Construction Of A New Laccase Expression System

Laccase are polyphenol oxidases with widespread application in various industry,but its application is limited by the low yield of enzyme.A high yield laccase producing strain Cerrena sp.HYB07 was obtained from the laboratory,and the laccase gene Lac7 in the strain can be highly expressed.In order to achieve high expression of laccase,a new laccase expression system containing Lac7 promoter was constructed.The main results are as follows:1.The maximum protoplast yield was 1×108 per milliliter when mycelium cultured 48 h were hydrolyzed at 30? for 3 h with 2%lywallzyme with mannitol of 0.6 mol/L as the osmotic stabilizer,and regeneration rate reached 1‰ at 2 type regeneration medium when sucrose of 0.8 mol/L as the osmotic stabilizer.2.Two strains of monokaryon were screened from regenerated protoplasts,12-12-34 and 11-10-9 respectively.The highest enzyme activity of 12-12-34 was 355.26 U/mL,while the highest enzyme activity of 11-10-9 was only 11.60 U/mL.Analysis of laccase isozyme gene showed that two monokaryons all had 13 laccase isozyme genes.The expression levels of Lac7 and Cufl genes in two monokaryons were analyzed.It was found that the expression levels of Lac7 and Cufl genes in 12-12-34 were 54.684 fold and 0.49 fold higher than those in 11-10-9,In order to obtain a laccase expressing host that with low laccase background and high expression system,we constructed a Lac7 gene knockout vector pGKO2-UP2000+Hyg+PD1500,and knocked out Lac7 gene in monokaryons by homologous substitution.But because of the low transformation efficiency and limited time,the gene knockout mutant was not screened.3.A laccase expression vector driven by Lac7 promoter(PLac7)and 35S promoter(P35s)was constructed.Heterologous expression of Lac7 gene from Cerrena sp.HYB07 strain in Cerrena unicolor 5.1011 strain.Three positive transformants PC1,PC5 and PC7 transformed from recombinant vector pCAMBIA1302-Lac7(P-cDNA)were obtained,and the enzyme activity was 93.22 U/mL?98.15 U/mL and 100.59 U/mL respectively,which were 2.68 fold?2.82 fold and 2.89 fold compared with 5.1011 strain,at twenty-second days fermentation.The four positive transformants IT2,IT3,IT4 and IT5 transformed from recombinant vector pCAMBIA1302-Lac7(35S-I+T)were obtained.The enzyme activities of IT2,IT3,IT4 and IT5 at fourth day were 3.65 U/mL?2.84 U/mL?1.22 U/mL and 1.56 U/mL respectively,which were 17.09 fold?13.32 fold?5.70 fold and7.29 fold compared with 5.1011 strains.A new laccase filamentous fungus expression system containing Lac7 promoter was successfully established.It provides a theoretical basis for the high expression of laccase,and lays a foundation for genetic manipulation and molecular biology research.