Study On Secretion Of Lytic Polysaccharide Monooxygenases And N-Acyl-homoserine Lactonase By Bacillus Subtilis

Bacillus subtilis is a Gram-positive bacterium which belongs to Bacillus with the ability of producing spores.Wild Bacillus subtilis doesn’t generate hazardous substances for human beings and contains no endotoxin on its cell wall, So Bacillus subtilis has been approved as a generally-recognized-as-safe(GRAS) microorganism by FDA and is one of the most widely used hosts in the production of recombinant proteins.As an attractive host, Bacillus subtilis is capable of secreting functional extracellular proteins efficiently to the culture medium which is very suitable for industrial production of heterologous protein. However, a major limitation hinders the wide application of B. subtilis which is a large amount of protease produced by Bacillus subtilis that causes serious degradation of heterologous protein. In order to achieve excellent enzyme production microorganism,it is an important way to optimize the B. subtilis expression system by using of genetic engineering methods.For the secretion of heterologous proteins commonly depend on the guidance of B. subtilis signal peptide itself, and studies have showed that there is suitability between the signal peptide and heterologous protein. So screening of signal peptides for optimizing the export of target protein in B. subtilis might be necessary. On the other hand, B. subtilis produces high level of proteases which include extracellular and intracellular proteases at the end of growth phase. Until now, research on reducing extracellular enzymatic activity has made great progress, but the studies about intracellular enzymes rarely involved.In the first part of this study, we chosen lytic polysaccharide monooxygenases gene CBP21 and N-Acyl-homoserine lactonase gene AiiO-AIO6 as reporter genes and cloned them into the vector pWB980. Then we cloned the signal peptides into the screening vector by new molecular cloning techniques such as seamless cloning, and finally we got the signal peptide screening vectors. The plasmid was transformed into B. subtilis 168 and we obtained a series of recombinant strains. The reporter proteins of the culture supernatant were detected after 24 h incubation. The results revealed that the recombinant strain containing YlqB signal peptide showed the highest secretion level of CBP21, but it also showed that AiiO-AIO6 does not depend on the guidance of B. subtilis signal peptide for it is capable of secreting into the culture medium by itself.In the second part of this study, two serine hydrolase gene YlbL and AprX, one cysteine hydrolase gene YwpE, one metalloenzyme AmpS gene were chosen to be knocked out. The mutants were obtained by inserting a resistant gene into the region between the upstream and the downstream of the target gene. Then we introduced the recombinant plasmids that each contained Lytic polysaccharide monooxygenases gene and N-Acyl-homoserine lactonase gene into the mutants. The reporter proteins of the culture supernatant were detected after 24 h incubation. The results revealed that compared with the wild type strain, the secretion level of CBP21 and AiiO-AIO6 were improved in the mutants △AprX,△YwpE and△AmpS, but as for △YlbL, CBP21 was not detected in the supernatant but the secretion level of AiiO-AIO6 was also improved in this mutant.Preliminary research on expression of heterologous proteins by B. subtilis revealed that the secretion mechanisms of different heterologous proteins are quite different, such as CBP21 and AiiO-AIO6. The secretion level of CBP21 can be improved by screening signal peptide, but AiiO-AIO6 can’t. And the knockout of intracellular proteases both improved the production of CBP21 and AiiO-AIO6.