Construction Of Homologous Co-expression System For Key Bio-degumming Enzyme Genes In Broad-spectrum And High-efficient Strain DCE-01

DCE-01 is a“broad spectrum”and“high efficient”degumming strain which can works well for degumming of a variety of herb fiber material,and can independently complete the degumming of ramie in 6 h without other chemically postprocessing.The broad-spectrum and high efficiency of the degumming characteristic is a leader at home and abroad.Taking the DCE-01 strain as the research object,cloned 3 key degumming enzyme genes from srtain DCE-01 and strung together by 2 different arrangements,and expressed in Escherichia coli and strain DCE-01.The results are as follows:1.The key degumming enzyme genes?pectinase gene,xylanase gene and mannanase gene?were cloned from the strain Dickeya sp.DCE-01 using the primers based on the whole genome sequencing annotation.The results of sequencing and bioinformatics analysis showed that the pelE was 1185 bp,encoded 395 amino acids,the predicted molecular weight of the protein was 43.8 kDa;the xyn was1251 bp,encoded 416 amino acids,the predicted molecular weight of the protein was 45.74 kDa;the man was 1137 bp,encoded 379 amino acids,the predicted molecular weight of the protein was41.85 kDa.2.According to the design of primers and PCR amplification,three degumming enzyme genes?pelE,xyn and man?with specific restriction enzyme sites is cloned,recombined with the expression vector pET-28a by restriction enzyme digestion,constructed single gene recombinant plasmids.The genes were tandem connected in the MCS region of the expression vector by PXM,XPM series to constructed multigene recombinant plasmids simultaneously.The recombinant plasmids were transformed into E.coli BL21 that successfully constructed single-gene recombinant strains pET-28a-P/BL21,pET-28a-M/BL21 and pET-28a-X/BL21 which expressed pectinase,mannanase and xylanase and multigene recombinant bacteria pET-28a-PXM/BL21 and pET-28a-XPM/BL21.3.The constructed recombinants was transformed into DCE-01?a key degumming enzyme gene-derived strain?,and 3 single-gene homologously expressed recombinant strains pET-28a-P/DCE-01,pET-28a-X/DCE-01 and pET-28a-M/DCE-01,and two multigene homology co-expressed recombinant strains pET-28a-PXM/DCE-01(or DCE-01^PXM)and pET-28a-XPM/DCE-01(or DCE-01^XPM)were successfully obtained.4.The results of the enzyme activity assay of the BL21 series of recombinant strains showed that:?1?The Pel E activity expressed by the pelE gene recombinant strain was 471.97 U/mL,while the corresponding PelE activity of pET-28a-PXM/BL21 and pET-28a-XPM/BL21 was 532.68 U/mL and121.43 U/mL when the pel E was ranked first and second after the promoter.?2?The Xyn enzyme activity expressed by the xyn gene recombinant strain was 44.32 U/mL,while the corresponding Xyn activity of pET-28a-XPM/BL21 and p ET-28a-PXM/BL21 was 47.43 U/mL and 31.42 U/mL when the xyn ranked in the first and second positions behind the promoter.?3?The Man activity expressed by the man gene recombinant strain reached 16882.86 U/mL,while the corresponding Man activity of pET-28a-PXM/BL21 and pET-28a-XPM/BL21 was 645.21 U/mL and 230.83 U/mL when the man was ranked far away from the promoter position.Therefore,it can be inferred that:?1?The promoter of the vector used to construct the multi-gene co-expression system has a significant promotion effect on the expression of pelE;?2?The expression effect of the genes and the distance between the insertion site to the promoter demonstrate a negative correlation in constructing multi-gene co-expression system.That is farther the distance,the worse the effect;?3?In the multi-gene coexpression system,the expression effect of man was significantly affected by the pelE insertion site.5.Taking the strains DCE-01 and DSM18020?model strains?as controls,the results of enzyme activity assays of multi-gene homologous co-expressing recombinant strains showed that:?1?The PelE enzyme activities of the recombinant strains DCE-01^PXM and DCE-01^XPM were 2.77 times,1.01 times of the strain DCE-01 and 10.91 times,4.11 times of the strain DSM18020,respectively;?2?The Xyn enzyme activities of the recombinant strains DCE-01^PXM and DCE-01^XPM were 1.28 times,1.82 times of the strain DCE-01 and 5.68 times,8.08 times of the strain DSM18020;?3?The Man enzyme activities of the recombinant strains DCE-01^PXM and DCE-01^XPM were 1.99 times,1.01 times of the strain DCE-01 and 6.23 times,3.16 times of the strain DSM18020.From this it can be concluded that the key degumming enzyme genes?pelE,xyn,man?from the strain DCE-01 were integrated into the expression vector?pET-28a?by the gene tandem method to form recombinants,whether introduced into the prokaryotic expression strain E.coli BL21?DE3?or the DCE-01 were successfully expressed,and the enzyme activity of PelE,Xyn,and Man in the multi-gene co-expressing recombinant strains was higher than that the strain DCE-01.6.The results of liquid chromatography analysis of the strains DCE-01,DSM18020,DCE-01^PXM,and DCE-01^XPM in the ramie degumming fermentation broth showed that the monosaccharide content detected in the fermentation broth of the strain DCE-01^PXM was significantly higher than other strains.The monosaccharide content of the strain DCE-01^PXM was slightly higher than the strain DCE-01,and the monosaccharide content of the strain DCE-01 was significantly higher than the strain DSM18020.This result is basically consistent with the results of the enzyme activity test which indicated that the way of multi-gene homology co-expression that key degumming enzyme gene of DCE-01 is tandemly arranged in the order of PXM and then introduced into the strain DCE-01 can somehow promote the degumming function of the strain DCE-01.