Method for constructing trehalose multi-enzyme complex in vitro mediated by artificial scaffold protein

A method for constructing a recombinant strain for expressing self-assembled tri-enzyme complex, wherein the method comprises the following steps of:

• Step 1:
• constructing recombinant bacteria WB800n-ScafCCR for self-assembled scaffold protein module, comprising:
• (1) designing primers and amplifying a full-length sequence of P43 promoter and a full-length sequence of phoD signal peptide through PCR, using the genome of Bacillus subtilis WB800n as a template;
• (2) designing primers and amplifying a full-length sequence of Rfcoh-Ctcoh-CBM-Cccoh through PCR, using a ScafCCR gene sequence in a bacterial culture of biosynthetic ScafCCR as a template, and the gene fragment Rfcoh-Ctcoh-CBM-Cccoh is designated as the gene fragment of scaffold protein ScafCCR;
• wherein the gene sequence of ScafCCR in the bacterial culture of ScafCCR is formed by ligating cohesins Rfcoh, Ctcoh and Cccoh, as well as a gene sequence of cellulose binding domain (CBD) to plasmid PUC57;
• (3) double digesting yeast episomal plasmid pHT01 with restriction endonucleases-ScaI and BamHI;
• (4) measuring concentrations of the gene fragment of P43 promoter, the gene fragment of phoD signal peptide obtained in step (1) and the gene fragment of scaffold protein ScafCCR in step (2) and the digested pHT01 plasmid in step (3), then ligating these gene fragments by using a multi-fragment seamless cloning technology, and transforming the ligated fragments into E. coli DH5a competent cells, and verifying for successful transformation to obtain a plasmid pHT01-P43-phoD-ScafCCR, wherein the obtained recombinant plasmid is designated as pHT01-ScafCCR;
• (5) transforming the recombinant plasmid pHT01-ScafCCR into bacterial cells of Bacillus subtilis WB800n to obtain a recombinant bacterium Bacillus subtilis WB800n which is designated as WB800n-ScafCCR;
• Step 2:
• constructing recombinant bacteria WB800n-P43-phoD-trey-Ccdoc for self-assembled catalytic module comprising steps of:
• a) designing primers for amplifying the gene fragment of P43 promoter and the gene fragment of phoD signal peptide through PCR, using the genome of Bacillus subtilis WB800n as a template;
• b) amplifying a full-length sequence of treY of malto-oligosaccharyl trehalose synthase (MTSase) through PCR, using the genome of Sulfolobus acidocaldarius having a accession number of ATCC 33909 as a template;
• c) designing primers and amplifying a gene sequence of dockerin Ccdoc through PCR, using the sequence of Ccdoc in a biosynthetic Ccdoc bacterial culture as a template;
• wherein in the Ccdoc bacterial culture, the gene sequence of dockerin Ccdoc is ligated to the plasmid PUC57;
• d) double digesting the yeast episomal plasmid pHT01 with restriction endonucleases ScaI and BamHI;
• e) measuring concentrations of the gene fragment of P43 promoter, the gene fragment of phoD signal peptide obtained in step a), the gene fragment treY of malto-oligosaccharyl trehalose synthase (MTSase) obtained in step b), the gene fragment of dockerin Ccdoc in step c) and the digested pHT01 plasmid in step d), then ligating these fragments by using a multi-fragment seamless cloning technology and transforming the resulting ligated fragments into E. coli DH5a competent cells, then verifying for successful transformation of the fragments to obtain a plasmid pHT01-P43-phoD-treY-Ccdoc;
• wherein the nucleotide sequence of the gene fragment of P43-phoD-treY-Ccdoc is set forth in SEQ ID NO:
• 2;
• f) transforming the recombinant plasmid pHT01-P43-phoD-treY-Ccdoc obtained in step e) into the genome of the bacterial cells of Bacillus subtilis WB800n to obtain a recombinant bacterium Bacillus subtilis WB800n which is designated as WB800n-P43-phoD-treY-Ccdoc;
• Step 3:
• constructing recombinant bacteria WB800n-P43-phoD-treZ-Ctdoc for self-assembled catalytic module, comprising steps of:
• I) designing primers and amplifying the gene fragment of P43 promoter through PCR, using the genome of Bacillus subtilis WB800n as a template,
• II) designing primers and amplifying full-length sequence treZ of phoD-malto-oligosaccharyl trehalose hydrolase (MTHase) through PCR to obtain a gene fragment phoD-treZ, using the genome of E. coli strain P43-phoD-MTHase constructed according to conventional techniques as a template;
• III) designing primers and amplifying a gene sequence of dockerin Ctdoc through PCR, using the sequence of a Ctdoc in a biosynthetic Ctdoc bacterial culture as a template;
• wherein in the bacterial culture of Ctdoc, the gene sequence of dockerin Ctdoc is ligated to the plasmid PUC57;
• IV) double digesting the yeast episomal plasmid pHT01 with restriction endonucleases ScaI and BamHI;
• V) measuring concentrations of the gene fragment of P43 promoter obtained in step I), the gene fragment of phoD-treZ obtained in step II), the gene fragment of dockerin Ctdoc in step III) and the digested pHT01 plasmid in step IV), then ligating these fragments by using a multi-fragment seamless cloning technology and transforming the ligated fragments into E. coli DH5a competent cells, and verifying for successful transformation of the fragments to obtain a plasmid pHT01-P43-phoD-treZ-Ctdoc;
• wherein the nucleotide sequence of the gene fragment P43-phoD-treZ-Ctdoc is set forth in SEQ ID NO:
• 3;
• VI) transforming the recombinant plasmid pHT01-P43-phoD-treZ-Ctdoc obtained in step V) into bacterial cells of Bacillus subtilis WB800n to obtain a recombinant bacterium Bacillus subtilis WB800n, and the recombinant bacterium is designated as WB800n-P43-phoD-treZ-Ctdoc;
• Step 4:
• constructing recombinant bacteria WB800n-P43-phoD-cgt-Rfdoc for self-assembled catalytic module, comprising:
• i) designing primers and amplifying the gene fragment cgt-Rfdoc of cyclodextrin glycosyltransferase (CGTase)-dockerin Rfdoc through PCR, using the sequence of biosynthetic CGTase-Rfdoc as a template;
• wherein CGTase-Rfdoc in the bacterial culture is formed by ligating gene sequences of the cyclodextrin glycosyltransferase (CGTase) and the dockerin Rfdoc to the plasmid PUC57;
• ii) designing primers and amplifying the gene fragment of pHT01-P43-phoD through reverse PCR, using the WB800n-P43-phoD-treZ-Ctdoc strain obtained in step 3) as a template,
• iii) measuring concentrations of the gene fragment cgt-Rfdoc obtained in step i) and the gene fragment pHT01-P43-phoD obtained in step ii), and ligating these fragments by using a single fragment seamless cloning technique and transforming the ligated fragments into E. coli DH5a competent cells, then verifying for successful transformation of the ligated fragments to obtain a plasmid pHT01-P43-phoD-cgt-Rfdoc;
• wherein the nucleotide sequence of the gene fragment P43-phoD-cgt-Rfdoc is set forth in SEQ ID NO:
• 4;
• iv) transforming the recombinant plasmid pHT01-P43-phoD-cgt-Rfdoc obtained in step iii) into bacterial cells of Bacillus subtilis WB800n to prepared a recombinant bacterium Bacillus subtilis WB800n, and the recombinant bacterium is designated as WB800n-P43-phoD-CGT-Rfdoc;
• secretorily expressing the recombinant bacterium WB800n-ScafCCR, WB800n-P43-phoD-treY-Ccdoc, WB800n-P43-phoD-treZ-Ctdoc, WB800n-P43-phoD-cgt-Rfdoc as constructed above, and performing self-assembling in vitro to obtain recombinant trehalose multi-enzyme complex;
• wherein in substep (2) of Step 1, the nucleotide sequence of the gene fragment of the scaffold protein ScafCCR is set forth in SEQ ID NO:
• 1.