. Moreover, we can not detect D-lactic dehydrogenase activity in ΔldhD112. As D-lactate was the terminal of cell wall peptidoglycan of P. acidilactici DQ2, remove the production of D-lactate reduce it’s resistant to vancomycin significantly. The successful construction of AldhD112makes the lactic acid more usefull and makes this strain more potential as industrail production strain. The above work is important to the pratical application of this strain in biorefinery and the further development of it’s fermentation ability.