High-level Expression Of Bacillus Stearothermophilus ?/?-cyclodextrin Glucosyltransferase In Bacillus Subtilis

Cyclodextrin glucotransferase is a multifunctional enzyme,widely used in AA-2G and other high value-added derivatives.The Bacillus stearothermophilus NO2?/?-CGTase with its excellent transglycosylation and high specificity,widely used in industrial production.Bacillus subtilis is a gram positive baculovirus,the host strain B.subtilis WSH11 capable of highly expressing foreign proteins was obtained through natural screening and genome orientation.In this study,B.stearothermophilus NO2?/?-CGTase was expressed in B.subtilis WSH11.Improvement of soluble expression of?/?-CGTase by screening for promoters,signal peptides and protein modification.Then investigate the enzymatic properties of?/?-CGTase wild-type and mutants.Targeted transformation of the expression host B.subtilis WSH11.The main results are demonstrated as follows:?1?Plasmid pHY300PLK was used to construct the expression plasmid of?/?-CGTase in B.subtilis expression system.In order to increase the expression of?/?-CGTase,9 single promoters were investigated,and fermentation showed that the optimal single promoter was P_amyQ'with?/?-CGTase enzyme activities 6.52 U·mL^-1,followed by promoters P_{hpa II} and P_nprE,and the enzyme activity corresponding to 5.80 U·m L^-1 and 5.73 U·m L^-1,respectively.Double promoter P_amyQ'-P_amyQ',P_hpaII-P_amyQ'and P_nprE-P_amyQ'were constructed and fermentation indicated that the best double promoter was P_hpaII-P_amyQ',and the expression of?/?-CGTase was 11.15 U·mL^-1,which was 1.71 times higher than that of single promoter P_amyQ'.Determination the?/?-CGTase mRNA transcription level of promoters P_{hpa II}-P_amyQ',PamyQ'-PamyQ',PamyQ'-PamyQ',PamyQ',P_gsiB,P_hpaII,the results showed that the enzyme activity increased with the increase of mRNA transcription.The highest?/?-CGTase activity of the fermentation supernatant was reached at 110.41 U·m L^-1 in 3-L bioreactor fermentation.?2?Useing commercial easily transformed and screened host strains B.subtilis RIK1285constructed recombinant?/?-CGTase,which containing B.subtilis Secretory Protein Expression System signal peptide library.Through a high-throughput screening technique,strains with high expression levels of?/?-CGTase were screened and their signal peptides were sequenced to obtain sp1 and sp2.sp1 was the signal peptide of extracellular endo-alpha-?1->5?-L-arabinanase 1 precursor and sp2 was the signal peptide of a hypothetical protein.Construction of recombinant strains CGTd2sp1 and CGTd2sp2 containing signal peptides sp1 and sp2 using B.subtilis WSH11 as a host.Fermentation results showed that the optimal signal peptide was sp1,?/?-CGTase extracellular enzyme activity was 16.73 U·m L^-1,1.50 times of the control signal peptide sp_amy.The recombinant CGTd2sp1 was used for 3-L bioreactor fermentation,the enzyme activity reached 151.93 U·m L^-1.?3??/?-CGTase was easy to from intracellular precipitate during the fermentation,but Bacillus circulans?-CGTase can be highly expressed in B.subtilis.Comparing the amino acid sequences of?/?-CGTase and?-CGTase.The unfolded amino acid content of?-CGTase N-terminus?56.3%?was significantly higher than that of?/?-CGTase?28.5%?,which conforming to the proportion of unfolded amino acids in the N-terminus of extracellular proteins was higher than that of cytoplasmic proteins.In order to examine whether the N-terminus affects the transport of?/?-CGTase,replacing the first 12 amino acids of the N-terminus of?/?-CGTase with the first 15 amino acids of the N-terminus of?-CGTase to obtain the recombinant gene?N-?/?-cgt,and the recombinant strain CGTd2sp1?N was constructed.The intracellular precipitate of?/?-CGTase in CGTd2sp1?N was significantly reduced compared with CGTd2sp1 by SDS-PAGE,indicating that the higher N-terminal unfolded amino acid contributed to?/?-CGTase transport.When the recombinant strain CGTd2sp1?N was used for 3-L bioreactor fermentation,the?/?-CGTase activity of the fermentation supernatant was the highest,reaching 249.35 U·m L^-1,which was 39 times than that of the shake flask fermentation of original strain.The wild-type?/?-CGTase and the mutant?N-?/?-CGTase were purified and their specific activities were 175.9 U·mg^-1 and185.3 U·mg^-1,respectively,indicating that the replacement of the N-terminus did not change?/?-CGTase enzyme activity,and the enzymatic properties of wild-type and mutants were similar.?4?Due to secretion of lipopeptide biosurfactants during the growth of the host strain B.subtilis WSH11,a large amount of foam accumulated in the 3-L bioreactor during fermentation.In order to reduce the foam yield of the host bacteria,CRISPR/Cas9 gene editing technology was used to knock out the lipopeptide biosurfactant-related genes ppsE and sfp from the B.subtilis WSH11 genome,resulting in the mutant strain B.subtilis WSH13.It was verified that during the fermentation of 3-L bioreactor,WSH11 and WSH13 were added defoamer thirteen times and three times,with a total amount of 0.565 mL and 0.167mL respectively,indicating that knocking out the gene ppsE and sfp reduced foam production was significantly reduced during fermentation.When WSH11 and WSH13 were used as the host bacteria,the?/?-CGTase activity was 249.3 U·mL^-1 and 255.9 U·mL^-1,respectively,and the OD₆₀₀ was 116 and 113 respectively.The knockout gene ppsE and sfp had no obvious effect on the enzyme production and growth of the host strain,and B.subtilis WSH13 can be used for fermentation.