Codon-optimized maltotetraglycoamylase gene, expression cassette, recombinant vector, genetically engineered bacterium, and construction method and application of codon-optimized maltotetraglycoamylase gene

Abstract

The invention relates to the technical field of gene engineering, in particular to a codon-optimized maltotetraglycoamylase gene, an expression cassette, a recombinant vector, a genetically engineered bacterium and a construction method and application of the codon-optimized maltotetraglycoamylase gene and the expression cassette, the recombinant vector and the genetically engineered bacterium. The genetically engineered bacterium containing the codon-optimized maltotetraose amylase gene provided by the invention is further bacillus subtilis, and maltotetraose can be produced under the condition that the original strain cannot produce the maltotetraose amylase in a genome integrated manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a codon-optimized maltotetraose amylase gene, an expression cassette, a recombinant vector and a genetically engineered bacterium, and a construction method and application thereof. BACKGROUND

[0002] Maltotetraose is a new type of linear malt oligosaccharide with low sweetness, good moisture retention, resistance to Maillard reaction and multiple beneficial physiological activities to the human body, and currently shows high application value in the fields of food, biological medicine and daily chemical products. In industry, maltotetraose is mainly prepared by maltotetraose amylase (Mta) using starch or maltodextrin as a substrate, and the use cost of Mta accounts for a large proportion in the industrial production of maltotetraose. It is of great significance to use food-grade microorganisms to construct a high-efficiency heterologous expression system for producing Mta, so as to reduce the production cost of maltotetraose and expand its market application. However, the existing Mta heterologous expression system mainly relies on free plasmids, which not only has limited stability in industrial production, but also needs to add antibiotics to maintain the plasmid. At the same time, the two-step biocatalytic process of first expressing the engineering bacteria and then using the separated enzyme for maltotetraose synthesis has many steps, high cost and low production efficiency.

[0003] The existing Mta heterologous expression system is mainly based on free plasmids, which not only relies on the addition of antibiotics to maintain the stability of the plasmid during the fermentation culture of the engineering bacteria, but also gradually loses the plasmid due to the limitations of the structure and instability of the plasmid itself during the large-scale fermentation tank culture, resulting in a continuous decrease in protein expression.

[0004] In summary, it is urgent to develop a codon-optimized maltotetraose amylase gene, an expression cassette, a recombinant vector and a genetically engineered bacterium, so as to produce maltotetraose amylase in Bacillus subtilis which cannot produce maltotetraose in the form of genomic integration, avoid the instability of the engineering bacteria obtained by using free plasmids in industrial production, and avoid the limitation of adding antibiotics to maintain the plasmid. SUMMARY

[0005] The present application is to solve the problem of how to provide a maltotetraose amylase gene, an expression cassette, a recombinant vector and a genetically engineered bacterium.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a codon-optimized maltotetraose amylase gene, wherein the coding gene nucleotide sequence of the maltotetraose amylase gene is shown in SEQ ID NO. 1.

[0007] The second aspect of the present application provides an expression cassette comprising the maltotetrahydrolase gene, wherein the nucleotide sequence of the coding gene of the expression cassette is shown in SEQ ID NO. 2.

[0008] The third aspect of the present application provides a recombinant vector comprising the expression cassette, wherein the intergenic region of the expression cassette knocked into the recombinant vector comprises one of the intergenic regions of ydeI / ydeJ, yyaP / tetB, ganA and sigF.

[0009] The fourth aspect of the present application provides a construction method of the recombinant vector.

[0010] The fifth aspect of the present application provides a genetically engineered bacterium comprising the recombinant vector, wherein the genetically engineered bacterium is Bacillus subtilis.

[0011] The sixth aspect of the present application provides a construction method of the genetically engineered bacterium.

[0012] The seventh aspect of the present application provides the maltotetrahydrolase gene, the expression cassette, the recombinant vector, the construction method of the recombinant vector, the genetically engineered bacterium or the construction method of the genetically engineered bacterium in the production of maltotetrahydrolase.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] The present application provides a recombinant vector targeting four sites of ydeI / ydeJ intergenic region, yyaP / tetB intergenic region, ganA and sigF of Bacillus subtilis genome respectively, and the correct recombinant vector is transferred into Bacillus subtilis to knock in the mta expression cassette into the above four different sites respectively through gene editing, and the amyE gene in the host genome is knocked out, thereby avoiding the pollution of the host's own extracellular amylase, improving the quality of the product enzyme preparation, and increasing the yield of maltotetrahydrolase. Finally, the engineering bacteria RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE and RIKmta4ΔamyE are obtained. The four strains of engineering bacteria are cultured in a shake flask, and the highest extracellular enzyme activity is 15.3 U / mL, 14.4 U / mL, 13.8 U / mL and 37.4 U / mL respectively.

[0015] Starting with the engineered bacterium RIKmta4ΔamyE, recombinant vectors targeting the intergenic regions of ganA, ydeI / ydeJ, and yyaP / tetB were sequentially transformed and gene-edited. The engineered bacteria were identified by colony PCR and gene sequencing, successfully constructing engineered bacteria RIKmta5ΔamyE, RIKmta6ΔamyE, and RIKmta7ΔamyE with 2-4 copies of the genome integrated. A series of engineered bacteria with different copy numbers of mta expression cassettes were cultured in shake flasks, and their extracellular enzyme activities were detected. The results showed that the highest extracellular enzyme activities of engineered bacteria RIKmta4ΔamyE, RIKmta5ΔamyE, RIKmta6ΔamyE, and RIKmta7ΔamyE were 46.3 U / mL, 70.4 U / mL, 98.8 U / mL, and 106.3 U / mL, respectively. Attached Figure Description

[0016] Figure 1 This is an electrophoresis image of the gel recovery products of the PCR amplified fragment. In the image: M: DNA marker; 1: Left homologous arm for knocking into the intergenic region of the ydeI / ydeJ genes; 2: Right homologous arm for knocking into the intergenic region of the ydeI / ydeJ genes; 3: Linearized pCas-ydeI / ydeJ vector; 4: P 43 P amyE -SP amyE -mta.

[0017] Figure 2 Colony PCR validation was performed on E. coli HST08 cells using a gene knock-in plasmid targeting the intergenic region of the ydeI / ydeJ genes. In the figure: M: DNA marker; 1-4: E. coli HST08 / pCas-ydeI / ydeJ-mta.

[0018] Figure 3 This is an electrophoresis image of the gel recovery product of the PCR amplified fragment. In the image: M: DNA marker; 1: left homologous arm for knocking in the intergenic region of the yyaP / tetB gene; 2: right homologous arm for knocking in the intergenic region of the yyaP / tetB gene; 3: linearized pCas-yyaP / tetB vector.

[0019] Figure 4 Colony PCR validation of E. coli HST08 cells using a gene knock-in plasmid targeting the intergenic region of the yyaP / tetB gene. In the figure: M: DNA marker; 1-3: E. coli HST08 / pCas-yyaP / tetB-mta.

[0020] Figure 5This is an electrophoresis image of the gel recovery product of the PCR amplified fragment. In the image: M: DNA marker; 1: left homologous arm for knocking in the ganA site; 2: right homologous arm for knocking in the ganA site; 3: linearized pCas-ganA vector.

[0021] Figure 6 Colony PCR validation of E. coli HST08 cells transformed with a plasmid targeting the ganA site gene knock-in. In the figure: M: DNA marker; 1-6: E. coli HST08 / pCas-ganA-mta.

[0022] Figure 7 This is an electrophoresis image of the gel recovery product of the PCR amplified fragment. In the image: M: DNA marker; 1: left homologous arm used for knocking in the sigF site; 2: right homologous arm used for knocking in the sigF site; 3: linearized pCas-sigF vector.

[0023] Figure 8 Colony PCR validation of E. coli HST08 cells transformed with a plasmid targeting the sigF site gene knock-in. In the figure: M: DNA marker; 1-6: E. coli HST08 / pCas-sigF-mta.

[0024] Figure 9 Colony PCR validation of engineered bacteria that integrate the mta expression cassette at the intergenic locus of the ydeI / ydeJ genes. In the figure: M: DNA marker; 1: RIK1285; 2: RIKmta1.

[0025] Figure 10 Colony PCR validation of engineered bacteria that integrate the mta expression cassette at the intergenic site of the yyaP / tetB gene. In the figure: M: DNA marker; 1: RIK1285; 2: RIKmta2.

[0026] Figure 11 Colony PCR validation of the RIKmta1 engineered bacteria with the amyE gene knocked out. In the figure: M: DNA marker; 1: RIKmta1; 2: RIKmta1ΔamyE.

[0027] Figure 12 Colony PCR verification of the RIKmta2 engineered bacteria with the amyE gene knocked out. In the figure: M: DNA marker; 1: RIKmta2; 2: RIKmta2ΔamyE.

[0028] Figure 13 Colony PCR validation of RIK1285 with the amyE gene knocked out. In the figure: M: DNA marker; 1: RIK1285; 2: RIK1285ΔamyE.

[0029] Figure 14 Colony PCR validation of engineered bacteria with integrated mta expression cassette at the ganA site. In the figure: M: DNA marker; 1: RIK1285ΔamyE; 2: RIKmta3ΔamyE.

[0030] Figure 15 Colony PCR validation of engineered bacteria with integrated mta expression cassette at the sigF site. In the figure: M: DNA marker; 1: RIK1285ΔamyE; 2: RIKmta4ΔamyE.

[0031] Figure 16 This image shows the detection of extracellular enzyme activity in a single-copy integrated engineered bacterium. In the figure: (A) extracellular enzyme activity; (B) bacterial cell OD. 600 .

[0032] Figure 17 This is a Western blotting analysis of the extracellular supernatant of a single-copy integrated engineered bacterium. In the figure: 1: RIKmta1ΔamyE extracellular supernatant; 2: RIKmt2ΔamyE extracellular supernatant; 3: RIKmta3ΔamyE extracellular supernatant; 4: RIKmta4ΔamyE extracellular supernatant.

[0033] Figure 18 Colony PCR validation of the double-copy integrated engineered bacterium RIKmta5ΔamyE. In the figure: M: DNA marker; 1: RIKmta4ΔamyE; 2: RIKmta5ΔamyE.

[0034] Figure 19 Colony PCR validation of the three-copy integrated engineered bacterium RIKmta6ΔamyE. In the figure: M: DNA marker; 1: RIKmta5ΔamyE; 2: RIKmta6ΔamyE.

[0035] Figure 20 Colony PCR validation of the four-copy integrated engineered bacterium RIKmta7ΔamyE. In the figure: M: DNA marker; 1: RIKmta6ΔamyE; 2: RIKmta7ΔamyE.

[0036] Figure 21 The extracellular enzyme activity of integrative engineered bacteria with different copy numbers was detected. In the figure: (A) extracellular enzyme activity; (B) bacterial cell OD. 600 .

[0037] Figure 22Western blotting analysis of extracellular supernatants from integrated engineered bacteria with different copy numbers. In the figure: 1: RIK1285ΔamyE extracellular supernatant; 2: RIKmta4ΔamyE extracellular supernatant; 3: RIKmta5ΔamyE extracellular supernatant; 4: RIKmta6ΔamyE extracellular supernatant; 5: RIKmta7ΔamyE extracellular supernatant. Detailed Implementation

[0038] 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.

[0039] Bacillus subtilis is widely used in the production of industrial enzymes due to its outstanding advantages, such as ease of large-scale fermentation culture, strong ability to secrete proteins into the extracellular space, and high safety, which have been certified as GRAS by the US-FDA.

[0040] The inventors discovered that by knocking a specific gene region into a specific expression cassette within a Bacillus subtilis vector as an integration target, Bacillus subtilis, which is unable to produce maltotetrasaccharide, can produce maltotetrasaccharide-producing ...

[0041] Based on this, the present invention expresses Mta in Bacillus subtilis in a genome-integrated manner, achieving for the first time genome integration of maltodextrin amylase and multi-copy genome integration in Bacillus subtilis. This enables engineered bacteria to produce maltodextrin amylase in a genome-integrated manner, thereby avoiding the limitations of insufficient stability of engineered bacteria obtained from free plasmids in industrial production and the need to add antibiotics to maintain the plasmid.

[0042] The first aspect of this invention provides a maltodextrin amylase gene with optimized codons, wherein the nucleotide sequence encoding the maltodextrin amylase gene is shown in SEQ ID NO.1.

[0043] A second aspect of the present invention provides an expression cassette comprising the above-mentioned maltotetrasaccharide amylase gene, wherein the nucleotide sequence of the encoding gene of the expression cassette is shown in SEQ ID NO.2.

[0044] A third aspect of the present invention provides a recombinant vector for the above-mentioned expression cassette, wherein the intergenic region of the expression cassette knocked into the recombinant vector includes one of the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA site, and the sigF site.

[0045] In this invention, the intergenic region refers to a sequence between the two genes. For example, the ydeI / ydeJ intergenic region is a sequence between the genes ydeI and ydeJ, and the yyaP / tetB intergenic region is a sequence between the genes yyaP and tetB.

[0046] A fourth aspect of the present invention provides a method for constructing the above-mentioned recombinant vector, wherein the steps of the construction method include:

[0047] S1. Phosphorylation and annealing reactions were performed on primer pairs P1 / P2, P3 / P4, P5 / P6, and P7 / P8, 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 DNA ligase reaction buffer, 1 μL of T4 PNK, and 6 μL of sterile water. The mixture was incubated at 37°C for 30 min, then incubated at 95°C for 5 min, and then cooled to 25°C at a rate of 0.1°C / s to obtain double-stranded oligonucleotides with sticky ends.

[0048] The nucleotide sequence of the gene encoding primer P1 is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding primer P2 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding primer P3 is shown in SEQ ID NO.5, the nucleotide sequence of the gene encoding primer P4 is shown in SEQ ID NO.6, the nucleotide sequence of the gene encoding primer P5 is shown in SEQ ID NO.7, the nucleotide sequence of the gene encoding primer P6 is shown in SEQ ID NO.8, the nucleotide sequence of the gene encoding primer P7 is shown in SEQ ID NO.9, and the nucleotide sequence of the gene encoding primer P8 is shown in SEQ ID NO.10.

[0049] 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;

[0050] S2. Digest the pJOE8999 vector using enzymes. The digestion conditions include: 1 μg of pJOE8999 vector, 1 μL of Bsa I-HFv2, and 10X rCutSmart enzyme. 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.

[0051] S3. The double-stranded oligonucleotide with sticky ends is mixed with the linearized pJOE8999 vector and ligated 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 sterile water is added to make the reaction system 10 μL. The mixture is incubated at 16°C for 2 h to obtain the ligation product.

[0052] S4. 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.

[0053] S5. 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 to extract the recombinant vectors pCas-ydeI / ydeJ, pCas-yyaP / tetB, pCas-ganA and pCas-sigF.

[0054] S6. Using the B. subtilis RIK1285 genome as a template, primers P9 / P10 and P11 / P12 were used to amplify the left and right homologous arms of the ydeI / ydeJ intergenic locus, primers P15 / P16 and P17 / P18 were used to amplify the left and right homologous arms of the yyaP / tetB intergenic locus, primers P21 / P22 and P23 / P24 were used to amplify the left and right homologous arms of the ganA locus, and primers P26 / P27 and P28 / P29 were used to amplify the left and right homologous arms of the sigF locus. 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 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.

[0055] In S6, 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;

[0056] The nucleotide sequences encoding the gene of primer P9 are shown in SEQ ID NO. 11, primer P10 in SEQ ID NO. 12, primer P11 in SEQ ID NO. 13, primer P12 in SEQ ID NO. 14, primer P15 in SEQ ID NO. 15, primer P16 in SEQ ID NO. 16, primer P17 in SEQ ID NO. 17, primer P18 in SEQ ID NO. 18, primer P21 in SEQ ID NO. 19, primer P22 in SEQ ID NO. 20, primer P23 in SEQ ID NO. 21, primer P24 in SEQ ID NO. 22, and primer P26 in SEQ ID NO. 19. As shown in NO.23, the nucleotide sequence of the gene encoding primer P27 is shown in SEQ ID NO.24, the nucleotide sequence of the gene encoding primer P28 is shown in SEQ ID NO.25, and the nucleotide sequence of the gene encoding primer P29 is shown in SEQ ID NO.26.

[0057] S7. 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.

[0058] The nucleotide sequence of the gene encoding primer P32 is shown in SEQ ID NO.27, and the nucleotide sequence of the gene encoding primer P33 is shown in SEQ ID NO.28.

[0059] S8. 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.

[0060] The nucleotide sequence of the gene encoding primer P34 is shown in SEQ ID NO.29, and the nucleotide sequence of the gene encoding primer P35 is shown in SEQ ID NO.30.

[0061] S9. 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 SnapAssembly 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 compared to the linearized pJOE8999 vector, twice the amount of the left homologous arm compared to the linearized pJOE8999 vector, twice the amount of the right homologous arm compared to the linearized pJOE8999 vector, and 5× Add 2 μL of SnapAssembly 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;

[0062] S10. 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℃ for 18 h. Single colonies were picked for colony PCR identification. Primers P9 / P12 were used to identify the ydeI-ydeJ intergenic region, primers P15 / P18 were used to identify the yyaP-tetB intergenic region, primers P21 / P24 were used to identify the ganA site, and primers P26 / P29 were used to identify the sigF site. Strains that were correctly identified by colony PCR were cultured at 30℃ for 14 h, and recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta, and pCas-sigF-mta were extracted.

[0063] In S10, the reaction conditions for colony PCR include: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of template, 2 μL of 10×Taq Buffer with Mg2+ plus, 1.6 μL of dNTP, 0.2 μL of Taq DNA Polymerase, and adding sterile water to a reaction volume of 20 μL. The reaction is carried out 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 for 30 cycles, followed by 72°C for 600 s.

[0064] In S10, 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.

[0065] The fifth aspect of the present invention provides a genetically engineered bacterium comprising the above-described recombinant vector, wherein the genetically engineered bacterium is Bacillus subtilis.

[0066] According to the present invention, in the genetically engineered bacteria, the intergenic region knocked into the expression cassette includes at least one intergenic region among the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA site, and the sigF site.

[0067] A sixth aspect of the present invention provides a method for constructing the above-mentioned genetically engineered bacteria, wherein the steps of the construction method include:

[0068] S1. The recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta and pCas-sigF-mta were respectively transformed into B. subtilis RIK1285 competent cells. The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30℃ for 18 h.

[0069] S2. 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.

[0070] S3. 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, to obtain strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4.

[0071] S4. The amyE gene was knocked out of strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4 using the gene knockout plasmid pCas-amyE-donor. Colony PCR was used for verification. Strains with the amyE gene knocked out were cultured to obtain strains RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE.

[0072] In S4, the knockout step includes: transforming the gene knockout plasmid pCas-amyE-donor into strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4 respectively, spreading the bacterial solution on LB plates containing 10 μg / mL Kan, and incubating it upside down at 30°C for 18 h.

[0073] 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.

[0074] Single colonies were picked and inoculated into antibiotic-free LB liquid medium and incubated at 50°C and 200 rpm for 12 h. The culture was then streaked onto antibiotic-free LB plates and incubated at 42°C until single colonies appeared.

[0075] The method for constructing the gene knockout plasmid pCas-amyE-donor includes:

[0076] Using the pJOE8999 vector with the inserted guide sequence as a template, PCR amplification was performed using primers P38 and P39 to obtain a linearized vector. The nucleotide sequence of the gene encoding the guide sequence inserted into the pJOE8999 vector is shown in SEQ ID NO.31, the nucleotide sequence of the gene encoding primer P38 is shown in SEQ ID NO.32, and the nucleotide sequence of the gene encoding primer P39 is shown in SEQ ID NO.33.

[0077] Using the Bacillus subtilis genome as a template, the left and right homologous arms were amplified using primers P40 and P41, and primers P42 and P43, respectively.

[0078] The nucleotide sequence of the gene encoding primer P40 is shown in SEQ ID NO.34, the nucleotide sequence of the gene encoding primer P41 is shown in SEQ ID NO.35, the nucleotide sequence of the gene encoding primer P42 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding primer P43 is shown in SEQ ID NO.37.

[0079] 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; the amount of the right homologous arm added was twice the amount of linearized pJOE8999 vector added; and 5× The amount of SnapAssembly 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.

[0080] 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.

[0081] Using pCas-amyE as a template, a linearized vector was obtained by PCR amplification using primers P38 and P39. The nucleotide sequence of the gene encoding plasmid pCas-amyE is shown in SEQ ID NO.31, the nucleotide sequence of the gene encoding primer P38 is shown in SEQ ID NO.32, and the nucleotide sequence of the gene encoding primer P39 is shown in SEQ ID NO.33.

[0082] Using the Bacillus subtilis genome as a template, the left and right homologous arms were amplified using primers P40 and P41, and primers P42 and P43, respectively.

[0083] The nucleotide sequence of the gene encoding primer P40 is shown in SEQ ID NO.34, the nucleotide sequence of the gene encoding primer P41 is shown in SEQ ID NO.35, the nucleotide sequence of the gene encoding primer P42 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding primer P43 is shown in SEQ ID NO.37.

[0084] 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; the amount of the right homologous arm added was twice the amount of linearized pJOE8999 vector added; and 5× The amount of SnapAssembly 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.

[0085] 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.

[0086] Alternatively, the steps of the construction method may include:

[0087] s1. The amyE gene in the genome of B. subtilis RIK1285 was knocked out using the gene knockout plasmid pCas-amyE-donor, resulting in strain RIK1285ΔamyE;

[0088] s2. The recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, 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.

[0089] 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.

[0090] 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, to obtain strains RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE.

[0091] In s1, the knockout step includes: transforming the gene knockout plasmid pCas-amyE-donor into B. subtilisRIK1285 competent cells, spreading the bacterial culture on LB plates containing 10 μg / mL Kan, and incubating upside down at 30°C for 18 h;

[0092] 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.

[0093] Single colonies were picked and inoculated into antibiotic-free LB liquid medium and incubated at 50°C and 200 rpm for 12 h. The culture was then streaked onto antibiotic-free LB plates and incubated at 42°C until single colonies appeared.

[0094] According to the present invention, the construction method further includes:

[0095] Starting with RIKmta4ΔamyE, the recombinant vector pCas-ganA-mta targeting the ganA site was transformed and gene-edited to obtain strain RIKmta5ΔamyE.

[0096] Using strain RIKmta5ΔamyE as the starting strain, the recombinant vector pCas-ydeI / ydeJ-mta targeting the intergenic region of ydeI / ydeJ was transformed and gene-edited to obtain strain RIKmta6ΔamyE.

[0097] Using strain RIKmta6ΔamyE as the starting strain, the recombinant vector pCas-yyaP / tetB-mta, which targets the intergenic region of yyaP / tetB, was transformed and gene-edited to obtain strain RIKmta7ΔamyE.

[0098] According to the present invention, in step S1, the preparation steps of the B. subtilis RIK1285 competent cells include:

[0099] a1. Take out the Bacillus subtilis stored at -80℃, streak it on LB agar plates, and then incubate it upside down in a 37℃ incubator for 12 hours.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] a5. Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain RIK1285ΔamyE competent cells;

[0104] a6. After adding 1 mL of 60% glycerol, freeze at -80°C;

[0105] According to the present invention, in S1, the conditions for transfection include: taking out B. subtilis RIK1285 competent cells, adding 1 μg of the recombinant vector, and culturing at 37°C and 90 rpm for 1.5 h;

[0106] According to the present invention, the preparation steps of RIK1285ΔamyE competent cells in s2 include:

[0107] b1. Take out the Bacillus subtilis stored at -80℃ and streak it on LB agar plates, then incubate it upside down in a 37℃ incubator for 12 hours.

[0108] b2. 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.

[0109] b3. Take 50 μL of bacterial culture and transfer it into 5 mL of SPI medium. Incubate at 37℃ and 150 rpm for 5 h.

[0110] b4. Transfer 500 μL of culture into 5 mL of SPII medium and continue to incubate at 37℃ and 150 rpm for 1.5 h.

[0111] b5. Add 50 μL of LEGTA solution and continue culturing at 37°C and 90 rpm for 10 min to obtain RIK1285ΔamyE competent cells;

[0112] b6. After adding 1 mL of 60% glycerol, freeze at -80°C;

[0113] According to the present invention, in s2, the step of transfer includes: taking out RIK1285ΔamyE competent cells, adding 1 μg of the recombinant vector, and culturing at 37°C and 90 rpm for 1.5 h.

[0114] The seventh aspect of the present invention provides the application of the above-mentioned maltotetrasaccharide amylase gene, the above-mentioned expression cassette, the above-mentioned recombinant vector, the above-mentioned method for constructing the recombinant vector, the above-mentioned genetically engineered bacteria, or the above-mentioned method for constructing genetically engineered bacteria in the production of maltotetrasaccharide.

[0115] E. coli HST08 and B. subtilis RIK1285 were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0116] SnapAssembly Master Mix, dNTP Mixture, HSDNAPolymerase, T4 DNALigase, and 10×T4DNALigase Buffer were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0117] Bsa I-HFv2, T4 polynucleotide kinase, and 10×T4 DNA ligase buffer were purchased from New England Biotechnology (Beijing) Co., Ltd.

[0118] Taq DNAPolymerase (5U / μL), 10×TaqBuffer (Mg 2+ (Plus) Purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0119] 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.

[0120] 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; CasaminoAcids / YeastExtract solution: 10g yeast extract, 2g CasaminoAcids, 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.

[0121] 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.

[0122] 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.

[0123] Assay methods for recombinant expression of mta in genetically engineered bacteria

[0124] 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:

[0125] 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.

[0126] Western blot

[0127] (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 sample by mixing 5× Loading Buffer with the protein sample and placing it in a boiling water bath for 5 min. After removing and cooling to room temperature, load the sample for electrophoresis. The SDS-PAGE electrophoresis conditions are as follows: First, electrophoresis is performed at 120V and 50mA. When the dye moves to the interface between the separating gel and the stacking gel, the voltage and current are changed to 170V and 60mA until the electrophoresis is completed.

[0128] Table 1. SDS-PAGE gel composition

[0129]

[0130] (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;

[0131] (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;

[0132] (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;

[0133] (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.

[0134] Example 1: Construction of Recombinant Vector

[0135] First, guide sequences targeting different sites are inserted into the pJOE8999 vector:

[0136] 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 DNA ligase 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.

[0137] 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;

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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:

[0143] 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.

[0144] 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 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 130 s as one cycle. The cycle was repeated 30 times, and then the reaction was carried out at 72°C for 600 s.

[0145] 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 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 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.

[0146] 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, twice the amount of the right homologous arm as the linearized pJOE8999 vector, and 5× 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;

[0147] 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.

[0148] In S10, the reaction conditions for colony PCR include: 2 μL of 10 μM upstream primer, 2 μL of 10 μM downstream primer, 1 μL of template, 2 μL of 10×Taq Buffer with Mg2+ plus, 1.6 μL of dNTP, 0.2 μL of Taq DNA Polymerase, and adding sterile water to a reaction volume of 20 μL. The reaction is carried out 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 for 30 cycles, followed by 72°C for 600 s.

[0149] 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.

[0150] Construction and validation of plasmid vectors targeting gene editing at different sites

[0151] A recombinant vector targeting the intergenic region of the ydeI / ydeJ genes was constructed. Using pJOE8999 plasmid as the starting vector, a guide sequence was first inserted to obtain the plasmid pCas-ydeI / ydeJ. Subsequently, the following fragments were obtained by PCR amplification: the pJOE8999 linearized plasmid vector containing the guide sequence inserted at the ydeI / ydeJ site, the left homologous arm, the right homologous arm, and the mta expression cassette. All fragments had a 20bp overlap region at the end for subsequent seamless cloning and ligation. The fragments were then recovered by gel electrophoresis. Figure 1 As shown, the PCR amplification products exhibited single bright bands at 5000-8000 bp, approximately 1000 bp, and approximately 2000 bp, respectively, consistent with the expected product sizes of 7424 bp (linearized vector), 1213 bp (left homologous arm), 1150 bp (right homologous arm), and 2076 bp (mta expression cassette). The four DNA fragments recovered from the gel were ligated using seamless cloning technology, then transformed into E. coli HST08 culture medium, plated, and subjected to colony PCR identification. The colony PCR results are shown below. Figure 2 As shown, the PCR amplification product of *E. coli* exhibits a single bright band at 3000-5000 bp, consistent with the expected product size of 4399 bp. After extraction of the recombinant vector, gene sequencing was performed, and the correctly identified recombinant vector was named pCas-ydeI / ydeJ-mta.

[0152] 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 linearized pJOE8999 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. Figure 3 As shown, the PCR amplification product exhibits single bright bands at 5000-8000 bp and approximately 1000 bp, consistent with the expected product sizes of 7424 bp (linearized vector), 1138 bp (left homologous arm), and 1051 bp (right homologous arm). The above three DNA fragments, along with the mta expression cassette fragment recovered earlier, were ligated using seamless cloning technology, then transformed into E. coli HST08 competent cells for identification. The colony PCR identification results are as follows... Figure 4 As shown, the amplified product exhibited a single bright band at 3000-5000 bp, consistent with the expected product size of 4225 bp. The recombinant vector was then extracted and sequenced. The correctly sequenced recombinant vector was named pCas-yyaP / tetB-mta.

[0153] 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. Figure 5 As shown, the amplified products exhibited single bright bands at 5000-8000 bp and 1000-1500 bp, respectively, consistent with the expected product sizes of 7424 bp (linearized vector), 1166 bp (left homologous arm), and 1079 bp (right homologous arm). The aforementioned DNA fragment and the mta expression cassette recovered earlier were ligated using an In-Fusion reaction, followed by transformation into E. coli HST08 culture. Single colonies were then identified by colony PCR. The identification results are as follows: Figure 6 As shown, the PCR product exhibited a single bright band at approximately 4500 bp, consistent with the expected product size of 4281 bp. Subsequently, gene sequencing was performed on the constructed recombinant vector, confirming its correctness; the recombinant vector was named pCas-ganA-mta.

[0154] 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 then recovered via gel electrophoresis. Figure 7As shown, the PCR amplification product exhibited single bright bands at 5000-8000 bp and 1000-1500 bp, consistent with the expected product sizes of 7424 bp (linearized vector), 1170 bp (left homologous arm), and 1162 bp (right homologous arm). The above DNA fragment and mta expression cassette fragment were ligated using In-Fusion seamless cloning technology and then transformed into E. coli HST08. After cultivation, single colonies were identified by colony PCR, and the results are as follows. Figure 8 As shown, the PCR product exhibited a single bright band at approximately 4500 bp, consistent with the expected product size of 4368 bp. Finally, the recombinant vector was extracted and sequenced, confirming its correctness; the recombinant vector was named pCas-sigF-mta.

[0155] Example 2: Construction and Validation of Engineered Bacteria with Single-Copy Genome Integration

[0156] Construction and validation of single-copy genome-integrated engineered bacteria RIKmta1ΔamyE and RIKmta2ΔamyE

[0157] (1) The successfully constructed gene knock-in plasmids pCas-ydeI / ydeJ-mta and pCas-yyaP / tetB-mta were respectively transformed into B. subtilis RIK1285 competent cells. The transformation method was as follows:

[0158] Bacillus subtilis stored at -80℃ was streaked on LB plates and then incubated 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 B. subtilis RIK1285 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 the recombinant vector, 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] (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 is 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, spread the bacterial solution on LB plates containing 10 μg / mL Kan and 0.2% (w / v) D-mannose and incubate at 30℃ for 18 h.

[0167] (3) Pick a single colony and inoculate it into LB liquid medium without antibiotics. Incubate at 50℃ and 200rpm for 12h. Continue to streak on LB plates without antibiotics and incubate at 42℃ until a single colony appears. 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.

[0168] (4) Colony PCR identification was performed using primers P13 / P14 and P19 / P20, respectively. The reaction conditions for the 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 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.

[0169] Genome extraction, specifically including:

[0170] 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.

[0171] Take 2 mL of the cultured bacterial solution and centrifuge at 8000 rpm for 5 min to collect the bacterial cell precipitate;

[0172] 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.

[0173] Add 400 μL of Buffer Digestion, and then incubate in a 65°C water bath for 1 hour, inverting the container every 10 minutes.

[0174] 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.

[0175] Add 200 μL of Buffer PB and let stand at -20℃ for 5 min;

[0176] Centrifuge the sample at 10,000 rpm for 5 min, and transfer the supernatant into a sterile 1.5 mL centrifuge tube;

[0177] 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.

[0178] 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;

[0179] 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.

[0180] The knock-in region is amplified and then sequenced. For example... Figure 9 As shown, the amplified product has a single bright band at approximately 5000 bp, consistent with the expected product size of 4792 bp. Genome sequencing was performed on the knock-in portion, and the strain whose gene sequencing confirmed its correctness was named RIKmta1. Colony PCR identification of the B. subtilis RIK1285 gene knock-in engineered strain at the yyaP / tetB site was performed, and the results are as follows... Figure 10 As shown, the amplified product has a single bright band at 3000-5000bp, consistent with the expected product size of 4815bp. The genome was extracted and the knock-in part was sequenced. The strain with correct gene sequencing was named RIKmta2.

[0181] The gene knockout plasmid pCas-amyE-donor was transformed into strains RIKmta1 and RIKmta2, respectively. The specific method is as follows:

[0182] 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 P38 and P39 to obtain a linearized vector. The nucleotide sequence of the gene encoding the guide sequence inserted into the pJOE8999 vector is shown in SEQ ID NO.31, the nucleotide sequence of the gene encoding primer P38 is shown in SEQ ID NO.32, and the nucleotide sequence of the gene encoding primer P39 is shown in SEQ ID NO.33.

[0183] Using the Bacillus subtilis genome as a template, the left and right homologous arms were amplified using primers P40 and P41, and primers P42 and P43, respectively.

[0184] The nucleotide sequence of the gene encoding primer P40 is shown in SEQ ID NO.34, the nucleotide sequence of the gene encoding primer P41 is shown in SEQ ID NO.35, the nucleotide sequence of the gene encoding primer P42 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding primer P43 is shown in SEQ ID NO.37.

[0185] 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; the amount of the right homologous arm added was twice the amount of linearized pJOE8999 vector added; and 5× The amount of SnapAssembly 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.

[0186] 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.

[0187] RIKmta1 and RIKmta2, stored at -80℃, were streaked on LB plates and then incubated upside down in a 37℃ incubator for 12 hours.

[0188] 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.

[0189] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0190] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0191] 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;

[0192] Add 1 mL of 60% glycerol and store at -80°C.

[0193] 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.

[0194] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0195] 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.

[0196] Single colonies were picked and inoculated into antibiotic-free LB broth and incubated at 50°C and 200 rpm for 12 hours. The culture was then continued by streaking onto antibiotic-free LB plates and incubating 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 results are shown in the attached instructions. Figure 11 , 12 As shown, all PCR products exhibited a single bright band at approximately 3000 bp, consistent with the expected product size of 2827 bp. Finally, the genomes of the engineered bacteria were extracted, and the knockout sites were amplified and sequenced, confirming that the correct engineered bacteria were RIKmta1ΔamyE and RIKmta2ΔamyE.

[0197] The primers used for colony PCR verification of RIKmta1ΔamyE and RIKmta2ΔamyE were P36 / P37.

[0198] The nucleotide sequence of the gene encoding primer P13 is shown in SEQ ID NO.38; the nucleotide sequence of the gene encoding primer P14 is shown in SEQ ID NO.39; the nucleotide sequence of the gene encoding primer P19 is shown in SEQ ID NO.40; and the nucleotide sequence of the gene encoding primer P20 is shown in SEQ ID NO.41.

[0199] The nucleotide sequence of the gene encoding primer P36 is shown in SEQ ID NO.42; the nucleotide sequence of the gene encoding primer P37 is shown in SEQ ID NO.43.

[0200] Construction and validation of single-copy genome-integrated engineered bacteria RIKmta3ΔamyE and RIKmta4ΔamyE

[0201] 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:

[0202] 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.

[0203] 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.

[0204] Single colonies were picked and inoculated into antibiotic-free LB broth, and cultured at 50℃ and 200 rpm for 12 h. The culture was then streaked onto antibiotic-free LB plates and cultured at 42℃ until single colonies appeared, yielding strain RIK1285ΔamyE. Results are as follows: Figure 13 As shown, the amplified product has 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 strain with the correct gene sequencing was RIK1285ΔamyE.

[0205] The transformation method for B. subtilis RIK1285 competent cells is as follows:

[0206] Bacillus subtilis stored at -80℃ was streaked on LB plates and then incubated upside down in a 37℃ incubator for 12 hours.

[0207] 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.

[0208] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0209] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0210] Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain competent Bacillus subtilis cells;

[0211] Add 1 mL of 60% glycerol and store at -80°C.

[0212] 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;

[0213] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0214] We obtain RIK1285ΔamyE;

[0215] 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.

[0216] The transformation method for RIK1285ΔamyE competent cells is as follows:

[0217] Remove RIK1285ΔamyE stored at -80℃ and streak it on an LB plate, then incubate it upside down in a 37℃ incubator for 12 hours.

[0218] 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.

[0219] Transfer 50 μL of bacterial culture into 5 mL of SPI medium and incubate at 37 °C and 150 rpm for 5 h.

[0220] Transfer 500 μL of culture into 5 mL of SPII medium and continue culturing at 37 °C and 150 rpm for 1.5 h.

[0221] Add 50 μL of EGTA solution and continue culturing at 37 °C and 90 rpm for 10 min to obtain RIK1285ΔamyE competent cells;

[0222] Add 1 mL of 60% glycerol and store at -80°C.

[0223] RIK1285ΔamyE competent cells were removed, and 1 μg of recombinant vectors pCas-ganA-mta and pCas-sigF-mta were added. The cells were then cultured at 37℃ and 90 rpm for 1.5 h.

[0224] The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30°C for 18 h.

[0225] 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.

[0226] 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.

[0227] The nucleotide sequence of the gene encoding primer P25 is shown in SEQ ID NO.44; the nucleotide sequence of the gene encoding primer P30 is shown in SEQ ID NO.45; and the nucleotide sequence of the gene encoding primer P31 is shown in SEQ ID NO.46.

[0228] Colony PCR identification was performed on the RIK1285ΔamyE gene knock-in engineered bacteria targeting the ganA site, and the results are as follows: Figure 14As shown, the amplified product exhibited a single bright band at 3000-5000 bp, consistent with the expected product size of 3322 bp. Genome sequencing was performed on the knock-in portion, and the correctly sequenced strain was named RIKmta3ΔamyE. Colony PCR identification of the RIK1285ΔamyE gene knock-in engineered strain targeting the sigF site was performed, and the results are as follows... Figure 15 As shown, the amplified product has a single bright band at approximately 5000 bp, consistent with the expected product size of 4926 bp. The genome was extracted and the knock-in portion was sequenced. The strain with the correct gene sequencing was named RIKmta4ΔamyE.

[0229] Enzyme production evaluation of single-copy integrated engineered bacteria

[0230] The four correctly constructed and validated single-copy genome-integrated engineered bacteria, RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE, were cultured in shake flasks, and their extracellular enzyme activities were detected and analyzed by Western blot. The results of the extracellular enzyme activity detection are as follows: Figure 16 As shown, the engineered strains RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE successfully achieved secretory expression of Mta. Furthermore, with prolonged culture time, the extracellular enzyme activity of the engineered strains continuously increased from day 1 to day 3, reaching its peak on day 3 at 15.3 U / mL, 14.4 U / mL, 13.8 U / mL, and 37.4 U / mL, respectively. Subsequent culture showed a decrease in extracellular enzyme activity. Western blot analysis of the extracellular supernatant from the fermentation culture of the four single-copy genome-integrated engineered strains after 3 days yielded the following results: Figure 17 As shown, the extracellular supernatant samples of each engineered bacterium all exhibited bands in the range of 42-55 kDa, consistent with the expected relative molecular mass of Mta of 47.4 kDa, thus further verifying the successful secretory expression of Mta by the engineered bacteria, and the expression level was consistent with the results of extracellular enzyme activity detection.

[0231] Example 3: Construction and Identification of Engineered Bacteria with Multi-Copy Genome Integration

[0232] 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.

[0233] Colony PCR identification was performed, and the results were as follows: Figure 18 As shown, the PCR product showed a single bright band at 3000-5000bp, consistent with the expected product size of 3322bp. The engineered bacteria were cultured and the genome was extracted. The gene knock-in region was amplified and the gene was sequenced. The strain that was correctly sequenced was named RIKmta5ΔamyE.

[0234] Colony PCR was performed to identify the engineered bacteria that had undergone gene knock-in at the ydeI / ydeJ intergenic locus. The results are as follows: Figure 19 As shown, a single bright band appears 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.

[0235] Colony PCR identification was performed on the engineered bacteria after the yyaP / tetB gene intergenic site knock-in was completed. The results are as follows: Figure 20 As shown, the amplified product exhibited a single bright band in the 4000-5000 bp range, consistent with the expected product size of 4815 bp. Further extraction of the engineered bacteria's genome and sequencing of the knock-in region were performed. The strain with correctly sequenced genome was named RIKmta7ΔamyE.

[0236] Enzyme production study of engineered bacteria with multi-copy genome integration

[0237] The engineered bacteria RIKmta4ΔamyE, RIKmta5ΔamyE, RIKmta6ΔamyE, and RIKmta7ΔamyE, which integrated 1-4 copies of the constructed mta expression cassette, were fermented in shake flasks, and extracellular enzyme activity was detected and Western blot analysis was performed. The results are as follows: Figure 21 As shown, the extracellular enzyme activities of the 1-4 copy integrated engineered bacteria RIKmta4ΔamyE, RIKmta5ΔamyE, RIKmta6ΔamyE, and RIKmta7ΔamyE continuously increased during the 1-3 day culture period, reaching their highest extracellular enzyme activities on day 3, at 46.3 U / mL, 70.4 U / mL, 98.8 U / mL, and 106.3 U / mL, respectively. Furthermore, the extracellular enzyme activity detection results indicated that the extracellular enzyme activity of the engineered bacteria continuously increased with the increase of the number of integrated mta copies. Western blot analysis was then performed on the extracellular supernatant of the above engineered bacteria on day 3. Figure 22 As shown, the results are consistent with the trend of extracellular enzyme activity detection, thus proving that increasing the copy number of Mta integrated into the genome can effectively improve the secretory expression level of Mta.

[0238] Because wild-type strains of Mta generally exhibit low expression levels (e.g., extracellular enzyme activities of 29 U / mL and 6.8 U / mL obtained using strains *Pseudomonas* spIMD353 and *P. stutzeri* AS22, respectively), current research focuses on recombinant expression of Mta using heterologous expression systems. In *E. coli*, Mta is primarily expressed intracellularly as inclusion bodies. However, extracellular secretory expression of Mta has been successfully achieved using various food-grade microorganisms. For example, using *Bacillus licheniformis* and a xylose-inducible promoter, an extracellular enzyme activity of 168.2 U / mL was obtained; using *Bacillus subtilis* WS11 as a host, and through culture medium optimization, an extracellular enzyme activity of 236 U / mL was obtained; using *Bacillus subtilis* WB600 as an expression host, recombinant expression of Mta and its mutants yielded extracellular enzyme activities of 160-310 U / mL; and through optimization of expression elements and fermentation conditions, an extracellular enzyme activity of 288.9 U / mL was obtained in *Bacillus subtilis*. This invention effectively improves the expression level of Mta in engineered bacteria through a multi-copy genome integration strategy. The extracellular enzyme activity of the four-copy integrated engineered bacteria reaches 106.3 U / mL. Although it is still lower than the expression system based on free plasmids mentioned above, it has the advantages of not requiring the addition of antibiotics and being more suitable for achieving stable expression in industrial production. It can avoid the limitations of adding antibiotics to maintain plasmids, which not only helps to reduce production costs, but also makes the production process more green and environmentally friendly.

[0239] 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 maltodextrose amylase gene with optimized codons, characterized in that, The nucleotide sequence encoding the maltodextrose amylase gene is shown in SEQ ID NO.

1.

2. An expression cassette comprising the maltodextrin amylase gene of claim 1, characterized in that, The nucleotide sequence of the gene encoding the expression cassette is shown in SEQ ID NO.

2.

3. A recombinant vector comprising the expression cassette of claim 2, characterized in that, The intergenic region into which the expression cassette is knocked into the recombinant vector includes one of the following intergenic regions: ydeI / ydeJ, yyaP / tetB, ganA, and sigF.

4. The method for constructing the recombinant vector according to claim 3, characterized in that, The steps of the construction method include: S1. Phosphorylation and annealing reactions were performed on primer pairs P1 / P2, P3 / P4, P5 / P6, and P7 / P8, 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 DNA ligase reaction buffer, 1 μL of T4 PNK, and 6 μL of sterile water. The mixture was incubated at 37°C for 30 min, then incubated at 95°C for 5 min, and then cooled to 25°C at a rate of 0.1°C / s to obtain double-stranded oligonucleotides with sticky ends. The nucleotide sequence of the gene encoding primer P1 is shown in SEQ ID NO.3, the nucleotide sequence of the gene encoding primer P2 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding primer P3 is shown in SEQ ID NO.5, the nucleotide sequence of the gene encoding primer P4 is shown in SEQ ID NO.6, the nucleotide sequence of the gene encoding primer P5 is shown in SEQ ID NO.7, the nucleotide sequence of the gene encoding primer P6 is shown in SEQ ID NO.8, the nucleotide sequence of the gene encoding primer P7 is shown in SEQ ID NO.9, and the nucleotide sequence of the gene encoding primer P8 is shown in SEQ ID NO.

10. 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; S2. Digest the pJOE8999 vector using enzymes. The digestion conditions include: 1 μg of pJOE8999 vector, 1 μL of BsaI-HFv2, and 10X rCutSmart enzyme. 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. S3. The double-stranded oligonucleotide with sticky ends is mixed with the linearized pJOE8999 vector and ligated 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 sterile water is added to make the reaction system 10 μL. The mixture is incubated at 16°C for 2 h to obtain the ligation product. S4. 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. S5. 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 to extract the recombinant vectors pCas-ydeI / ydeJ, pCas-yyaP / tetB, pCas-ganA and pCas-sigF. S6. Using the B. subtilis RIK1285 genome as a template, primers P9 / P10 and P11 / P12 were used to amplify the left and right homologous arms of the ydeI / ydeJ intergenic locus, primers P15 / P16 and P17 / P18 were used to amplify the left and right homologous arms of the yyaP / tetB intergenic locus, primers P21 / P22 and P23 / P24 were used to amplify the left and right homologous arms of the ganA locus, and primers P26 / P27 and P28 / P29 were used to amplify the left and right homologous arms of the sigF locus. 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 HS DNA 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. In S6, 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; The nucleotide sequences encoding the gene of primer P9 are shown in SEQ ID NO. 11, primer P10 in SEQ ID NO. 12, primer P11 in SEQ ID NO. 13, primer P12 in SEQ ID NO. 14, primer P15 in SEQ ID NO. 15, primer P16 in SEQ ID NO. 16, primer P17 in SEQ ID NO. 17, primer P18 in SEQ ID NO. 18, primer P21 in SEQ ID NO. 19, primer P22 in SEQ ID NO. 20, primer P23 in SEQ ID NO. 21, primer P24 in SEQ ID NO. 22, and primer P26 in SEQ ID NO.

19. As shown in NO.23, the nucleotide sequence of the gene encoding primer P27 is shown in SEQ ID NO.24, the nucleotide sequence of the gene encoding primer P28 is shown in SEQ ID NO.25, and the nucleotide sequence of the gene encoding primer P29 is shown in SEQ ID NO.

26. S7. 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 HS DNA 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 30 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 130 s. After 30 cycles, the reaction was carried out at 72°C for 600 s. The nucleotide sequence of the gene encoding primer P32 is shown in SEQ ID NO.27, and the nucleotide sequence of the gene encoding primer P33 is shown in SEQ ID NO.

28. S8. 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 HS DNA 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 30 cycles of 94°C for 30 s, 57°C for 45 s, and 72°C for 470 s. After 30 cycles, the reaction was carried out at 72°C for 600 s. The nucleotide sequence of the gene encoding primer P34 is shown in SEQ ID NO.29, and the nucleotide sequence of the gene encoding primer P35 is shown in SEQ ID NO.

30. S9. 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; S10. 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℃ for 18 h. Single colonies were picked for colony PCR identification. Primers P9 / P12 were used to identify the ydeI-ydeJ intergenic region, primers P15 / P18 were used to identify the yyaP-tetB intergenic region, primers P21 / P24 were used to identify the ganA site, and primers P26 / P29 were used to identify the sigF site. Strains that were correctly identified by colony PCR were cultured at 30℃ for 14 h, and recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta, and pCas-sigF-mta were extracted. 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. In S10, 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.

5. A genetically engineered bacterium comprising the recombinant vector of claim 3, characterized in that, The genetically engineered bacterium is Bacillus subtilis.

6. The genetically engineered bacterium according to claim 5, characterized in that, In the genetically engineered bacteria, the intergenic region knocked into the expression cassette includes at least one intergenic region among the ydeI / ydeJ intergenic region, the yyaP / tetB intergenic region, the ganA site, and the sigF site.

7. The method for constructing the genetically engineered bacteria according to claim 5 or 6, characterized in that, The steps of the construction method include: S1. The recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, pCas-ganA-mta and pCas-sigF-mta were respectively transformed into B. subtilis RIK1285 competent cells. The bacterial culture was spread on LB plates containing 10 μg / mL Kan and incubated upside down at 30℃ for 18 h. S2. 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. S3. 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, to obtain strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4. S4. The amyE gene was knocked out of strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4 using the gene knockout plasmid pCas-amyE-donor. Colony PCR was used for verification. Strains with the amyE gene knocked out were cultured to obtain strains RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE. In S4, the knockout step includes: transforming the gene knockout plasmid pCas-amyE-donor into strains RIKmta1, RIKmta2, RIKmta3, and RIKmta4 respectively, spreading the bacterial solution on LB plates containing 10 μg / mL Kan, and incubating it upside down at 30°C for 18 h. 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. Single colonies were picked and inoculated into antibiotic-free LB liquid medium and incubated at 50°C and 200 rpm for 12 h. The culture was then streaked onto antibiotic-free LB plates and incubated at 42°C until single colonies appeared. The method for constructing the gene knockout plasmid pCas-amyE-donor includes: Using the pJOE8999 vector with inserted guide sequence as a template, PCR amplification was performed using primers P38 and P39 to obtain a linearized vector. The nucleotide sequence of the gene encoding the guide sequence inserted into the pJOE8999 vector is shown in SEQ ID NO.31, the nucleotide sequence of the gene encoding primer P38 is shown in SEQ ID NO.32, and the nucleotide sequence of the gene encoding primer P39 is shown in SEQ ID NO.

33. Using the Bacillus subtilis genome as a template, the left and right homologous arms were amplified using primers P40 and P41, and primers P42 and P43, respectively. The nucleotide sequence of the gene encoding primer P40 is shown in SEQ ID NO.34, the nucleotide sequence of the gene encoding primer P41 is shown in SEQ ID NO.35, the nucleotide sequence of the gene encoding primer P42 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding primer P43 is shown in SEQ ID NO.

37. The linearized pJOE8999 vector, left homologous arm, and right homologous arm PCR amplification products were recovered by gel extraction using In- 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. 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. Alternatively, the steps of the construction method may include: s1. The amyE gene in the genome of B. subtilis RIK1285 was knocked out using the gene knockout plasmid pCas-amyE-donor, resulting in strain RIK1285ΔamyE; s2. The recombinant vectors pCas-ydeI / ydeJ-mta, pCas-yyaP / tetB-mta, 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. 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. 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, to obtain strains RIKmta1ΔamyE, RIKmta2ΔamyE, RIKmta3ΔamyE, and RIKmta4ΔamyE. In s1, the knockout step includes: transforming the gene knockout plasmid pCas-amyE-donor into B. subtilisRIK1285 competent cells, spreading the bacterial culture on LB plates containing 10 μg / mL Kan, and incubating upside down at 30°C for 18 h; 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. Single colonies were picked and inoculated into antibiotic-free LB liquid medium and incubated at 50°C and 200 rpm for 12 h. The culture was then streaked onto antibiotic-free LB plates and incubated at 42°C until single colonies appeared.

8. The construction method according to claim 7, characterized in that, The construction method also includes: Starting with RIKmta4ΔamyE, the recombinant vector pCas-ganA-mta targeting the ganA site was transformed and gene-edited to obtain strain RIKmta5ΔamyE. Using strain RIKmta5ΔamyE as the starting strain, the recombinant vector pCas-ydeI / ydeJ-mta targeting the intergenic region of ydeI / ydeJ was transformed and gene-edited to obtain strain RIKmta6ΔamyE. Using strain RIKmta6ΔamyE as the starting strain, the recombinant vector pCas-yyaP / tetB-mta, which targets the intergenic region of yyaP / tetB, was transformed and gene-edited to obtain strain RIKmta7ΔamyE.

9. The construction method according to claim 7, characterized in that, In S1, the preparation steps of the B. subtilis RIK1285 competent cells include: a1. Take out the Bacillus subtilis stored at -80℃, streak it on LB agar plates, and then incubate it upside down in a 37℃ incubator for 12 hours. 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. 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. 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. a5. Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain B. subtilis RIK1285 competent cells; a6. After adding 1 mL of 60% glycerol, freeze at -80°C; In S1, the conditions for transfection include: taking out B. subtilis RIK1285 competent cells, adding 1 μg of the recombinant vector, and culturing at 37°C and 90 rpm for 1.5 h; In s2, the preparation steps of RIK1285ΔamyE competent cells include: b1. Take out the Bacillus subtilis stored at -80℃ and streak it on LB agar plates, then incubate it upside down in a 37℃ incubator for 12 hours. b2. 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. b3. Take 50 μL of bacterial culture and transfer it into 5 mL of SPI medium. Incubate at 37℃ and 150 rpm for 5 h. b4. Transfer 500 μL of culture into 5 mL of SPII medium and continue to incubate at 37℃ and 150 rpm for 1.5 h. b5. Add 50 μL of EGTA solution and continue culturing at 37℃ and 90 rpm for 10 min to obtain RIK1285ΔamyE competent cells; b6. After adding 1 mL of 60% glycerol, freeze at -80°C; In s2, the step of transfer includes: taking out RIK1285ΔamyE competent cells, adding 1 μg of the recombinant vector, and culturing at 37°C and 90 rpm for 1.5 h.

10. The application of the maltotetrasaccharide amylase gene of claim 1, the expression cassette of claim 2, the recombinant vector of claim 3, the method for constructing the recombinant vector of claim 4, the genetically engineered bacteria of claim 5, or the method for constructing the genetically engineered bacteria of any one of claims 6-9 in the production of maltotetrasaccharide.

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