Recombinant plasmid and genetically engineered bacterium as well as construction method and application of recombinant plasmid and genetically engineered bacterium

Abstract

The invention relates to the technical field of genetic engineering, in particular to a recombinant plasmid, a genetically engineered bacterium and a construction method and application of the genetically engineered bacterium, and genes knocked out by the recombinant plasmid are yvdF and mdxE genes; the recombinant plasmid is composed of a pJOE8999 vector, a 20bp guide sequence and a knockout box, the insertion position of the 20bp guide sequence is the insertion position of the exogenous guide sequence in the pJOE8999 vector; the nucleotide sequence of the knockout box is as shown in SEQ ID NO. 1; the nucleotide sequence of the 20bp guide sequence is as shown in SEQ ID NO.2. The genetically engineered bacterium containing the recombinant plasmid can avoid the situation that a host bacterium self maltodextrin transport and decomposition pathway competes with a substrate in a maltotetraglycoamylase culture medium and decomposes a product maltotetraose formed by a maltotetraglycoamylase catalytic substrate, and the maltotetraose yield is increased.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant plasmid and genetically engineered bacteria, their construction method, and their applications. Background Technology

[0002] Reported engineered bacteria producing maltodextrin amylase primarily rely on free plasmids for cloning and expressing exogenous genes. During fermentation, antibiotics are needed to maintain the plasmids. Furthermore, in industrial production using large fermenters, the free plasmids are gradually lost during fermentation due to their structural instability and separation instability, leading to reduced protein yield. Current methods, such as using engineered bacteria to first produce the enzyme and then using purified maltodextrin (Mta) crude or pure enzyme preparations for maltodextrin biotransformation, involve separate enzyme preparation and conversion, resulting in a more complex process, lower efficiency, and higher costs. Bacillus subtilis itself decomposes and consumes the substrate maltodextrin and the catalytic product maltodextrin, further reducing usable substrate levels and product yield.

[0003] In summary, there is an urgent need to develop a Bacillus subtilis strain that produces maltotetrasaccharide in a genome-integrated manner for efficient synthesis of maltotetrasaccharide. Summary of the Invention

[0004] This invention addresses the problem of how to provide a genetically engineered bacterium that efficiently synthesizes maltodextrose.

[0005] To achieve the above objectives, the first aspect of the present invention provides a recombinant plasmid, wherein the genes knocked out by the recombinant plasmid are the yvdF and mdxE genes;

[0006] The recombinant plasmid consists of the pJOE8999 vector, a 20bp guide sequence, and a knockout cassette.

[0007] The 20bp guide sequence is inserted at the location where the exogenous guide sequence is inserted into the pJOE8999 vector;

[0008] The nucleotide sequence of the knockout cassette is shown in SEQ ID NO.1;

[0009] The nucleotide sequence of the 20bp guide sequence is shown in SEQ ID NO.2.

[0010] A second aspect of the present invention provides a method for constructing the above-mentioned recombinant plasmid.

[0011] A third aspect of the present invention provides a genetically engineered bacterium comprising the above-mentioned recombinant plasmid, wherein the genetically engineered bacterium is a strain that has knocked out the yvdF and mdxE genes in the operon mdxR-yvdF-mdxE-mdxF-mdxG-yvdJ-yvdK-malL-pgcM containing maltodextrin metabolism.

[0012] The fourth aspect of this invention provides a method for constructing the above-mentioned genetically engineered bacteria.

[0013] The fifth aspect of the present invention provides the application of the above-mentioned recombinant plasmid, the method for constructing the above-mentioned recombinant plasmid, the above-mentioned genetically engineered bacteria, or the method for constructing the above-mentioned genetically engineered bacteria in the production of maltodextrin.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention targets the maltose amylase gene yvdF and the maltodextrin-binding protein gene mdxE in the genome of engineered bacteria, which are capable of decomposing maltodextrin oligosaccharides. Guide sequences and homology repair templates were designed to construct a gene-editing plasmid vector for gene knockout. The yvdF-mdxE gene in the genome of the engineered bacteria was knocked out using CRISPR / Cas9 technology. The knocked-out engineered bacteria were then cultured in media containing high concentrations of maltodextrin at concentrations of 80 g / L, 120 g / L, and 200 g / L. The engineered bacteria secreted an extracellular Mta enzyme to directly biotransform the maltodextrin in the culture medium to synthesize maltodextrin. The results showed that the engineered strain RIKmta7ΔamyEΔym could efficiently synthesize maltodextrin in maltodextrin media at concentrations of 80 g / L, 120 g / L, and 200 g / L, with the highest yields being 64.32%, 66.25%, and 69.37%, respectively. These yields were higher than those of the control strain without knockout of genes related to the maltodextrin metabolic pathway, thus demonstrating promising prospects for industrial application. Attached Figure Description

[0016] Figure 1 This is an electrophoresis image of the gel recovery product of the PCR amplified fragment. In the image: (A) M: DNA marker; 1: linearized vector; (B) M: DNA marker; 1: left homologous arm used to knock out the yvdF-mdxE gene; 2: right homologous arm used to knock out the yvdF-mdxE gene.

[0017] Figure 2 Colony PCR validation was performed on E. coli HST08 cells transformed with a plasmid targeting the yvdF / mdxE gene knockout site. In the figure: M: DNA marker; 1-6: E. coli HST08 / pCas-yvdF / mdxE-donor.

[0018] Figure 3Colony PCR validation of the yvdF-mdxE gene knockout engineered bacterium RIKmta7ΔamyEΔym. In the figure: M: DNA marker; 1: RIKmta7ΔamyE; 2: RIKmta7ΔamyEΔym.

[0019] Figure 4 The DNA sequencing chromatogram of the yvdF-mdxE region of RIKmta7ΔamyEΔym is shown.

[0020] Figure 5 The time process for engineered bacteria to produce maltodextrin. In the figure: (A): maltodextrin concentration 80 g / L; (B): maltodextrin concentration 120 g / L; (C): maltodextrin concentration 200 g / L. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] The inventors discovered that the maltodextrin-specific ABC transport complex (composed of MdxE, MdeF, MdxG, and MsmX) is used to transport maltodextrin into cells. Among these, the maltodextrin-binding protein MdxE has a high affinity for maltodextrin oligosaccharides such as maltotetrasaccharide and maltopentose, but a low affinity for maltose, playing a crucial role in the transmembrane transport of maltodextrin. Once inside the cell, maltodextrin is further broken down into glucose and glucose-1-phosphate by maltose amylase (yvdF), maltose phosphorylase (yvdK), and α-glucosidase (Mal), and then further converted to glucose-6-phosphate, subsequently undergoing glycolysis. Furthermore, yvdF is also present between the cell wall and cell membrane of Bacillus subtilis, and can directly hydrolyze maltodextrin oligosaccharides, thereby accelerating substrate degradation and utilization.

[0023] Based on this, the inventors established a system for the direct biosynthesis of maltodextrin using engineered bacteria in a culture medium containing a high concentration of the substrate maltodextrin. By knocking out the yvdF / mdxE gene, the host bacteria's own maltodextrin transport and degradation pathways are prevented from competing with Mta for the substrate in the culture medium, as well as for the transport and degradation of the product maltodextrin, thereby increasing the yield of maltodextrin.

[0024] The first aspect of the present invention provides a recombinant plasmid, wherein the genes knocked out by the recombinant plasmid are the yvdF and mdxE genes;

[0025] The recombinant plasmid consists of the pJOE8999 vector, a 20bp guide sequence, and a knockout cassette.

[0026] The 20bp guide sequence is inserted at the location where the exogenous guide sequence is inserted into the pJOE8999 vector;

[0027] The nucleotide sequence of the knockout cassette is shown in SEQ ID NO.1;

[0028] The nucleotide sequence of the 20bp guide sequence is shown in SEQ ID NO.2.

[0029] A second aspect of the present invention provides a method for constructing the above-mentioned recombinant plasmid, wherein the steps of the construction method include:

[0030] S1. The pJOE8999 linearized vector with the guide sequence targeting the yvdF / mdxE site, the left homologous arm upstream and downstream of the yvdF / mdxE gene, and the right homologous arm were amplified separately and recovered by gel extraction. The pJOE8999 linearized vector, the left homologous arm, and the right homologous arm each have a 20bp overlapping region at their ends. They were then ligated by a seamless cloning reaction to obtain the ligation reaction product.

[0031] S2. Using the B. subtilis RIK1285 genome as a template, the left and right homologous arms were amplified using primers P38 / P39 and P40 / P41, respectively. The PCR reaction conditions included: 50 ng of genomic DNA, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 10 μL of 5×PrimeSTAR Buffer, and 4 μL of dNTPs. The amount of HSDNA Polymerase added was 1 μL. Sterile water was added to make the reaction system 50 μL. The reaction was carried out at 95°C for 300 s, followed by 94°C for 30 s, 57°C for 45 s, and 72°C for 90 s as one cycle. The cycle was repeated 30 times, and then the reaction was carried out at 72°C for 600 s.

[0032] In S2, the upstream primer is one of P38 and P40, and the downstream primer is one of P39 and P41;

[0033] The nucleotide sequence of the gene encoding primer P38 is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding primer P39 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding primer P40 is shown in SEQ ID NO.5, and the nucleotide sequence of the gene encoding primer P41 is shown in SEQ ID NO.6.

[0034] S3. The PCR amplification products were recovered from the gel and then used... Ligation was performed using the SnapAssembly MasterMix kit, with the following conditions: 100 ng of linearized plasmid vector pCas-yvdF / mdxE added; the amount of the left homologous arm added was twice the amount of the linearized pJOE8999 vector added; and the amount of the right homologous arm added was twice the amount of the linearized pJOE8999 vector added. The amount of Snap Assembly Master Mix added was 2 μL, and sterile water was added to bring the reaction system to 10 μL. The reaction conditions were: incubation at 50°C for 30 min, followed by incubation in an ice-water mixture for 15 min. Then, the cells were transferred into E. coli HST08 competent cells, plated on LB plates containing 30 μg / mL Kan, and cultured at 30°C for 18 h. Single colonies were picked and activated for culture. The activation culture conditions included: culture at 30°C for 14 h. Extraction was performed to obtain the recombinant plasmid pCas-yvdF / mdxE-donor.

[0035] A third aspect of the present invention provides a genetically engineered bacterium comprising the above-mentioned recombinant plasmid, wherein the genetically engineered bacterium is a strain that has knocked out the yvdF and mdxE genes in the operon mdxR-yvdF-mdxE-mdxF-mdxG-yvdJ-yvdK-malL-pgcM containing maltodextrin metabolism.

[0036] According to the present invention, the genetically engineered bacterium is Bacillus subtilis, and the genetically engineered bacterium contains a recombinant vector, the recombinant vector is knocked into an expression cassette, and the intergenic regions knocked into the expression cassette are the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA and sigF sites;

[0037] The expression cassette contains a codon-optimized maltotetrasaccharide amylase gene;

[0038] The nucleotide sequence encoding the maltodextrose amylase gene is shown in SEQ ID NO. 9.

[0039] The nucleotide sequence of the gene encoding the expression cassette is shown in SEQ ID NO.10.

[0040] A fourth aspect of the present invention provides a method for constructing the above-mentioned genetically engineered bacteria, wherein the construction method includes the following steps:

[0041] S1. The engineered strain RIKmta7ΔamyE containing the recombinant vector with the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA and sigF site knock-in expression cassette was prepared into competent cells.

[0042] S2. Take out RIKmta7ΔamyE competent cells, add 1μg of recombinant plasmid and culture at 37℃ and 90rpm for 1.5h. Spread the bacterial culture on LB plates containing 10μg / mL Kan and incubate upside down at 30℃ for 18h.

[0043] S3. Pick a single colony and inoculate it into LB liquid medium containing 10 μg / mL Kan. Incubate at 30℃ and 200 rpm until the OD600 reaches 0.4-0.6. Add D-mannose to a final concentration of 0.2% (w / v). Gene editing is performed by inducing Cas9 nuclease expression through mannose. After culturing for another 12 h, dilute the bacterial solution and spread it on LB plates containing 10 μg / mL Kan and 0.2% (w / v) D-mannose. Incubate at 30℃ for 18 h.

[0044] S4. Pick a single colony and inoculate it into antibiotic-free LB liquid medium. Incubate at 50℃ and 200rpm for 12h. Continue to streak on antibiotic-free LB plates and incubate at 42℃ until a single colony appears, thus obtaining the genetically engineered bacterium RIKmta7ΔamyEΔym.

[0045] Single colonies were picked and inoculated into LB liquid medium containing Kan and LB liquid medium without antibiotics, respectively, to test the susceptibility of the strains to Kan.

[0046] Colony PCR identification was performed using primers P42 / P43. The reaction conditions included: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of template, and Mg... 2+ The following reaction mixture was prepared: 2 μL of 10×TaqBuffer, 1.6 μL of dNTPs, 0.2 μL of Taq DNA Polymerase, and 20 μL of sterile water. The mixture was kept at 95°C for 300 s, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C at 1000 bp / min. After 30 cycles, the mixture was kept at 72°C for 600 s.

[0047] The upstream primer was P42, and the downstream primer was P43; the template was strain RIKmta7ΔamyEΔym.

[0048] The nucleotide sequence of the gene encoding primer P42 is shown in SEQ ID NO.7, and the nucleotide sequence of the gene encoding primer P43 is shown in SEQ ID NO.8;

[0049] The genome was then extracted, and the extraction steps included:

[0050] The genome of Bacillus subtilis was extracted using a rapid bacterial genomic DNA extraction kit. Bacillus subtilis was inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 h.

[0051] Take 2 mL of the cultured bacterial solution and centrifuge at 8000 rpm for 5 min to collect the bacterial cell precipitate;

[0052] Resuspend the bacterial cells in 200 μL of 40 mg / mL lysozyme solution, then incubate in a 37°C water bath for 1 h, inverting the container every 10 min.

[0053] Add 400 μL of Buffer Digestion, and then incubate in a 65°C water bath for 1 hour, inverting the container every 10 minutes.

[0054] After removing the sample and cooling it to room temperature, add 20 μL of RNase A (10 mg / mL) and let it stand at room temperature for 5 min.

[0055] Add 200 μL of Buffer PB and let stand at -20℃ for 5 min;

[0056] Centrifuge the sample at 10,000 rpm for 5 min, and transfer the supernatant into a sterile 1.5 mL centrifuge tube;

[0057] Add an equal volume of isopropanol and mix 6 times. Let stand at room temperature for 3 minutes, then centrifuge at 10,000 rpm for 5 minutes and discard the supernatant.

[0058] Add 1 mL of 75% ethanol to rinse the sample, then centrifuge at 10000 rpm for 2 min, discard the supernatant, and repeat this step once;

[0059] Open the centrifuge tube cap and allow the residual ethanol to evaporate at room temperature. Then add 50 μL of TE Buffer to dissolve the precipitate and obtain genomic DNA. The concentration of the extracted genomic DNA sample was determined using a NanoVue plus ultraviolet spectrophotometer.

[0060] After amplifying the sequence of the knock-in region, gene sequencing was performed. The strain with the correct sequencing was identified as the genetically engineered bacterium RIKmta7ΔamyEΔym.

[0061] According to the present invention, the method for preparing the engineered bacterium RIKmta7ΔamyE competent cells includes:

[0062] a1. Take out the engineered bacteria RIKmta7ΔamyE stored at -80℃, streak it on LB agar plates, and then incubate it upside down in a 37℃ incubator for 12 hours.

[0063] a2. Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0064] a3. Take 50 μL of bacterial culture and transfer it into 5 mL of SPI medium. Incubate at 37℃ and 150 rpm for 5 h.

[0065] a4. Transfer 500 μL of culture into 5 mL of SPⅡ medium and continue to incubate at 37℃ and 150 rpm for 1.5 h.

[0066] a5. Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain RIKmta7ΔamyE competent cells;

[0067] a6. Add 1 mL of 60% glycerol and freeze at -80°C.

[0068] In this invention, the method for constructing the engineered bacterium RIKmta7ΔamyE includes the following steps:

[0069] Construction of recombinant vectors

[0070] First, guide sequences targeting different sites are inserted into the pJOE8999 vector:

[0071] Step 1: Phosphorylation and annealing reactions were performed on primer pairs P1 / P2 (ydeI / ydeJ intergenic region), P3 / P4 (yyaP / tetB intergenic region), P5 / P6 (ganA site), and P7 / P8 (sigF site), respectively. The phosphorylation and annealing conditions included: 1 μL of 100 μM upstream primer, 1 μL of 100 μM downstream primer, 1 μL of 10×T4 DNAligase reaction buffer, 1 μL of T4PNK, and 6 μL of sterile water. The mixture was incubated at 37°C for 30 min, then at 95°C for 5 min, followed by cooling to 25°C at a rate of 0.1°C / s to obtain double-stranded oligonucleotides with sticky ends.

[0072] The nucleotide sequence of the gene encoding primer P1 is shown in SEQ ID NO.11, the nucleotide sequence of the gene encoding primer P2 is shown in SEQ ID NO.12, the nucleotide sequence of the gene encoding primer P3 is shown in SEQ ID NO.13, the nucleotide sequence of the gene encoding primer P4 is shown in SEQ ID NO.14, the nucleotide sequence of the gene encoding primer P5 is shown in SEQ ID NO.15, the nucleotide sequence of the gene encoding primer P6 is shown in SEQ ID NO.16, the nucleotide sequence of the gene encoding primer P7 is shown in SEQ ID NO.17, and the nucleotide sequence of the gene encoding primer P8 is shown in SEQ ID NO.18.

[0073] In S1, the upstream primer of the guide sequence is one of P1, P3, P5 and P7, and the downstream primer of the guide sequence is one of P2, P4, P6 and P8;

[0074] Step 2: Digest the pJOE8999 vector using the restriction enzyme BsaI. The digestion conditions include: 1 μg of pJOE8999 vector, 1 μL of BsaI-HFv2, and 10X rCutSmart. TM The amount of buffer added was 5 μL, and sterile water was added to make the reaction system 50 μL. The mixture was incubated at 37 °C for 10 min, and then inactivated at 80 °C for 20 min after incubation to obtain the linearized pJOE8999 vector.

[0075] Step 3: Mix the double-stranded oligonucleotide with sticky ends with the linearized pJOE8999 vector and ligate using T4 DNA ligase. The reaction conditions are as follows: 100 ng of linearized pJOE8999 vector, 2 μL of double-stranded oligonucleotide, 1 μL of T4 DNA Ligase, 1 μL of 10×T4 DNA Ligase Buffer, and add sterile water to make the reaction volume 10 μL. Incubate at 16°C for 2 h to obtain the ligation product.

[0076] Step 4: Transform the ligation product into E. coli HST08 competent cells, spread them on LB plates containing 30 μg / mL Kan, and incubate at 30°C for 18 h.

[0077] Step 5: Pick a single colony and inoculate it into LB liquid medium containing 30 μg / mL Kan. Incubate at 30℃ and 200 rpm for 18 h, then extract plasmids for gene sequencing. The correctly sequenced plasmids were named pCas-ydeI / ydeJ, pCas-yyaP / tetB, pCas-ganA, and pCas-sigF, respectively.

[0078] We continued by amplifying and recovering a series of DNA fragments with 20bp overlap regions via PCR, and then ligating them using seamless cloning technology:

[0079] Step 1: Using the B. subtilis RIK1285 genome as a template, amplify the left and right homologous arms of the ydeI / ydeJ intergenic locus using primers P9 / P10 and P11 / P12, respectively; amplify the left and right homologous arms of the yyaP / tetB intergenic locus using primers P15 / P16 and P17 / P18, respectively; amplify the left and right homologous arms of the ganA locus using primers P21 / P22 and P23 / P24, respectively; and amplify the left and right homologous arms of the sigF locus using primers P26 / P27 and P28 / P29, respectively. The PCR reaction conditions for amplification include: 50 ng of genomic DNA, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 10 μL of 5×PrimeSTAR Buffer, and 4 μL of dNTPs. The amount of HSDNAPolymerase added was 1 μL. Sterile water was added to make the reaction system 50 μL. The reaction was carried out at 95°C for 300 s, followed by 94°C for 30 s, 57°C for 45 s, and 72°C for 90 s as one cycle. The cycle was repeated 30 times, and then the reaction was carried out at 72°C for 600 s.

[0080] The upstream primer is one of P9, P11, P15, P17, P21, P23, P26 and P28, and the downstream primer is one of P10, P12, P16, P18, P22, P24, P27 and P29;

[0081] The nucleotide sequences encoding the gene of primer P9 are shown in SEQ ID NO. 19, primer P10 in SEQ ID NO. 20, primer P11 in SEQ ID NO. 21, primer P12 in SEQ ID NO. 22, primer P15 in SEQ ID NO. 23, primer P16 in SEQ ID NO. 24, primer P17 in SEQ ID NO. 25, primer P18 in SEQ ID NO. 26, primer P21 in SEQ ID NO. 27, primer P22 in SEQ ID NO. 28, primer P23 in SEQ ID NO. 29, primer P24 in SEQ ID NO. 30, and primer P26 in SEQ ID NO. 29. As shown in NO.31, the nucleotide sequence of the gene encoding primer P27 is shown in SEQ ID NO.32, the nucleotide sequence of the gene encoding primer P28 is shown in SEQ ID NO.33, and the nucleotide sequence of the gene encoding primer P29 is shown in SEQ ID NO.34.

[0082] Step 2: Amplify the expression cassette using primers P32 / P33. The PCR reaction conditions for amplification include: 50 ng of expression cassette, 2 μL of 10 μM primer P32, 2 μL of 10 μM primer P33, 10 μL of 5×PrimeSTAR Buffer, and 4 μL of dNTPs. The amount of HSDNAPolymerase added was 1 μL. Sterile water was added to make the reaction system 50 μL. The reaction was carried out at 95°C for 300 s, followed by 94°C for 30 s, 57°C for 45 s, and 72°C for 130 s as one cycle. The cycle was repeated 30 times, and then the reaction was carried out at 72°C for 600 s.

[0083] The nucleotide sequence of the gene encoding primer P32 is shown in SEQ ID NO.35, and the nucleotide sequence of the gene encoding primer P33 is shown in SEQ ID NO.36.

[0084] Step 3: Using recombinant vectors pCas-ydeI / ydeJ, pCas-yyaP / tetB, pCas-ganA, and pCas-sigF as templates, PCR amplification was performed using primers P34 / P35 to obtain linearized vectors of pJOE8999 carrying guide sequences targeting different sites. The PCR reaction conditions included: 20 ng of the recombinant vector, 2 μL of 10 μM primer P34, 2 μL of 10 μM primer P35, 10 μL of 5×PrimeSTAR Buffer, and 4 μL of dNTPs. The amount of HSDNAPolymerase added was 1 μL. Sterile water was added to make the reaction system 50 μL. The reaction was carried out at 95°C for 300 s, followed by 30 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 470 s. After that, the reaction was carried out at 72°C for 600 s.

[0085] The nucleotide sequence of the gene encoding primer P34 is shown in SEQ ID NO.37, and the nucleotide sequence of the gene encoding primer P35 is shown in SEQ ID NO.38.

[0086] Step 4: The linearized vector, expression cassette, left homologous arm, and right homologous arm PCR amplification products of pJOE8999 targeting different site guide sequences were recovered by gel extraction and used... The Snap Assembly Master Mix kit was used for ligation to obtain the ligation reaction product. The ligation conditions included: 100 ng of the linearized pJOE8999 vector, twice the amount of the expression cassette fragment as the linearized pJOE8999 vector, twice the amount of the left homologous arm as the linearized pJOE8999 vector, and twice the amount of the right homologous arm as the linearized pJOE8999 vector. Add 2 μL of Snap Assembly Master Mix, add sterile water to make the reaction system 10 μL, incubate at 50°C for 30 min, then in an ice bath for 15 min;

[0087] Step 5: Transform the ligation reaction product into E. coli HST08 competent cells, then plate them on LB plates containing 30 μg / mL Kan, and incubate at 30°C for 18 h. Pick single colonies for colony PCR identification. Use primers P9 / P12 to identify the ydeI-ydeJ intergenic region, primers P15 / P18 to identify the yyaP-tetB intergenic region, primers P21 / P24 to identify the ganA site, and primers P26 / P29 to identify the sigF site. Incubate the colony PCR-identified strains at 30°C for 14 h to extract the recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta, and pCas-sigF-mta.

[0088] In S10, the reaction conditions for colony PCR include: adding 2 μL of 10 μM upstream primer, adding 2 μL of 10 μM downstream primer, adding 1 μL of template, and adding Mg... 2+ The following steps were performed: Add 2 μL of 10×Taq Buffer, 1.6 μL of dNTPs, and 0.2 μL of Taq DNA Polymerase. Add sterile water to bring the reaction volume to 20 μL. Hold at 95°C for 300 s, then repeat the cycle of 95°C for 30 s, 58°C for 30 s, and 72°C at 1000 bp / min for 30 cycles. After each cycle, hold at 72°C for 600 s.

[0089] The upstream primer is one of P9, P15, P21 and P26, and the downstream primer is one of P12, P18, P24 and P29; the template is the bacterial culture obtained by culturing the correctly identified strain in LB liquid medium containing 30 μg / mL Kan at 30°C for 18 h.

[0090] The bacterial strains that were correctly identified by colony PCR were cultured, and then plasmids were extracted for gene sequencing. The correctly sequenced plasmids were named pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta, and pCas-sigF-mta.

[0091] Construction and validation of plasmid vectors targeting gene editing at different sites

[0092] A recombinant vector targeting the intergenic locus of ydeI / ydeJ was constructed. Using pJOE8999 plasmid as the starting vector, a guide sequence was inserted to obtain plasmid pCas-ydeI / ydeJ. Subsequently, the following fragments were obtained by PCR amplification: the pJOE8999 linearized plasmid vector with the guide sequence inserted at the ydeI / ydeJ locus, the left homologous arm, the right homologous arm, and the mta expression cassette. All fragments had a 20bp overlap region at the ends for subsequent seamless cloning ligation. The fragments were then recovered by gel cloning. The four recovered DNA fragments were ligated using seamless cloning technology, then transformed into E. coli HST08, plated, and identified by colony PCR. The recombinant vector was extracted and sequenced. The correctly identified recombinant vector was named pCas-ydeI / ydeJ-mta.

[0093] A recombinant vector targeting the intergenic region of the yyaP / tetB gene was constructed. First, the guide sequence was inserted into the pJOE8999 vector to obtain the plasmid pCas-yyaP / tetB. The following fragments were obtained by PCR amplification: the pJOE8999 linearized plasmid vector containing the guide sequence at the yyaP / tetB site, the left homologous arm, and the right homologous arm. These fragments were then recovered via gel electrophoresis. The three DNA fragments and the recovered mta expression cassette fragment were ligated using seamless cloning technology, then transformed into E. coli HST08 competent cells and identified. The recombinant vector was then extracted and sequenced. The correctly sequenced recombinant vector was named pCas-yyaP / tetB-mta.

[0094] A recombinant vector targeting the ganA site in the genome was constructed. First, the guide sequence was inserted into the pJOE8999 vector to obtain the plasmid pCas-ganA. The left and right homologous arms of the guide sequence inserted at the ganA site were obtained by PCR amplification, followed by gel recovery. The DNA fragment and the recovered mta expression cassette were ligated using an In-Fusion reaction, then transformed into E. coli HST08, and single colonies were identified by colony PCR. The constructed recombinant vector was then sequenced, and the correctly identified recombinant vector was named pCas-ganA-mta.

[0095] A recombinant vector targeting the sigF site was constructed. First, a guide sequence for binding to the genomic sigF site was inserted into the pJOE8999 vector to obtain the plasmid pCas-sigF. The left and right homologous arms were obtained by PCR amplification, and the fragments were recovered via gel electrophoresis. The DNA fragments and the mta expression cassette fragment were ligated using In-Fusion seamless cloning technology and then transformed into E. coli HST08. Finally, the recombinant vector was extracted and sequenced. The correctly identified recombinant vector was named pCas-sigF-mta.

[0096] Construction and validation of engineered bacteria with single-copy genome integration

[0097] Construction and validation of single-copy genome-integrated engineered bacteria RIKmta1ΔamyE and RIKmta2ΔamyE

[0098] Step 1: Transform the successfully constructed gene knock-in plasmids pCas-ydeI / ydeJ-mta and pCas-yyaP / tetB-mta into B. subtilis RIK1285 competent cells, respectively. The transformation method is as follows:

[0099] Bacillus subtilis stored at -80℃ was streaked on LB plates and then incubated upside down in a 37℃ incubator for 12 hours.

[0100] Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0101] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0102] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0103] Add 50 μL of EGTA solution and continue culturing at 37 °C and 90 rpm for 10 min to obtain B. subtilis RIK1285 competent cells;

[0104] Add 1 mL of 60% glycerol and store at -80°C.

[0105] Remove B. subtilis RIK1285 competent cells, add 1 μg of the recombinant vector, and culture at 37℃ and 90 rpm for 1.5 h;

[0106] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0107] Step 2: Select single colonies with good morphology and inoculate them into LB liquid medium containing 10 μg / mL Kan. Incubate at 30℃ and 200 rpm until the OD600 reaches 0.4-0.6. Add D-mannose to a final concentration of 0.2% (w / v) to induce Cas9 nuclease expression for gene editing. Continue culturing for 12 h and then spread the bacterial culture onto LB plates containing 10 μg / mL Kan and 0.2% (w / v) D-mannose. Incubate at 30℃ for 18 h.

[0108] Step 3: Pick a single colony and inoculate it into antibiotic-free LB liquid medium. Incubate at 50℃ and 200rpm for 12h. Continue to streak on antibiotic-free LB plates and incubate at 42℃ until single colonies appear. Pick a single colony and inoculate it into LB liquid medium containing Kan and LB liquid medium without antibiotics, respectively, to test the sensitivity of the strain to Kan.

[0109] Step 4: Colony PCR identification was performed using primers P13 / P14 and P19 / P20, respectively. The reaction conditions for colony PCR included: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of template, and Mg... 2+ The following reaction mixture was prepared: 2 μL of 10×Taq Buffer, 1.6 μL of dNTPs, and 0.2 μL of Taq DNA Polymerase. Sterile water was added to bring the reaction volume to 20 μL. The mixture was held at 95°C for 300 s, followed by a cycle of 95°C for 30 s, 58°C for 30 s, and 72°C at 1000 bp / min. This cycle was repeated 30 times, followed by a 600 s holding time at 72°C. The template was a bacterial culture obtained by culturing the strain in LB liquid medium containing 30 μg / mL Kan at 30°C for 18 h.

[0110] Genome extraction, specifically including:

[0111] The genome of Bacillus subtilis was extracted using a rapid bacterial genomic DNA extraction kit. Bacillus subtilis was inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 h.

[0112] Take 2 mL of the cultured bacterial solution and centrifuge at 8000 rpm for 5 min to collect the bacterial cell precipitate;

[0113] Resuspend the bacterial cells in 200 μL of 40 mg / mL lysozyme solution, then incubate in a 37°C water bath for 1 h, inverting the container every 10 min.

[0114] Add 400 μL of Buffer Digestion, and then incubate in a 65°C water bath for 1 hour, inverting the container every 10 minutes.

[0115] After removing the sample and cooling it to room temperature, add 20 μL of RNase A (10 mg / mL) and let it stand at room temperature for 5 min.

[0116] Add 200 μL of Buffer PB and let stand at -20℃ for 5 min;

[0117] Centrifuge the sample at 10,000 rpm for 5 min, and transfer the supernatant into a sterile 1.5 mL centrifuge tube;

[0118] Add an equal volume of isopropanol and mix 6 times. Let stand at room temperature for 3 minutes, then centrifuge at 10,000 rpm for 5 minutes and discard the supernatant.

[0119] Add 1 mL of 75% ethanol to rinse the sample, then centrifuge at 10000 rpm for 2 min, discard the supernatant, and repeat this step once;

[0120] Open the centrifuge tube cap and allow the residual ethanol to evaporate at room temperature. Then add 50 μL of TE Buffer to dissolve the precipitate and obtain genomic DNA. The concentration of the extracted genomic DNA sample was determined using a NanoVue plus ultraviolet spectrophotometer.

[0121] The knock-in region sequence was amplified and then sequenced. The genome was extracted and the knock-in region was sequenced; the strain whose sequence was correctly verified was named RIKmta1. The genome was extracted and the knock-in region was sequenced; the strain whose sequence was correctly verified was named RIKmta2.

[0122] The gene knockout plasmid pCas-amyE-donor was transformed into strains RIKmta1 and RIKmta2, respectively. The specific method is as follows:

[0123] The method for constructing the gene knockout plasmid pCas-amyE-donor includes: using the pJOE8999 vector with inserted guide sequence as a template, named pCas-amyE, and performing PCR amplification using primers P44 and P45 to obtain a linearized vector. The nucleotide sequence of the gene encoding plasmid pCas-amyE is shown in SEQ ID NO.39, the nucleotide sequence of the gene encoding primer P44 is shown in SEQ ID NO.40, and the nucleotide sequence of the gene encoding primer P45 is shown in SEQ ID NO.41.

[0124] Using the Bacillus subtilis genome as a template, the left and right homologous arms were amplified using primers P46 and P47, and primers P48 and P49, respectively.

[0125] The nucleotide sequence of the gene encoding primer P46 is shown in SEQ ID NO.42, the nucleotide sequence of the gene encoding primer P47 is shown in SEQ ID NO.43, the nucleotide sequence of the gene encoding primer P48 is shown in SEQ ID NO.44, and the nucleotide sequence of the gene encoding primer P49 is shown in SEQ ID NO.45.

[0126] The linearized pJOE8999 vector, the PCR amplification products of the left and right homologous arms were recovered by gel extraction, and then... The Snap Assembly Master Mix kit was used for ligation to obtain the ligation reaction product. The ligation conditions included: 100 ng of linearized pJOE8999 vector added; the amount of the left homologous arm added was twice the amount of linearized pJOE8999 vector added; and the amount of the right homologous arm added was twice the amount of linearized pJOE8999 vector added. The amount of Snap Assembly Master Mix added was 2 μL, and sterile water was added to make the reaction system 10 μL. After incubation at 50°C for 30 min, the mixture was placed in an ice bath for 15 min to obtain the ligation product.

[0127] The ligation reaction product was transformed into E. coli HST08 competent cells, then plated on LB plates containing 30 μg / mL Kan, and cultured at 30°C for 18 h. Single colonies were picked for colony PCR identification. Strains that were correctly identified by colony PCR were cultured at 30°C for 14 h, and the gene knockout plasmid pCas-amyE-donor was extracted.

[0128] RIKmta1 and RIKmta2, stored at -80℃, were streaked on LB plates and then incubated upside down in a 37℃ incubator for 12 hours.

[0129] Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0130] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0131] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0132] Add 50 μL of EGTA solution and continue culturing at 37 °C and 90 rpm for 10 min to obtain RIKmta1 and RIKmta2 competent cells;

[0133] Add 1 mL of 60% glycerol and store at -80°C.

[0134] The competent cells were removed, and 1 μg of gene knockout plasmid pCas-amyE-donor was added before culturing at 37℃ and 90 rpm for 1.5 h.

[0135] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0136] Single colonies were picked and inoculated into LB liquid medium containing 10 μg / mL Kan and cultured at 30℃ and 200 rpm until the OD600 reached 0.4-0.6. D-mannose was added to a final concentration of 0.2% w / v to induce Cas9 nuclease expression and perform gene editing. After culturing for another 12 h, the bacterial culture was spread on LB plates containing 10 μg / mL Kan and 0.2% w / v D-mannose and cultured at 30℃ for 18 h.

[0137] Single colonies were picked and inoculated into antibiotic-free LB broth and cultured at 50°C and 200 rpm for 12 h. The culture was then continued on antibiotic-free LB plates and incubated at 42°C until single colonies appeared. Finally, the genome of the engineered bacteria was extracted, and the knockout region sequence was amplified by PCR followed by gene sequencing. The PCR products all showed a single bright band at approximately 3000 bp, consistent with the expected product size of 2827 bp. Finally, the genome of the engineered bacteria was extracted, and the knockout site was amplified and sequenced, confirming that the correct engineered bacteria were RIKmta1ΔamyE and RIKmta2ΔamyE.

[0138] The primers used for colony PCR verification of RIKmta1ΔamyE and RIKmta2ΔamyE were P36 / P37.

[0139] The nucleotide sequence of the gene encoding primer P13 is shown in SEQ ID NO.46; the nucleotide sequence of the gene encoding primer P14 is shown in SEQ ID NO.47; the nucleotide sequence of the gene encoding primer P19 is shown in SEQ ID NO.48; and the nucleotide sequence of the gene encoding primer P20 is shown in SEQ ID NO.49.

[0140] The nucleotide sequence of the gene encoding primer P36 is shown in SEQ ID NO.50; the nucleotide sequence of the gene encoding primer P37 is shown in SEQ ID NO.51.

[0141] Construction and validation of single-copy genome-integrated engineered bacteria RIKmta3ΔamyE and RIKmta4ΔamyE

[0142] s1. The amyE gene in the B. subtilis RIK1285 genome was knocked out using the gene knockout plasmid pCas-amyE-donor. The specific knockout method included:

[0143] The gene knockout plasmid pCas-amyE-donor was transformed into B. subtilis RIK1285 competent cells, and the bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0144] Single colonies were picked and inoculated into LB liquid medium containing 10 μg / mL Kan and cultured at 30℃ and 200 rpm until the OD600 reached 0.4-0.6. D-mannose was added to a final concentration of 0.2% w / v to induce Cas9 nuclease expression and perform gene editing. After culturing for another 12 h, the bacterial culture was spread on LB plates containing 10 μg / mL Kan and 0.2% w / v D-mannose and cultured at 30℃ for 18 h.

[0145] Single colonies were picked and inoculated into antibiotic-free LB liquid medium and cultured at 50°C and 200 rpm for 12 h. The culture was then continued on antibiotic-free LB plates and cultured at 42°C until single colonies appeared, yielding strain RIK1285ΔamyE. The amplified product showed a single bright band at approximately 3000 bp, consistent with the expected product size of 2827 bp. The genome was extracted, and the knockout portion was sequenced. The correctly sequenced strain was identified as RIK1285ΔamyE.

[0146] The transformation method for B. subtilis RIK1285 competent cells is as follows:

[0147] Bacillus subtilis stored at -80℃ was streaked on LB plates and then incubated upside down in a 37℃ incubator for 12 hours.

[0148] Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0149] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0150] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0151] Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain competent Bacillus subtilis cells;

[0152] Add 1 mL of 60% glycerol and store at -80°C.

[0153] Remove B. subtilis RIK1285 competent cells, add 1 μg of gene knockout plasmid pCas-amyE-donor and culture at 37℃ and 90 rpm for 1.5 h;

[0154] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0155] We obtain RIK1285ΔamyE;

[0156] s2. The recombinant vectors pCas-ganA-mta and pCas-sigF-mta were respectively transformed into RIK1285ΔamyE competent cells. The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30℃ for 18 h.

[0157] The transformation method for RIK1285ΔamyE competent cells is as follows:

[0158] Remove RIK1285ΔamyE stored at -80℃ and streak it on an LB plate, then incubate it upside down in a 37℃ incubator for 12 hours.

[0159] Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0160] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0161] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0162] Add 50 μL of EGTA solution and continue culturing at 37 °C and 90 rpm for 10 min to obtain RIK1285ΔamyE competent cells;

[0163] Add 1 mL of 60% glycerol and store at -80°C.

[0164] Remove B. subtilis RIK1285 competent cells, add 1 μg of gene knockout plasmid pCas-amyE-donor and culture at 37℃ and 90 rpm for 1.5 h;

[0165] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0166] s3. Pick a single colony and inoculate it into LB liquid medium containing 10 μg / mL Kan. Incubate at 30℃ and 200 rpm until the OD600 reaches 0.4-0.6. Add D-mannose to a final concentration of 0.2% w / v to induce Cas9 nuclease expression and perform gene editing. After culturing for another 12 h, spread the bacterial culture onto LB plates containing 10 μg / mL Kan and 0.2% w / v D-mannose and incubate at 30℃ for 18 h.

[0167] s4. Pick a single colony and inoculate it into antibiotic-free LB liquid medium. Incubate at 50℃ and 200rpm for 12h. Continue to streak on antibiotic-free LB plates and incubate at 42℃ until single colonies appear. Perform colony PCR verification on the engineered bacteria that have completed gene editing and plasmid elimination. Use primers P32 / P25 and P30 / P31. The reaction system and reaction procedure are the same as those for strains RIKmta1ΔamyE and RIKmta2ΔamyE. The difference is that primers P32 / P25 and P30 / P31 are used respectively.

[0168] The nucleotide sequence of the gene encoding primer P25 is shown in SEQ ID NO.52; the nucleotide sequence of the gene encoding primer P30 is shown in SEQ ID NO.53; and the nucleotide sequence of the gene encoding primer P31 is shown in SEQ ID NO.54.

[0169] Colony PCR was performed to identify the RIK1285ΔamyE gene knock-in engineered bacteria targeting the ganA site. The strain with correct gene sequencing was named RIKmta3ΔamyE. Colony PCR was also performed to identify the RIK1285ΔamyE gene knock-in engineered bacteria targeting the sigF site. The strain with correct gene sequencing was named RIKmta4ΔamyE.

[0170] Construction and identification of engineered bacteria with multi-copy genome integration

[0171] The construction process of Bacillus subtilis engineered strains with integrated multi-copy mta expression cassettes is as follows: Starting with strain RIKmta4ΔamyE, gene editing plasmids pCas-ganA-mta, pCas-ydeI / ydeJ-mta, and pCas-yyaP / tetB-mta targeting the ganA site, the ydeI / ydeJ intergenic region, and the yyaP / tetB intergenic region, respectively, were sequentially transformed and gene-edited to obtain engineered strains with integrated 1-4 copy mta expression cassettes. The specific gene editing experimental methods are the same as those used for constructing the single-copy integrated engineered strains RIKmta1ΔamyE and RIKmta2ΔamyE. The colony PCR reaction system and procedure are the same as for strains RIKmta1ΔamyE and RIKmta2ΔamyE. The difference lies in the primers used for colony PCR identification of the 2-4 copy engineered strains: P32 / P25, P13 / P14, and P19 / P20, respectively. The successfully constructed 2-4 copy genome-integrating engineered bacteria were named RIKmta5ΔamyE, RIKmta6ΔamyE, and RIKmta7ΔamyE, respectively.

[0172] Colony PCR identification was performed. The PCR product showed a single bright band at 3000-5000 bp, consistent with the expected product size of 3322 bp. The engineered bacteria were cultured and the genome was extracted. The gene knock-in region was amplified and the gene was sequenced. The correctly sequenced strain was named RIKmta5ΔamyE.

[0173] Colony PCR was performed on the engineered bacteria that successfully knocked into the ydeI / ydeJ intergenic region. A single bright band was observed at approximately 5000 bp, consistent with the expected product size of 4792 bp. Further genomic extraction and PCR amplification of the knock-in region sequence were performed, followed by gene sequencing. The correctly sequenced strain was named RIKmta6ΔamyE.

[0174] Colony PCR was performed on the engineered bacteria after the yyaP / tetB gene knock-in site was completed. The amplified product showed a single bright band at 4000-5000 bp, consistent with the expected product size of 4815 bp. The genome of the engineered bacteria was then extracted and sequenced to identify the knock-in region. The strain with correctly sequenced genome was named RIKmta7ΔamyE.

[0175] The fifth aspect of the present invention provides the application of the above-mentioned recombinant plasmid, the method for constructing the above-mentioned recombinant plasmid, the above-mentioned genetically engineered bacteria, or the method for constructing the above-mentioned genetically engineered bacteria in the production of maltodextrin.

[0176] E. coli HST08 and B. subtilis RIK1285 were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0177] Snap Assembly Master Mix, dNTP Mixture, HSDNAPolymerase, T4 DNA Ligase, and 10×T4DNALigase Buffer were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0178] Bsa I-HFv2, T4 polynucleotide kinase, and 10×T4 DNA ligase buffer were purchased from New England Biotechnology (Beijing) Co., Ltd.

[0179] Taq DNAPolymerase (5U / μL), 10×Taq Buffer (Mg 2+ (Plus) Purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0180] LB medium: 10g sodium chloride, 10g tryptone, 5g yeast extract, bring to a final volume of 1L, pH 7.2, sterilize at 121℃ for 20min. LB solid medium: Add 2% agar powder to the LB medium, sterilize under the same conditions as LB medium.

[0181] Reagents for preparing Bacillus subtilis competent cells: SPI salts solution: 14g K₂HPO₄, 2g (NH₄)₂SO₄, 6g KH₂PO₄·3H₂O, 0.2g MgSO₄·7H₂O, 1g Na₃-Citrate·2H₂O, diluted to 1L; Casamino Acids / Yeast Extract solution: 10g yeast extract, 2g Casamino Acids, diluted to 100mL; 50% glucose solution: 50g glucose, diluted to 100mL; 50mM CaCl₂ solution: 0.555g CaCl₂, diluted to 100mL; 250mM MgCl₂ solution: 5.083g MgCl₂·6H₂O, diluted to 100mL; 100mM EGTA solution: 3.804g EGTA, diluted to 100mL, pH 7.0. The above solution was sterilized at 115°C for 30 minutes.

[0182] SPI medium: 10 mL SPI salt, 0.1 mL 50% glucose, 0.1 mL Casaminoacids / Yeast Extract; SPII medium: 5 mL SPI medium, 0.05 mL 50 mM CaCl2, 0.05 mL 250 mM MgCl2. All solutions were prepared in a clean bench.

[0183] Unless otherwise specified in the following examples and comparative examples, all conditions were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0184] Assay methods for recombinant expression of mta in genetically engineered bacteria

[0185] Glyceryl spores of engineered Bacillus subtilis, stored at -80℃, were streaked onto antibiotic-free LB agar plates and incubated upside down at 37℃ for 12 h. Single colonies were picked and inoculated into 10 mL of antibiotic-free LB liquid medium and incubated at 37℃ for 12 h in a shaker. Subsequently, 1 mL of activated seed culture was added to 50 mL of TB medium and incubated at 37℃ for 2.5 h, followed by incubation at 30℃ and 200 rpm for 96 h. During this period, bacterial culture samples were collected every 24 h for OD measurement. 600 And extracellular enzyme activity, enzyme activity assay methods include:

[0186] The fermentation broth of engineered Bacillus subtilis was centrifuged at 10,000 rpm for 10 min, and the supernatant was the crude enzyme solution from the engineered bacteria fermentation. The reaction system was then prepared by mixing 0.5 mL of 1% (w / v) soluble starch solution with 0.45 mL of 10 mM pH 7.0 Na₂HPO₄-NaH₂PO₄ buffer and 0.05 mL of the crude enzyme solution, and immediately incubating in a 55°C water bath for 10 min. Subsequently, the enzyme-catalyzed reaction solution was treated in a boiling water bath for 15 min to inactivate the enzyme, and then centrifuged at 10,000 rpm for 10 min. The supernatant was used to quantitatively determine the amount of reducing sugar produced using the 3,5-dinitrosalicylic acid (DNS) method, and the enzyme activity was calculated. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the above reaction conditions.

[0187] Western blot

[0188] (1) SDS-PAGE: First, prepare a 12% protein separating gel, as shown in Table 1. Add the 12% separating gel solution to the protein gel plate, seal with deionized water, and incubate at 37°C for 30 min until the separating gel is completely solidified. Then, prepare a 5% protein stacking gel, as shown in Table 1. Pour out the deionized water from the protein gel plate, add the 5% stacking gel solution, insert a comb, and continue incubating at 37°C until the stacking gel is completely solidified. Subsequently, prepare the electrophoresis samples. Mix 5× Loading Buffer with the protein samples evenly and place them in a boiling water bath for 5 min. After removing and cooling to room temperature, load the samples for electrophoresis. The SDS-PAGE electrophoresis conditions are: first, electrophoresis at 120V and 50mA. When the dye moves to the interface between the separating gel and the stacking gel, change the voltage and current to 170V and 60mA until the electrophoresis is complete.

[0189] Table 1. SDS-PAGE gel composition

[0190]

[0191] (2) Transfer: Soak the filter paper thoroughly in electrotransfer buffer. Before transfer, soak the PVDF membrane in methanol for 15 seconds and double-distilled water for 2 minutes, and finally soak it in electrotransfer buffer for 5 minutes. Stack the filter paper, PVDF membrane, SDS-PAGE gel and filter paper neatly from bottom to top and electrotransfer for 50 minutes;

[0192] (3) Blocking: Take out the PVDF membrane and put it into PBST solution, wash it on a low-speed shaker for 5 min, and then put it into blocking solution and block it overnight at 4°C;

[0193] (4) Antibody incubation: Immerse the blocked PVDF membrane in PBST solution and wash on a low-speed shaker for 10 min. Dilute 6×His-Tag Monoclonal antibody with blocking buffer at a ratio of 1:10000 (v / v) to obtain a primary antibody solution, then transfer the PVDF membrane into it and continue incubation on a low-speed shaker for 1 h. Subsequently, transfer the PVDF membrane into PBST solution and wash on a low-speed shaker for 10 min, repeating the washing three times. Dilute HRP GoatAnti-MouselgG (H&L) with blocking buffer at a ratio of 1:20000 (v / v) to obtain a secondary antibody solution, transfer the PVDF membrane into it, and incubate on a low-speed shaker for 1 h. Finally, wash the PVDF membrane three times with PBST solution, each time placing it on a low-speed shaker for 10 min;

[0194] (5) Development: The PVDF membrane was placed in Tanon™ High-sig ECL Western Blotting Substrate and incubated for 1 min. Then, the membrane was developed and the bands were observed using ImageQuant LAS4000.

[0195] Example 1 Construction of recombinant plasmid

[0196] S1. The pJOE8999 linearized vector with the guide sequence targeting the yvdF / mdxE site, the left homologous arm upstream and downstream of the yvdF / mdxE gene, and the right homologous arm were amplified separately and recovered by gel extraction. The pJOE8999 linearized vector, the left homologous arm, and the right homologous arm each have a 20bp overlapping region at their ends. They were then ligated by a seamless cloning reaction to obtain the ligation reaction product.

[0197] S2. Using the B. subtilis RIK1285 genome as a template, the left and right homologous arms were amplified using primers P38 / P39 and P40 / P41, respectively. The PCR reaction conditions included: 50 ng of genomic DNA, 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 10 μL of 5×PrimeSTAR Buffer, and 4 μL of dNTPs. The amount of HSDNA Polymerase added was 1 μL. Sterile water was added to make the reaction system 50 μL. The reaction was carried out at 95°C for 300 s, followed by 94°C for 30 s, 57°C for 45 s, and 72°C for 90 s as one cycle. The cycle was repeated 30 times, and then the reaction was carried out at 72°C for 600 s.

[0198] S3. The PCR amplification products were recovered from the gel and then used... Ligation was performed using the SnapAssembly MasterMix kit, with the following conditions: 100 ng of linearized plasmid vector pCas-yvdF / mdxE added; the amount of the left homologous arm added was twice the amount of the linearized pJOE8999 vector added; and the amount of the right homologous arm added was twice the amount of the linearized pJOE8999 vector added. The amount of Snap Assembly Master Mix added was 2 μL, and sterile water was added to bring the reaction system to 10 μL. The reaction conditions were: incubation at 50°C for 30 min, followed by incubation in an ice-water mixture for 15 min. Then, the cells were transferred into E. coli HST08 competent cells, plated on LB plates containing 30 μg / mL Kan, and cultured at 30°C for 18 h. Single colonies were picked and activated for culture. The activation culture conditions included: culture at 30°C for 14 h. Extraction was performed to obtain the recombinant plasmid pCas-yvdF / mdxE-donor.

[0199] As per the instruction manual Figure 1As shown, the PCR amplification product exhibited single bright bands at 5000-10000 bp and 1000-1500 bp, consistent with the expected product sizes of 7424 bp (linearized vector), 1416 bp (left homologous arm), and 1198 bp (right homologous arm). The three DNA fragments were ligated using seamless cloning technology, and the ligation product was then transformed into E. coli HST08. After culturing, single colonies were picked for colony PCR identification. The results are shown in the attached instructions. Figure 2 As shown, the PCR amplification product exhibited a single bright band at 2250-3000 bp, consistent with the expected product size of 2594 bp. After activating and culturing the correctly identified *E. coli* colonies, the plasmid was extracted and sequenced, confirming the plasmid as pCas-yvdF / mdxE-donor.

[0200] Example 2: Construction and Identification of Genetically Engineered Bacteria

[0201] S1. The engineered strain RIKmta7ΔamyE containing the recombinant vector with the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA and sigF site knock-in expression cassette was prepared into competent cells.

[0202] The preparation methods for engineered bacterium RIKmta7ΔamyE competent cells include:

[0203] a1. Take out the engineered bacteria RIKmta7ΔamyE stored at -80℃, streak it on LB agar plates, and then incubate it upside down in a 37℃ incubator for 12 hours.

[0204] a2. Pick a single colony and inoculate it into 2 mL of LB liquid medium. Continue to incubate in a constant temperature shaker at 28°C and 150 rpm for 16 h.

[0205] a3. Take 50 μL of bacterial culture and transfer it into 5 mL of SPI medium. Incubate at 37℃ and 150 rpm for 5 h.

[0206] a4. Transfer 500 μL of culture into 5 mL of SPⅡ medium and continue to incubate at 37℃ and 150 rpm for 1.5 h.

[0207] a5. Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain RIKmta7ΔamyE competent cells;

[0208] a6. After adding 1 mL of 60% glycerol, freeze at -80°C;

[0209] S2. Take out RIKmta7ΔamyE competent cells, add 1μg of recombinant plasmid and culture at 37℃ and 90rpm for 1.5h. Spread the bacterial culture on LB plates containing 10μg / mL Kan and incubate upside down at 30℃ for 18h.

[0210] S3. Pick a single colony and inoculate it into LB liquid medium containing 10 μg / mL Kan. Incubate at 30℃ and 200 rpm until the OD600 reaches 0.4-0.6. Add D-mannose to a final concentration of 0.2% (w / v). Gene editing is performed by inducing Cas9 nuclease expression through mannose. After culturing for another 12 h, dilute the bacterial solution and spread it on LB plates containing 10 μg / mL Kan and 0.2% (w / v) D-mannose. Incubate at 30℃ for 18 h.

[0211] S4. Pick a single colony and inoculate it into antibiotic-free LB liquid medium. Incubate at 50℃ and 200rpm for 12h. Continue to streak on antibiotic-free LB plates and incubate at 42℃ until a single colony appears, thus obtaining the genetically engineered bacterium RIKmta7ΔamyEΔym.

[0212] Finally, the engineered bacteria that successfully knocked out the virus were validated by colony PCR. Primers P42 / P43 were used, and the reaction conditions included: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of template, and Mg... 2+ The following steps were performed: 2 μL of 10×Taq Buffer, 1.6 μL of dNTPs, 0.2 μL of Taq DNA Polymerase, and 20 μL of sterile water. The reaction mixture was kept at 95°C for 300 s, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C at 1000 bp / min. After 30 cycles, the mixture was kept at 72°C for 600 s.

[0213] The upstream primer was P42, and the downstream primer was P43; the template was strain RIKmta7ΔamyEΔym.

[0214] Finally, the genome of the engineered bacteria was extracted and the genome knockout region sequence was amplified for gene sequencing. The strain with correct gene sequencing was RIKmta7ΔamyEΔym.

[0215] Colony PCR identification was performed on the metabolically modified engineered bacteria, and the results are shown in the attached instructions. Figure 3 As shown, the PCR amplification product exhibited a single bright band at approximately 3000 bp, consistent with the expected product size of 3017 bp. After further activation and culture of the engineered bacteria, the genome was extracted, and the knockout region was then amplified and sequenced. The sequencing results are shown in the attached instructions. Figure 4 As shown, the strain with correct gene sequencing is RIKmta7ΔamyEΔym.

[0216] Test Example: Synthesis of Maltodextrose by Engineered Bacillus subtilis in Culture Medium System

[0217] The engineered bacteria RIKmta7ΔamyE and RIKmta7ΔamyEΔym glycerol culture stock stored at -80℃ were retrieved and streaked onto antibiotic-free LB agar plates at 37℃ for 12 hours. Subsequently, morphologically sound single colonies were picked and inoculated into 10 mL of antibiotic-free LB liquid medium and activated in a shaker at 37℃ for 12 hours to obtain seed culture. Then, 1 mL of the activated seed culture was inoculated into 50 mL of TB medium containing 80 g / L, 120 g / L, and 200 g / L maltodextrin, respectively, and cultured in a shaker at 30℃ and 200 rpm. Samples were taken at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The sample was first centrifuged at 10,000 rpm for 10 min, the supernatant was then treated in a boiling water bath for 15 min, centrifuged again, and the supernatant was filtered through a 0.22 μm filter membrane for high performance liquid chromatography analysis.

[0218] The engineered bacteria RIKmta7ΔamyEΔym (obtained through metabolic engineering) and RIKmta7ΔamyE (without knockout of maltodextrin metabolism-related genes) were respectively inoculated into a culture medium containing a high concentration of maltodextrin. The recombinant Mta secreted extracellularly by the engineered bacteria during growth and enzyme production converted the maltodextrin in the culture medium into maltotetrasaccharide, and the results were quantitatively detected by HPLC. The results are shown in the attached figure. Figure 5As shown, the engineered bacterium RIKmta7ΔamyEΔym, in 80 g / L, 120 g / L, and 200 g / L maltodextrin media, gradually converted maltodextrin to maltotetrasaccharide with increasing culture time, achieving yields of 59.84%, 58.85%, and 65.42% at 24 h, 36 h, and 48 h, respectively. Further extension of culture time did not significantly increase the yield. In contrast, the control engineered bacterium, while also converting maltodextrin to maltotetrasaccharide in 80 g / L, 120 g / L, and 200 g / L maltodextrin media within 24 h, 36 h, and 48 h, showed a continuous decrease in maltotetrasaccharide content with further extended culture time. Meanwhile, under the aforementioned conditions of 80 g / L, 120 g / L, and 200 g / L maltodextrin, the highest maltotetrasaccharide yields of the engineered strain RIKmta7ΔamyEΔym were 64.32%, 66.25%, and 69.37%, respectively, while the yields of the control engineered strain were 59.05%, 52.49%, and 51.88%, respectively. Therefore, through metabolic engineering modification strategies, an engineered bacterial system for the efficient conversion of maltotetrasaccharides from high-concentration maltodextrin was successfully established, which has good prospects for industrial application.

[0219] In industrial production, using engineered bacteria to directly prepare maltodextrin via biotransformation in a medium containing high-concentration substrates simplifies the production process and reduces costs. Existing research mainly utilizes crude or pure Mta enzymes for biotransformation. Using crude enzyme solutions containing recombinant Mta obtained from Bacillus subtilis engineered bacteria fermentation yielded a maximum yield of 70.3%; using Mta expressed in Bacillus subtilis WB600 and its mutants for maltodextrin biotransformation yielded a maximum conversion rate of 73.1%; and using recombinant Mta expressed in Bacillus licheniformis yielded a maximum maltodextrin yield of 72.4%. This invention utilizes the metabolically engineered bacterium RIKmta7ΔamyEΔym, which can directly biotransform in a high-concentration maltodextrin medium, achieving a maximum yield of 69.37%. This yield is not only similar to the levels obtained using enzyme preparations but also eliminates the extraction and purification steps of the Mta enzyme preparation, simplifying the process and reducing production costs.

[0220] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A recombinant plasmid, characterized in that, The knocked-out gene in the recombinant plasmid is yvdF and mdxE genes; The recombinant plasmid is composed of a pJOE8999 vector, a 20 bp guide sequence and a knockout cassette; The insertion position of the 20 bp guide sequence is the position of inserting the exogenous guide sequence in the pJOE8999 vector; The nucleotide sequence of the knockout cassette is shown as SEQ ID NO. 1; The nucleotide sequence of the 20 bp guide sequence is shown as SEQ ID NO.

2.

2. The method of constructing the recombinant plasmid of claim 1, characterized in that, The steps of the construction method comprise: S1, respectively amplifying the pJOE8999 linearized vector with the target yvdF / mdxE site guide sequence, the left and right homologous arms upstream and downstream of the yvdF / mdxE gene, carrying out gel recovery, and then carrying out ligation through a seamless cloning reaction to obtain a ligation reaction product, wherein the ends of the pJOE8999 linearized vector, the left homologous arm and the right homologous arm respectively carry 20 bp overlapping regions; S2, using the B. subtilis RIK1285 genome as a template, left and right homologous arms were amplified by primers P38 / P39 and P40 / P41 respectively, and the PCR reaction conditions included: the addition amount of genomic DNA was 50 ng, the addition amount of upstream primer with a concentration of 10 μM was 2 μL, the addition amount of downstream primer with a concentration of 10 μM was 2 μL, the addition amount of 5×PrimeSTAR Buffer was 10 μL, the addition amount of dNTP was 4 μL, The addition amount of HSDNA Polymerase was 1 μL, sterilized water was added to the reaction system to 50 μL, 95°C was kept for 300 s, 94°C was kept for 30 s, 57°C was kept for 45 s, 72°C was kept for 90 s for one cycle, and the cycle was repeated for 30 times, followed by keeping at 72°C for 600 s. In S2, the upstream primer is one of P38 and P40, and the downstream primer is one of P39 and P41; The coding gene nucleotide sequence of the primer P38 is shown as SEQ ID NO. 3, the coding gene nucleotide sequence of the primer P39 is shown as SEQ ID NO. 4, the coding gene nucleotide sequence of the primer P40 is shown as SEQ ID NO. 5, and the coding gene nucleotide sequence of the primer P41 is shown as SEQ ID NO. 6; S3, the PCR amplification product was recovered, and then the recovered product was ligated with Snap Assembly Master Mix kit, the ligation conditions included: the amount of linearized plasmid vector pCas-yvdF / mdxE was 100 ng, the amount of left homologous arm was twice the amount of linearized pJOE8999 vector, the amount of right homologous arm was twice the amount of linearized pJOE8999 vector, Snap Assembly Master Mix kit, the ligation conditions included: the amount of linearized plasmid vector pCas-yvdF / mdxE was 100 ng, the amount of left homologous arm was twice the amount of linearized pJOE8999 vector, the amount of right homologous arm was twice the amount of linearized pJOE8999 vector, the amount of Snap Assembly Master Mix was 2 μL, sterilized water was added to the reaction system to 10 μL, the reaction conditions were incubation at 50°C for 30 min, then incubation in ice water mixture for 15 min, then transferred into E. coli HST08 competent cells, coated on LB plate containing 30 μg / mL Kan, and cultured at 30°C for 18 h, a single colony was picked and activated, the activation conditions included: 30°C, culture for 14 h, extraction, and the recombinant plasmid pCas-yvdF / mdxE-donor was obtained.

3. The genetically engineered bacteria comprising the recombinant plasmid of claim 1, characterized in that, The genetically engineered bacteria are strains in which the yvdF and mdxE genes in the operon mdxR-yvdF-mdxE-mdxF-mdxG-yvdJ-yvdK-malL-pgcM for metabolizing maltodextrin are knocked out.

4. The genetically engineered bacteria according to claim 3, characterized in that, The genetically engineered bacteria are Bacillus subtilis, and the genetically engineered bacteria comprise a recombinant vector, the recombinant vector is knocked into an expression cassette, and the intergenic region knocked into the expression cassette is the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA and sigF site; The expression cassette comprises a maltotetrahydrolase gene with optimized codons; The coding gene nucleotide sequence of the maltotetrahydrolase gene is shown as SEQ ID NO. 9 The coding gene nucleotide sequence of the expression cassette is shown as SEQ ID NO.

10.

5. The method for constructing the genetically engineered bacteria according to claim 3 or 4, characterized in that, The construction method comprises the following steps: S1, preparing the engineering bacteria RIKmta7ΔamyE comprising the recombinant vector with the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA and sigF site knocked into the expression cassette into competent cells; S2, taking out the RIKmta7ΔamyE competent cells, adding 1 μg of the recombinant plasmid, and culturing at 37℃ and 90 rpm for 1.5 h, then coating the bacterial solution on an LB plate containing 10 μg / mL Kan, and culturing at 30℃ for 18 h. S3, picking single colony and inoculating in LB liquid medium containing 10 μg / mL Kan, culturing at 30℃, 200 rpm until OD600 is 0.4-0.6, adding D-mannose with final concentration of 0.2% (w / v) to induce Cas9 nuclease expression by mannose and then to edit gene, diluting and spreading on LB plate containing 10 μg / mL Kan and 0.2% (w / v) D-mannose after 12h culture, culturing at 30℃ for 18h; S4, picking single colony and inoculating in LB liquid medium without antibiotic, culturing at 50℃, 200 rpm for 12h, streaking on LB plate without antibiotic and culturing at 42℃ until single colony appears, obtaining genetic engineering bacteria RIKmta7ΔamyEΔym.

6. The construction method of claim 5, wherein, The method for preparing the competent cell of the engineering bacteria RIKmta7ΔamyE comprises: a1, streaking the engineering bacteria RIKmta7ΔamyE preserved at-80℃ on LB plate, then inverting and culturing in 37℃ constant temperature incubator for 12h; a2, picking single colony and inoculating in 2mL LB liquid medium, continuing to culture in constant temperature shaker at 28℃, 150 rpm for 16h; a3, transferring 50 μL bacterial liquid into 5mL SPI medium, culturing at 37℃, 150 rpm for 5h; a4, transferring 500 μL culture into 5mL SPⅡ medium, continuing to culture at 37℃, 150 rpm for 1.5h; a5, adding 50 μL EGTA solution, continuing to culture at 37℃, 90 rpm for 10min, obtaining RIKmta7ΔamyE competent cell; a6, adding 1mL 60% glycerol and then freezing at-80℃.

7. Application of the recombinant plasmid of claim 1, the method for constructing the recombinant plasmid of claim 2, the genetic engineering bacteria of claim 3 or 4 or the method for constructing the genetic engineering bacteria of claim 5 or 6 in production of maltotetraose.

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