A method for constructing a Bacillus subtilis chassis strain with improved recombinant protein expression ability

Abstract

The present invention discloses a method for constructing a Bacillus subtilis chassis strain with improved recombinant protein expression ability, which belongs to the technical field of enzyme process and microbial transformation. The present invention increases the expression of medium-temperature amylase AmyS, β-galactosidase, ultra-high temperature amylase, and chitinase produced by Bacillus subtilis in shake flask fermentation by 4.32 times, 2.05 times, 1.54 times, and 1.12 times by regulating one or more genes among the genes rodZ, lytS, hrcA, ypmS, and ftsZ. Combined with co-expression of PonA and fermentation optimization, the enzyme activity of AmyS was increased to 102,893U·mL in high-density fermentation in a 3-L bioreactor. ‑1 , which is the highest level of AmyS expression so far.

Description

Technical Field

[0001] The invention relates to a method for constructing a Bacillus subtilis chassis strain with improved recombinant protein expression ability, and belongs to the technical field of enzyme engineering and microbial transformation. Background Art

[0002] Bacillus subtilis is a Gram-positive strain of the genus Bacillus. Since B. subtilis does not produce toxins and heat-sensitizing proteins, it is listed as a certified strain on the US FDA's GRAS (Generally Recognized as Safe) list. It is easy to isolate and culture, can be fermented at high density, and has strong protein secretion capabilities, making Bacillus subtilis able to produce a variety of exogenous recombinant proteins and metabolites. At the same time, as a model microorganism, Bacillus subtilis has mature molecular biological gene editing methods, which is conducive to genetic modification to enhance the expression ability of Bacillus subtilis as a host for exogenous protein production.

[0003] There are many strategies to transform Bacillus subtilis to obtain higher exogenous protein production, such as overexpressing Sec pathway-related factors, knocking out intracellular or extracellular proteases, increasing chaperone protein content; rationally allocating energy to make energy distribution more reasonable, thereby promoting the expression of exogenous proteins. However, the selection of these transformation targets depends largely on the existing annotations of Bacillus subtilis functional genes, and the synthesis and secretion of proteins is not regulated by a single pathway. Studies have shown that overexpression of molecular chaperones can significantly increase the expression level of recombinant proteins. For example, Song et al. overexpressed the chaperone protein GroESL complex to increase the expression of β-mannanase in B. subtilis by 25%. However, the current annotations of protein synthesis and secretion-related pathways are not perfect, which means that the current transformation strategy is relatively one-sided, thus limiting the further development of Bacillus subtilis as an expression host. Due to the diversity and complexity of gene functions and the small number of genes currently known to directly regulate protein expression, it is urgent to use irrational screening in B. subtilis to identify more genes that can improve the expression level of recombinant proteins and further improve the expression level of recombinant proteins.

[0004] At the same time, Bacillus stearothermophilus α-amylase has extremely high potential industrial application value, but the current maximum enzyme activity in B. subtilis can only reach 38,314U / mL, which cannot meet the growing needs of industrial production. How to find the key genes that affect the expression of recombinant proteins on the basis of existing ones and construct a B. subtilis chassis strain with improved expression ability is of great significance for improving the production and application of recombinant proteins. Summary of the invention

[0005] The invention provides a method for increasing the expression amount of foreign protein in Bacillus subtilis. The method comprises the steps of co-expressing the foreign protein and the class A penicillin binding protein PonA and inhibiting the expression of key genes affecting the expression of the recombinant protein.

[0006] In one embodiment, the key gene affecting the expression of the recombinant protein is any one of the following: cytoskeletal protein gene mreB (Gene ID: 936759), cell shape determining gene rodZ (Gene ID: 939591), spore germination related gene yndF (Gene ID: 939547), methionine glutamine aminotransferase gene mtnE (Gene ID: 939330), pyruvate utilization controlling gene lytS (Gene ID: 937404), tRNA modification related gene tsaD (Gene ID: 938020), phosphoserine phosphorylase gene rsbX (Gene ID: 938155), ferric citrate transporter gene yfmF (Gene ID: 936117), negative control molecular chaperone expression gene hrcA (Gene ID: 937843), Z ring forming gene ftsZ (Gene ID: 935971) during division, unknown function gene yoaQ (Gene ID: 940010), ypmS (Gene ID: 938020 ...tRNA modification related gene tsaD (Gene ID: 938020), tRNA modification related gene tsaD (Gene ID: ID:939100) or more.

[0007] In one embodiment, the class A penicillin-binding protein PonA is derived from Bacillus subtilis, and the amino acid sequence is shown in SEQ ID NO.1.

[0008] In one embodiment, the class A penicillin-binding protein PonA is expressed via a plasmid, and the key gene inhibiting the expression of the recombinant protein is integrated with its targeting sgRNA on the genome to inhibit the expression of the corresponding gene.

[0009] In one embodiment, the gene expression is inhibited by integrating the targeted sgRNA of the key gene affecting the expression of the recombinant protein into the lacA, mpr or spoIIAC site.

[0010] In one embodiment, the targeting sgRNA of the key gene affecting the expression of the recombinant protein is integrated and expressed on the genome, using the promoter P 43 Regulate expression.

[0011] In one embodiment, pAD123 is used as an expression vector and promoter P glv , P HpaII or P aprERegulate the expression of the class A penicillin-binding protein gene ponA shown in SEQ ID NO.1.

[0012] In one embodiment, the class A penicillin binding protein PonA is expressed in pAD123, using promoter P HpaII Regulate expression, inhibit the key genes affecting the expression of recombinant proteins, the cell shape determining gene rodZ and the pyruvate utilization controlling gene lytS, by integrating and expressing the targeted sgRNA at the lacA site on the genome; inhibit the key genes affecting the expression of recombinant proteins, the negative control molecular chaperone expression gene hrcA and the unknown function gene ypmS by integrating and expressing the targeted sgRNA at the mpr site on the genome; inhibit the key genes affecting the expression of recombinant proteins, the Z ring forming gene ftsZ during division by integrating and expressing the targeted sgRNA at the spoIIAC site on the genome.

[0013] In one embodiment, the exogenous protein includes but is not limited to α-amylase, β-galactosidase, ultra-high temperature amylase or chitinase.

[0014] In one embodiment, the gene sequence encoding the α-amylase is shown as SEQ ID NO.2; the gene sequence encoding the ultra-high temperature α-amylase is shown as SEQ ID NO.18; the gene sequence encoding the chitinase is shown as SEQ ID NO.19; and the gene sequence encoding the β-galactosidase is shown as SEQ ID NO.20.

[0015] In one embodiment, the nucleotide sequence of the targeting sgRNA that determines cell shape rodZ is shown as SEQ ID NO.3.

[0016] In one embodiment, the nucleotide sequence of the targeting sgRNA that controls pyruvate utilization lytS is shown as SEQ ID NO.4.

[0017] In one embodiment, the nucleotide sequence of the sgRNA targeting hrcA expression of the negative control molecular chaperone is shown in SEQ ID NO.5.

[0018] In one embodiment, the nucleotide sequence of the targeting sgRNA of the unknown functional gene ypmS is shown as SEQ ID NO.6.

[0019] In one embodiment, the nucleotide sequence of the targeting sgRNA that forms the Z-ring ftsZ upon cleavage is as shown in SEQ ID NO.7.

[0020] In one embodiment, the method is to express the foreign protein and the class A penicillin-binding protein PonA in the same host, and inhibit the expression of the key gene affecting the expression of the recombinant protein.

[0021] In one embodiment, the host is Bacillus subtilis.

[0022] In one embodiment, the recombinant Bacillus subtilis is based on WS9 and has integrated the competent regulatory protein ComK (WS9C).

[0023] The present invention also provides a Bacillus subtilis chassis cell with improved recombinant protein expression ability, wherein the Bacillus subtilis chassis cell inhibits key genes that affect the expression of recombinant proteins; the key genes that affect the expression of recombinant proteins are: cytoskeleton protein mreB, cell shape determining rodZ, spore germination related yndF, methionine glutamine aminotransferase mtnE, pyruvate utilization controlling lytS, tRNA modification related tsaD, phosphoserine phosphorylase rsbX, ferric citrate transporter yfmF, negative control of molecular chaperone expression hrcA, Z ring formed during division ftsZ, and one or more of genes yoaQ and ypmS of unknown function.

[0024] In one embodiment, the Bacillus subtilis chassis cells suppress the expression of the rodZ and lytS genes.

[0025] In one embodiment, the Bacillus subtilis chassis cells suppress the expression of rodZ, lytS, hrcA, and ympS genes.

[0026] In one embodiment, the Bacillus subtilis chassis cells suppress the expression of the rodZ, lytS, hrcA, ympS and ftsZ genes.

[0027] In one embodiment, the Bacillus subtilis chassis cells further express the class A penicillin-binding protein PonA derived from Bacillus subtilis.

[0028] In one embodiment, the Bacillus subtilis chassis cells use pAD123 as an expression vector and use P HpaII Regulate the expression of the class A penicillin-binding protein gene ponA shown in SEQ ID NO.1.

[0029] In one embodiment, the Bacillus subtilis chassis cell uses Bacillus subtilis 1A976 or WS9 as a host cell.

[0030] The present invention also provides the use of the Bacillus subtilis chassis cell in expressing foreign proteins.

[0031] In one embodiment, the exogenous protein is expressed using pUB110 as an expression vector.

[0032] In one embodiment, the exogenous protein includes but is not limited to α-amylase, β-galactosidase, ultra-high temperature amylase or chitinase.

[0033] The present invention also provides a recombinant Bacillus subtilis expressing foreign proteins in the chassis cells.

[0034] The invention also provides the use of the recombinant Bacillus subtilis in fermentation and enzyme production.

[0035] In one embodiment, the enzyme includes but is not limited to alpha-amylase.

[0036] In one embodiment, the gene encoding the α-amylase is shown as SEQ ID NO.2.

[0037] In one embodiment, the application is to culture the recombinant Bacillus subtilis in TB medium for a period of time and induce enzyme production with maltose.

[0038] In one embodiment, the application is to first culture at 35°C to 40°C for a period of time, and after adding maltose, culture at 32°C to 34°C for at least 48 hours.

[0039] In one embodiment, the use is to first culture at 37°C for 2 hours and then culture at 33°C for at least 48 hours.

[0040] In one embodiment, fermentation is carried out using one or more of soy peptone, yeast extract, industrial Angel yeast extract, corn steep liquor, and industrial corn steep liquor as nitrogen sources.

[0041] In one embodiment, the concentration of the nitrogen source is 20-44 g / L.

[0042] In one embodiment, soy peptone and industrial corn steep liquor are used as nitrogen sources; the concentration of soy peptone is 22 g / L, and the concentration of industrial corn steep liquor is 18 g / L.

[0043] In one embodiment, one or more of potato starch, corn starch, maltose, and soluble starch are used as the carbon source.

[0044] In one embodiment, the concentration of the carbon source is 5 to 30 g / L.

[0045] In one embodiment, Fe is added to the culture medium. 2+ and / or Ca 2+ .

[0046] In one embodiment, the fermentation is carried out at 25-42°C.

[0047] In one embodiment, the fermentation pH is 5.5 to 8.5.

[0048] The present invention also provides a method for producing alpha-amylase by fermentation, wherein the method comprises fermenting at 35-38°C in a fermentation medium containing corn steep liquor, soy peptone, potato, calcium salt, magnesium salt and ammonium salt.

[0049] In one embodiment, the method further comprises feeding during the fermentation process.

[0050] In one embodiment, the feed uses maltose, glycerol or glucose as the carbon source of the feed.

[0051] In one embodiment, the feed medium further contains corn steep liquor and soy peptone.

[0052] The present invention also provides application of the method or the Bacillus subtilis in the fields of food, chemical industry, sugar production, feed, etc.

[0053] Beneficial effects:

[0054] 1. The present invention integrates sgRNA targeting key genes affecting recombinant protein expression into the Bacillus subtilis genome, including rodZ, which determines cell shape, lytS, which controls pyruvate utilization, hrcA, which is a negative control chaperone, and ypmS, which is an unknown functional gene, or ftsZ, which forms a Z ring during division, and knocks out hrcA, which is a negative control chaperone, to construct a Bacillus subtilis chassis cell with improved protein expression ability. β-galactosidase β-gal from Bacillus circulans, ultra-high temperature α-amylase Pfa from extreme thermophilic archaea (Pyrococcus furiosus), and chitinase BlChiA2 from Bacillus licheniformis are expressed in the chassis cell, and the enzyme activities of the recombinant strains are 2.05, 1.54, and 1.12 times of those of the corresponding control strains, respectively.

[0055] 2. The present invention overexpresses the penicillin-binding protein PonA from Bacillus subtilis on the basis of inhibiting rodZ, which determines cell shape, lytS, which controls pyruvate utilization, hrcA, which negatively controls the expression of molecular chaperones, and ypmS, a gene of unknown function, or ftsZ, a gene that forms a Z ring during division, so that the expression level of α-amylase in the shake flask level in Bacillus subtilis is increased by more than 6.38 times, and the enzyme activity at the 3-L level reaches 102,893U·mL -1 , which is 10.73 times the highest enzyme activity in the shake flask and is currently the highest level of AmyS expression.

[0056] 3. The present invention also optimizes the fermentation conditions of the recombinant bacteria. The recombinant bacteria WS9KA4 is fermented in a nitrogen source of 22 g / L soy peptone and 18 g / L corn steep liquor; a carbon source of 10 g / L potato starch; and a metal ion of 3.5 mmol·L -1 Fe 2+ The enzyme activity can reach 9582U·mL in the medium at 37℃ and 6.5℃ for 48h. -1 , which is 1.15 times that before optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of shake flask bacterial concentration and enzyme activity of recombinant strains containing different sgRNAs expressing α-amylase.

[0058] Figure 2 Schematic diagram of the shake flask bacterial concentration and enzyme activity of the recombinant strain expressing α-amylase before and after knocking out yndF.

[0059] Figure 3 Schematic diagram of shake flask bacterial concentration and enzyme activity of α-amylase expressed by the recombinant strain before and after attenuation of the RBS of lytS and rodZ.

[0060] Figure 4 Schematic diagram of the effect of integrating a single sgRNA on the shake flask bacterial concentration and enzyme activity of the recombinant strain expressing α-amylase.

[0061] Figure 5 Schematic diagram of shake flask bacterial concentration and enzyme activity of recombinant strains expressing α-amylase that integrate multiple sgRNAs.

[0062] Figure 6 Schematic diagram of shake flask bacterial concentration and enzyme activity of α-amylase expressed by the recombinant strain co-expressing PonA.

[0063] Figure 7 The curves of bacterial concentration and enzyme activity of the strain with the optimal sgRNA combination for co-expressing PonA and α-amylase in a 3-L bioreactor.

[0064] Figure 8 Schematic diagram of shake flask bacterial concentration and enzyme activity of α-amylase expressed in the strain with the optimal sgRNA combination before and after knocking out the negative regulatory molecular chaperone hrcA.

[0065] Fig. 9 Schematic diagram of shake flask enzyme activity of recombinant strains expressing ultra-high temperature amylase, β-galactosidase and chitinase in strain WS9K, which is the optimal sgRNA combination for knocking out the negative regulatory molecular chaperone hrcA.

[0066] Fig.10Schematic diagram of shake flask bacterial concentration and enzyme activity of α-amylase expressed by the recombinant strain PonA from Bacillus subtilis co-expressed in WS9K.

[0067] Fig.11 The cell concentration and enzyme activity curves of α-amylase expressed by the recombinant strain of Bacillus subtilis PonA co-expressed in WS9K in a 3-L bioreactor with different carbon sources: (a) 250 g / L maltose and 250 g / L glycerol; (b) 500 g / L glucose; (c) 500 g / L maltose.

[0068] Fig.12 This is a graph showing the bacterial concentration and enzyme activity curve of α-amylase expressed by the recombinant strain PonA from Bacillus subtilis in WS9K in a 3-L bioreactor with a carbon-nitrogen ratio of 4:1. DETAILED DESCRIPTION

[0069] (I) Abbreviation and full name of the invention, as well as Chinese and English translations

[0070] CRISPRi: Clustered Regularly Interspaced Short Palindromic Repeatsinterfence, gene suppression.

[0071] (II) Detection method

[0072] (1) Biomass determination: The bacterial concentration (OD 600 After sampling, the sample was diluted with deionized water by an appropriate multiple and placed in a spectrophotometer to measure the absorbance at 600 nm.

[0073] (2) α-amylase activity assay: The assay method was based on the paper “Enhanced extracellular expression of Bacillus stearothermophilus alpha-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and alpha-amylase mutant selection”. The enzyme activity assay formula is: U (U mL -1 )=2×n×(6.0539×△540+0.3494), where U is the enzyme activity unit, n is the enzyme dilution multiple, △540 is the sample at OD 540 The absorbance value of the blank group was subtracted from the absorbance value measured.

[0074] (3) Determination of ultra-high temperature amylase activity: For specific operations, please refer to the literature (Tan Ruiting, Zhang Kang, Wu Jing. Secretory expression of ultra-high temperature α-amylase from thermophilic archaea in Bacillus subtilis [J]. Genomics and Applied Biology).

[0075] (4) Determination of β-galactosidase activity: For specific operations, please refer to the literature (Xu Junyong. Specific molecular modification and fermentation optimization of Bacillus circulans ATCC31382 β-galactosidase transglycoside products [D]: [Master's thesis]. Wuxi: Jiangnan University, 2022.).

[0076] (5) Chitinase activity determination: For specific operations, please refer to the literature (Yuan Yuan, Su Lingqia, Zhang Kang, Zhu Xuyang, Xia Wei, Wu Jing. Study on the recombinant expression of Bacillus licheniformis chitinase in Bacillus subtilis and its preparation of amino oligosaccharides [J]. Southern Fisheries Science. 2022.018(002):39-47).

[0077] (III) Culture medium

[0078] The culture medium was prepared with ddH2O and sterilized at 121°C for 15-20 min.

[0079] LB liquid medium (g / L): yeast powder 5.00, sodium chloride 10.00, peptone 10.00.

[0080] LB solid medium: add 1.5% (w·v) -1 ) of agar powder.

[0081] TB medium (g / L): yeast powder 24.00, peptone 12.00, glycerol 5.00, potassium dihydrogen phosphate 2.31, potassium hydrogen phosphate 12.54.

[0082] Trace element solution (g / L): manganese sulfate monohydrate 0.10, cobalt chloride hexahydrate 0.18, calcium chloride 0.50, copper sulfate pentahydrate 0.16, zinc sulfate heptahydrate 0.18, ferric chloride 8.35, disodium ethylenediaminetetraacetic acid 10.05.

[0083] (IV) Preparation of competent cells of B. subtilis 1A976 and its derivatives

[0084] (1) Streak the Bacillus subtilis strain from the glycerol tube onto a plate with the corresponding resistance and culture at 37°C for 8-10 h.

[0085] (2) Pick a single colony from the streaked plate and place it in 10 mL of LB medium. Culture it at 37°C and 200 rpm for 10 h.

[0086] (3) The above LB fermentation broth was transferred to another 10 mL LB medium at a 5% inoculum volume and cultured at 37°C and 200 rpm for 4 h.

[0087] (4) Use a pre-cooled pipette tip to draw 170 μL of bacterial cells into a pre-cooled 1.5 mL EP tube, add 30 μL of 70% (w / v) glycerol, and store in a -80°C refrigerator.

[0088] The steps for preparing competent cells of B. subtilis WS9C and its derivatives are the same as above.

[0089] (V) Culture conditions

[0090] Seed liquid culture: Place the glycerol tube stored in a -80℃ refrigerator on ice to melt, use an inoculation loop to dip a small amount of bacterial liquid and streak on a plate containing the corresponding resistance, invert the plate and culture it in a 37℃ constant temperature incubator for 10 hours, then pick a single colony and inoculate it in 10mL LB medium containing the corresponding resistance, incubate at 37℃, 200r·min -1 Cultured for 10 h under the same conditions.

[0091] Shake flask culture: The seed solution cultured in LB medium was transferred to TB medium at a 5% inoculum volume and incubated at 37°C and 200 r·min. -1 The culture was carried out under the same conditions for 2 h, and then the fermentation temperature was changed to 33°C and the culture was continued for 48 h.

[0092] 96-well plate culture: Aliquot 140 μL of 50 μg mL into each well of a sterile 96-well plate. -1 Kanamycin and 5 μg mL -1 Chloramphenicol LB medium, use a sterile toothpick or pipette tip to pick a single colony from the corresponding resistance plate into a 96-well plate, set a diagonal line for inoculating the control strain, and ensure that there is a control strain in each row and column. Incubate at 37°C, 750r / min -1 The cells were cultured in a 96-well plate constant temperature vortex shaking incubator for 10 h. Then 40 μL of LB seed culture solution was transferred to a 96-well deep-well plate containing 600 μL of TB medium (with corresponding antibiotics added), and the deep-well plate was sealed with sterile gauze and incubated at 37°C, 750 r·min -1 The cells were cultured in a 96-well plate constant temperature vortex shaking incubator for 2 h under the same conditions and then cultured at 33°C for 48 h.

[0093] Example 1 Screening of key genes affecting recombinant protein expression

[0094] Using α-amylase (55kDa) as the exogenous target protein expression object, referring to the strategy of application number 202410699284.1, CRISPRi technology and sgRNA library targeting the coding region of the whole genome were used in B. subtilis 1A976 to screen single genotype expression inhibition strains. The specific steps are:

[0095] (1) Integrating dCas9 protein into the lacA site of B. subtilis 1A976 genome (the strain was disclosed in the paper “Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis”) using Cre / lox: First, the dCas9 expression cassette and the upstream and downstream homologous regions of lacA amplified from the B. subtilis 1A976 genome were amplified using PCR, and then the ligated product lacA-dCas9 (shown in SEQ ID NO. 21) was transformed into B. subtilis 1A976 genome using Overlap PCR. 1A976 competent state, ice bath for 20 minutes, 37°C water bath for 20 minutes, 37°C, 200 rpm recovery for 1.5 hours, spread on LB plates containing spectinomycin (50 μg / mL), cultured overnight, and the grown single colonies were verified by colony PCR for successful integration, and then transformed with the pBE-ST recombinant plasmid containing Cre recombinase and a thermosensitive type (disclosed in the patent application with publication number CN117821485A), spread on LB plates containing kanamycin resistance (40 μg / mL), cultured overnight, and the grown single colonies were verified to have eliminated tetracycline resistance. The strain was named SCK6D.

[0096] (2) The α-amylase gene amyS (shown in SEQ ID NO.2) and the vector fragment were amplified from the plasmid containing the α-amylase gene (the plasmid was disclosed in "Identification and optimization of genes potentially related to protein expression for enhancing α-amylase production in Bacillus subtilis") and the pUB110 vector by PCR. After gel recovery, they were connected by POE-PCR and transformed into B. subtilis 1A976. After verification by sequencing the extracted plasmid, the correct recombinant plasmid pUB110-amyS was obtained.

[0097] (3) The plasmid pUB110-amyS constructed in step (2) was transformed into the strain SCK6D into which the dCas9 protein had been integrated, and then the plasmid library containing sgRNA was transformed into the strain. The resulting strain was named SCK6DA, and nearly 3,500 single clones were selected for initial screening in a 96-well plate.

[0098] (4) Select a strain with higher enzyme activity than the control strain SCKDA0 (containing pAD123-empty N20 plasmid in the strain SCK6DA) for shake flask verification; the specific steps are:

[0099] Seed liquid culture: Place the glycerol tube stored in a -80℃ refrigerator on ice to melt, use an inoculation loop to dip a small amount of bacterial liquid and streak on a plate containing the corresponding resistance, invert the plate and culture it in a 37℃ constant temperature incubator for 10 hours, then pick a single colony and inoculate it in 10mL LB medium containing the corresponding resistance, incubate at 37℃, 200r·min -1 Cultured for 10 h under the same conditions.

[0100] Shake flask culture: The seed solution cultured in LB medium was transferred to TB medium at a 5% inoculum and incubated at 37°C and 200 r / min. -1 The culture was carried out under the same conditions for 2 h, and then the fermentation temperature was changed to 33°C and the culture was continued for 48 h.

[0101] The results showed that the bacterial concentrations of the strains after 48 hours of shake flask fermentation were relatively consistent and did not show significant differences. Compared with the empty control strain SCKDA, a total of 13 recombinant strains could increase the enzymatic activity of α-amylase by 1.18-1.68 times.

[0102] The sgRNA of the recombinant strain with improved expression ability was sequenced and identified; after comparison of the sequencing results, a total of 13 key genes for enhancing protein expression were screened, as shown in Table 1: cytoskeletal protein gene mreB (Gene ID: 936759), cell shape determining gene rodZ (Gene ID: 939591), spore germination related gene yndF (Gene ID: 939547), methionine glutamine aminotransferase gene mtnE (Gene ID: 939330), pyruvate utilization control gene lytS (Gene ID: 937404), tRNA modification related gene tsaD (Gene ID: 938020), phosphoserine phosphorylase gene rsbX (Gene ID: 938155), citrate iron transporter gene yfmF (Gene ID: 936117), negative control molecular chaperone expression gene hrcA (Gene ID: 937843), Z ring formation gene ftsZ (Gene ID: 937843) during division. ID: 935971), one of the genes with unknown functions, yoaQ (Gene ID: 940010), and ypmS (Gene ID: 939100).

[0103] Table 1 Functional annotations of key genes screened and their sgRNA sequences

[0104]

[0105] Example 2 Construction of a recombinant strain with knockout of the spore germination gene yndF

[0106] Using α-amylase (55 kDa) as the exogenous target protein for expression, the yndF knockout strain SCK7A was constructed based on B. subtilis 1A976 using CRISPR / Cas9.

[0107] The specific steps are:

[0108] (1) Using CRISPR / Cas9 to knock out yndF: First, the PCR method was used to replace the targeting sgRNA using the laboratory-preserved plasmid pHYcas9aprE (published in the master's thesis "Study on the transformation of Bacillus subtilis strains, promoter optimization and efficient preparation of pullulanase") as a template. Subsequently, the vector backbone was amplified using the successfully replaced plasmid as a template. At the same time, the B. subtilis 1A976 genome was used as a template, and primers were used to add a 19bp random insertion sequence. The upper and lower homologous arms of yndF were then amplified using the primers.

[0109] (2) The vector backbone and homologous repair sequence were connected using a one-step cloning kit, transformed into E. coli JM109 competent cells, and plated on ampicillin-resistant plates. After overnight culture, a single colony was picked from the plate to extract the plasmid and verify the enzyme digestion. If correct, it was sent to the company for sequencing. The correctly sequenced plasmid obtained was pHYcas9yndF.

[0110] (3) pHYcas9yndF was transformed into B. subtilis 1A976 competent cells, spread on tetracycline-resistant plates, and colony PCR was performed with primers. After the PCR product was verified to be correct by XhoI digestion, it was sequenced and identified. After verification of successful editing, the colony was picked up and placed in LB medium without antibiotics at 51°C and 200 r / min. -1 The cells were cultured under the same conditions for 10 h, and the obtained colonies were printed onto resistance-free plates and tetracycline-resistant plates, respectively. Single colonies that could only grow on resistance-free plates but not on tetracycline-resistant plates were picked out, and the obtained strain without plasmid was SCK7.

[0111] (4) The plasmid pUB110-amyS was transformed into SCK7 competent cells and spread on kanamycin-resistant plates. After overnight culture, a single colony was picked from the plate to extract the plasmid and verified by enzyme digestion. If it was correct, it was sent to the company for sequencing. The strain with correct sequencing was SCK7A.

[0112] The strain SCK7A was cultured under the culture conditions of Example 1. The results showed that after 48 h of shake flask fermentation, compared with the control strain SCK6A, knocking out the spore germination gene yndF increased the enzymatic activity of α-amylase by 1.30 times.

[0113] Example 3 Construction of recombinant strains with reduced RBS strength of lytS and rodZ

[0114] α-Amylase (55kDa) was used as the exogenous target protein for expression, and based on SCK7, CRISPR / Cas9 was used to construct the RBS attenuated strains SCK8A and SCK9A of lytS and rodZ.

[0115] The specific steps are:

[0116] (1) Using CRISPR / Cas9 to weaken the RBS of lytS: First, the PCR method was used to replace the targeting sgRNA using the plasmid pHYcas9aprE as a template. Then, the vector backbone was amplified using the successfully replaced plasmid as a template. At the same time, the upper and lower homologous arms of lytS were amplified using primers using the B. subtilis1A976 genome as a template.

[0117] (2) The vector backbone and homologous repair sequence were connected using a one-step cloning kit, transformed into E. coli JM109 competent state, and plated on an ampicillin-resistant plate. After overnight culture, a single colony was picked from the plate to extract the plasmid and verify the enzyme digestion. After verification, it was sent to the company for sequencing. The obtained plasmid with correct sequencing was pHYcas9lytS.

[0118] (3) pHYcas9lytS was transformed into B. subtilis 1A976 competent cells, spread on tetracycline-resistant plates, and colony PCR was performed with primers. After verification, sequencing was performed. After verification of successful editing, the colony was picked up and placed in LB medium without antibiotics at 51°C and 200 r / min. -1 The cells were cultured under the same conditions for 10 h, and the obtained colonies were printed onto resistance-free plates and tetracycline-resistant plates, respectively. Single colonies that could only grow on resistance-free plates but not on tetracycline-resistant plates were picked out, and the obtained strain without plasmid was SCK8.

[0119] (4) The plasmid pUB110-amyS was transformed into SCK8 competent cells and spread on kanamycin-resistant plates. After overnight culture, a single colony was picked from the plate to extract the plasmid and verified by enzyme digestion. If it was correct, it was sent to the company for sequencing. The strain with correct sequencing was SCK8A.

[0120] The same strategy as above was used to construct the strain SCK9A with attenuated RBS of gene rodZ.

[0121] The strains SCK8A and SCK9A were cultured for 48 h under the shake flask culture conditions of Example 1. The results showed that compared with the control strain SCK7A, reducing the RBS strength of lytS and rodZ did not further increase the α-amylase activity. This may be due to the inconsistency between the RBS strength and the CRISPRi inhibition strength, resulting in no further increase in enzyme activity.

[0122] Table 2 RBS strength and sequence of lytS and rodZ

[0123]

[0124]

[0125] Example 4 Construction of a recombinant strain integrating a single sgRNA

[0126] α-Amylase (55kDa) was used as the exogenous target protein for expression. Based on B. subtilis WS9C, the dCas9 protein was integrated at the epr site to obtain the strain WS9D. Subsequently, the sgRNA expression frame targeting the key gene affecting the expression of the recombinant protein was integrated at the lacA site in WS9D to construct the integrated Bacillus subtilis recombinant bacteria WS9F0A-WS9F13A.

[0127] The specific steps are:

[0128] (1) Integrate the sgRNA expression frame into the lacA site of the WS9D genome using Cre / lox: First, use the PCR method to amplify the upstream and downstream homologous regions of lacA on the sgRNA expression frame and the B. subtilis WS9C genome. After Overlap PCR connection, the connection product is transformed into the WS9C competent cell, ice bathed for 20 minutes, 37°C water bath for 20 minutes, 37°C, 200 rpm for 1.5 hours, and spread on LB plates containing tetracycline (20 μg / mL) for overnight culture. After the single colonies grown are verified by colony PCR for successful integration, they are transformed with the pBE-ST recombinant plasmid containing Cre recombinase and temperature-sensitive type, and spread on LB plates containing kanamycin resistance (40 μg / mL) for overnight culture. After the single colonies grown are verified to have eliminated tetracycline resistance, the strains are named WS9F0-WS9F13.

[0129] (2) The plasmid pUB110-amyS was transformed into WS9F0-WS9F13 competent cells and spread on kanamycin-resistant plates. After overnight culture, a single colony was picked from the plate to extract the plasmid and verify by enzyme digestion. If it was correct, it was sent to the company for sequencing. The strains with correct sequencing were WS9F0A-WS9F13A.

[0130] Among them, strains WS9C, WS9D and plasmid pBE-ST were previously preserved in the laboratory.

[0131] Preparation of competent cells of WS9C and its derivatives:

[0132] (1) Streak the Bacillus subtilis strain from the glycerol tube onto a plate with the corresponding resistance and culture at 37°C for 8-10 h.

[0133] (2) Pick a single colony from the streaked plate and place it in 10 mL of LB medium. Culture it at 37°C and 200 rpm for 10 h.

[0134] (3) The above LB fermentation broth was transferred to another 10 mL LB medium at a 5% inoculum volume and cultured at 37°C and 200 rpm for 4 h.

[0135] (4) Use a pre-cooled pipette tip to draw 170 μL of bacterial cells into a pre-cooled 1.5 mL EP tube, add 30 μL of 70% (w / v) glycerol, and store in a -80°C refrigerator.

[0136] Table 3 List of recombinant strains integrating a single sgRNA

[0137]

[0138]

[0139] Note: The relevant sgRNA sequences are shown in Table 1.

[0140] The strains SCK8A and SCK9A were cultured in shake flasks for 48 h according to the culture conditions of Example 1. The results showed that compared with the control strain WS9F0A, the results showed that the integration of sgRNA lytS , sgRNA ypmS , sgRNA ftsZ , sgRNA hrcA and sgRNA rodZ The enzyme activities of the corresponding recombinant strains WS9F5A, WS9F10A, WS9F13A, WS9F12A and WS9F2A were 1665, 1755, 2295, 2310 and 2640 U·mL -1 , which were 1.11, 1.17, 1.53, 1.54 and 1.76 times that of the control strain WS9F0A (non-targeted N20 was integrated at the same site).

[0141] Example 5 Construction of recombinant strains integrating multiple sgRNAs

[0142] α-Amylase (55 kDa) was used as the exogenous target protein for expression. Based on B. subtilis WS9F2, sgRNA expression frames targeting key genes affecting recombinant protein expression were integrated at the lacA site, mpr site, and spoIIAC site (the sgRNA sequences are shown in Table 1) to construct the integrated Bacillus subtilis recombinant bacteria WS9GA-WS9JA.

[0143] The specific steps are:

[0144] (1) Taking the construction of WS9GA as an example, the sgRNA expression cassette was integrated into the lacA site of the WS9D genome using Cre / lox: First, the sgRNA expression cassette (sgRNA) was integrated into the lacA site of the WS9D genome using the PCR method. rodZ and sgRNA lytS) and B. subtilis WS9C genomes were amplified with lacA upstream and downstream homologous regions, and connected by Overlap PCR. The ligation products were transformed into WS9C competent cells, incubated on ice for 20 min, in a 37°C water bath for 20 min, and revived at 37°C, 200 rpm for 1.5 h. The cells were spread on LB plates containing tetracycline (20 μg / mL) and cultured overnight. After the single colonies grown were verified for successful integration by colony PCR, they were transformed with the recombinant plasmid containing Cre recombinase and the thermosensitive pBE-ST, and spread on LB plates containing kanamycin resistance (40 μg / mL). After overnight culture, the single colonies grown were verified to have eliminated tetracycline resistance, and the strain was named WS9G.

[0145] (2) The plasmid pUB110-amyS was transformed into WS9F0-WS9F13 competent cells and spread on kanamycin-resistant plates. After overnight culture, a single colony was picked from the plate to extract the plasmid and verify by enzyme digestion. If it was correct, it was sent to the company for sequencing. The strain with correct sequencing was WS9GA.

[0146] The construction of WS9G0A-WS9JA was the same as above, and the strain characteristics are shown in Table 4.

[0147] Table 4 Recombinant bacteria integrating different sgRNA combinations

[0148]

[0149]

[0150] Note: The relevant sgRNA sequences are shown in Table 1.

[0151] The strain was cultured in a shake flask for 48 h according to the culture conditions of Example 1. The results showed that the sgRNA was integrated. rodZ -sgRNA lytS The enzyme activity of WS9GA strain reached 3270 U·mL -1 , which is 2.18 times that of the control strain WS9F0A, and the biomass has increased; sgRNA has been integrated rodZ -sgRNA ypmS The enzyme activity of WS9G0A reached 2925 U·mL -1 , which is 1.95 times that of the control strain WS9F0A. Strain WS9G was selected as the basis to continue to superimpose sgRNA hrcA , integrating sgRNA hrcA The WS9HA strain not only further increased the expression of AmyS, but also increased the enzyme activity to 3630 U·mL -1 , which is 2.42 times that of the control strain WS9F0A, and the biomass has increased. Strain WS9H was selected as the basis to continue to superimpose sgRNAypmS and sgRNA ftsZ , integrating sgRNA ympS The WS9IA strain further increased the expression of AmyS, with the enzyme activity reaching 3735 U·mL -1 , which is 2.49 times that of the control strain WS9F0A; integrated sgRNA ftsZ The enzyme activity of WS9I0A strain reached 3675 U·mL -1 .

[0152] Based on the four sgRNA integration strain B. subtilis WS9I with the most significant improvement effect, superimposed sgRNA ftsZ . Integration of sgRNA ftsZ The WS9JA strain not only further increased the expression of AmyS, but also increased the enzyme activity to 4215 U·mL -1 , which is 2.81 times that of the control strain WS9F0A, and the biomass increased.

[0153] Example 6 Construction of recombinant strains co-expressing PonA

[0154] α-Amylase (55kDa) was used as the exogenous target protein for expression. Based on B. subtilis WS9JA, a recombinant plasmid containing ponA was co-expressed to construct a WS9JA derivative with enhanced recombinant protein production capacity.

[0155] The specific steps are:

[0156] (1) constructing a recombinant plasmid containing ponA; the specific steps are: amplifying the gene fragment shown in SEQ ID NO.8, connecting it to the pAD123 vector through one-step cloning after gel recovery, and using the maltose-inducible promoter P glv The expression of ponA was regulated and then transformed into Escherichia coli JM109, and the correct recombinant plasmid was obtained after sequencing verification.

[0157] The empty plasmids pAD123-CK and pAD123-P were respectively transformed into WS9JA. glv -ponA, construct strains WS9JA0 and WS9JA1.

[0158] The strain was cultured under the shake flask culture conditions of Example 1 for 48 h, and the enzyme activity of the recombinant strain WS9JA1 reached 4770 U·mL -1 , which was 1.13 times that of the control strain WS9JA0, and the biomass was also increased.

[0159] Example 7 Strain WS9JA1 produces AmyS enzyme in a 3-L fermenter

[0160] α-Amylase (55 kDa) was used as the exogenous target protein expression object. In order to further verify the ability of the recombinant strain WS9JA1 to produce AmyS, the enzyme production ability of the strain was verified at the 3-L tank level. The specific steps are:

[0161] (1) Seed liquid culture: Place the glycerol tube stored in a -80℃ refrigerator on ice to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on a plate containing the corresponding resistance, invert the plate and incubate it in a 37℃ constant temperature incubator for 10 h, then pick a single colony and inoculate it in 10 mL LB medium containing the corresponding resistance, incubate at 37℃, 200 r·min -1 Cultured for 10 h under the same conditions.

[0162] (2) 3-L tank fermentation: 100 mL of seed solution was quickly added to a 3-L fermentation tank using the flame-sealed inoculation method. The dissolved oxygen was maintained at 30%. The dissolved oxygen coupling was set. The rotation speed and oxygen ventilation were adjusted according to the dissolved oxygen. The system temperature was 37°C. When the dissolved oxygen showed an upward trend, feed was started and pH coupling was turned on. The pH value was set to 6.5. 20% (v·v -1 ) phosphoric acid and pure ammonia water to adjust the pH. When feeding, the feed rate was 0.01 mL min -1 Feed medium was added to the fermenter at a flow rate of 100 mL / min. According to the changes in bacterial growth during the fermentation process, the feed flow rate was gradually increased until it reached 0.1 mL / min. -1 , then, keep the feed flow rate constant until the end of fermentation.

[0163] Fermentation medium (g / L): corn steep liquor 10.00, soy peptone 10.00, sucrose 5.00, anhydrous calcium chloride 0.35, magnesium phosphate heptahydrate 1.00, ammonium citrate 1.00, ammonium sulfate 2.68, sodium sulfite 2.00, dipotassium hydrogen phosphate 14.60, sodium dihydrogen phosphate monohydrate 4.00. 3.00 mL of trace element solution should be added to each liter of basic culture medium.

[0164] Feed medium (g / L): corn steep liquor 35.00, soy peptone 35.00, glucose 500.00. 40.00 mL of trace element solution should be added per liter of feed medium.

[0165] Trace element solution (g / L): manganese sulfate monohydrate 0.10, cobalt chloride hexahydrate 0.18, calcium chloride 0.50, copper sulfate pentahydrate 0.16, zinc sulfate heptahydrate 0.18, ferric chloride 8.35, disodium ethylenediaminetetraacetic acid 10.05.

[0166] The results showed that the AmyS enzyme activity of the recombinant strain WS9JA1 reached the highest level of 16,906 U·mL after 120 h of fermentation. -1 , which is 3.54 times the shake bottle level.

[0167] Example 8 Construction of WS9J derivatives with hrcA knockout

[0168] Referring to the strategy of Example 3, based on the B. subtilis WS9J constructed in Example 6, hrcA was knocked out to construct a WS9J derivative of the Bacillus subtilis chassis strain with improved recombinant protein expression ability, named WS9K.

[0169] The strain WS9K was fermented under the same conditions as in Example 1. The results showed that after 48 h of shake flask fermentation, the AmyS enzyme activity of the hrcA knockout strain WS9KA was 6491 U·mL -1 , which is 1.54 times that of WS9JA.

[0170] Example 9 Evaluation of the effect of chassis strains on the expression of recombinant proteins

[0171] Ultrahigh temperature α-amylase Pfa from Pyrococcus furiosus (shown in SEQ ID NO.18), chitinase BlChiA2 from Bacillus licheniformis (shown in SEQ ID NO.19) and β-galactosidase β-gal from Bacillus circulans ATCC 31382 (shown in SEQ ID NO.20) were used as the exogenous target protein expression objects to verify the ability of the chassis strain WS9K to express other recombinant proteins.

[0172] The fermentation time of the recombinant bacteria expressing Pfa, BlChiA2, and β-gal was 60, 48, and 24 h, respectively, and the other fermentation conditions were consistent with the shake flask fermentation conditions in Example 1. The expression of the extracellular enzyme Pfa, BlChiA2, and the intracellular enzyme β-gal in WS9K were 1.54, 1.12, and 2.05 times that of the corresponding WS9C control strain, respectively.

[0173] Example 10 Construction of recombinant strains for optimizing the promoter of ponA

[0174] α-Amylase (55k Da) was used as the exogenous target protein for expression. Based on the B. subtilis WS9KA constructed in Example 6, recombinant plasmids containing ponA mediated by different promoters were co-expressed to construct recombinant protein capacity-enhanced strains WS9KA4 and WS9KA5.

[0175] The specific steps are as follows: referring to the plasmid construction and strain construction steps of Example 4, the derived strains obtained are shown in Table 3.

[0176] The strain was kept in the same shake flask fermentation conditions as in Example 1. The results showed that after 48 h of fermentation, the promoter P HpaII The enzyme activity of strain WS9KA4 mediated by ponA overexpression reached 8308 U·mL -1 , which is 1.28 times that of the control strain WS9KA0.

[0177] Table 5 Recombinant bacteria expressing AmyS

[0178] Recombinant bacteria feature WS9KA0 WS9KA derivative strain containing pAD123 empty plasmid WS9KA3 <![CDATA[WS9KA-derived strain, containing pAD123-P glv -ponA plasmid]]> WS9KA4 <![CDATA[WS9KA-derived strain, containing the pAD123-P HpaII -ponA plasmid]]> WS9KA5 <![CDATA[WS9KA-derived strain, containing the pAD123-P aprE -ponA plasmid]]>

[0179] Example 11 Optimization of shake flask fermentation of strain WS9KA4

[0180] Taking α-amylase (55kDa) as the exogenous target protein expression object, in order to improve the ability of recombinant bacteria WS9KA4 to produce AmyS by fermentation, the fermentation medium components and culture conditions were first optimized at the shake flask level. Among them, the optimization of medium components includes the optimization of the types and concentrations of nitrogen sources, carbon sources and metal ions; the optimization of culture conditions includes the optimization of fermentation temperature and pH.

[0181] (1) Nitrogen source optimization

[0182] On the basis of TB medium, the concentration of 36g / L tryptone and yeast powder in TB medium was replaced with the same concentration of porcine bone peptone, soy peptone (marginal peptide), soy peptone (Wokai), corn steep liquor (Angel), yeast extract, Angel yeast extract powder, polypeptone, tryptone, beef powder, nutrient broth (NB), bacteriological peptone, beef peptone, yeast powder, industrial corn steep liquor (Vita), soybean meal powder, cottonseed powder and industrial Angel yeast extract, and the replaced medium was used to shake flask ferment the recombinant bacteria WS9KA4. The shake flask fermentation conditions were consistent with those in Example 1. The results showed that after 48h of fermentation culture, when Angel yeast extract powder, polypeptone or yeast powder was used as the single nitrogen source, the AmyS enzyme activity was 5996U·mL -1 、5981U·mL -1 、5843U·mL -1 When soybean peptone (Wokai), yeast extract, industrial Angel yeast extract, corn steep liquor and industrial corn steep liquor were used as single nitrogen sources, the recombinant strain WS9KA4 could also obtain relatively high AmyS enzyme activity, which was 4811 U·mL -1 、4343U·mL -1 、4272U·mL -1 、4058U·mL -1 and 3671U·mL -1 Therefore, the concentration of the above 8 nitrogen sources will be optimized in the future.

[0183] (2) Optimization of nitrogen source concentration

[0184] Based on the optimization results of nitrogen source types, the production and growth of AmyS by recombinant bacteria WS9KA4 were investigated under shake flask fermentation conditions of Angel yeast extract powder, polypeptone, yeast powder, soy peptone (Wokai), yeast extract, industrial Angel yeast extract, corn steep liquor or industrial corn steep liquor at concentrations of 20, 28, 36 and 44 g / L, respectively. The fermentation conditions were consistent with those in Example 1. The results showed that after 48 h of shake flask fermentation, when the optimal yeast powder concentration was 44 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 6866 U·mL -1 And OD 600 When the optimum concentration of polypeptone was 44 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 6144 U·mL -1 And OD 600 When the optimum concentration of soybean peptone (Wokai) was 28 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 5625 U·mL -1 And OD 600 When the optimum concentration of Angel yeast extract powder was 28 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 4862 U·mL -1 And OD 600 When the optimum concentration of industrial Angel yeast extract powder was 44 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 4455 U·mL -1 And OD 600 When the optimum concentration of corn steep liquor was 44 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 4261 U·mL -1 And OD 600 is 6.32; the optimum concentration of yeast extract is 44g / L and the enzyme activity is 3977U·mL -1 And OD 600 When the optimum concentration of industrial corn syrup was 36 g / L, the enzyme activity of recombinant bacteria WS9KA4 was 3671 U·mL -1 And OD 600 The value is 11.52. Since the enzyme activity at lower concentrations using polypeptone or yeast powder as a single nitrogen source is not much different from that using soy peptone (Wokai) or Angel yeast extract powder as a single nitrogen source, and the cost of polypeptone or yeast powder is relatively high, polypeptone and yeast powder are no longer used in subsequent nitrogen source compounding. At the same time, except for industrial corn syrup as a single carbon source, the biomass of recombinant bacteria WS9KA4 cultured in other culture media is relatively low. Therefore, in order to balance enzyme production and growth and reduce the cost of industrial application, the ratio of industrial corn syrup and soy peptone (Wokai) or Angel yeast extract powder composite nitrogen source is subsequently optimized.

[0185] (3) Optimization of compound nitrogen sources

[0186] Soybean peptone (Wokai) and Angel yeast extract powder were compounded with industrial corn steep liquor, and orthogonal experiments were conducted with soybean peptone (Wokai) and Angel yeast extract powder at concentrations of 10, 14, 18 and 22 g / L and industrial corn steep liquor at concentrations of 10, 14, 18 and 22 g / L, respectively. The rest of the fermentation conditions were the same as in Example 1. The results showed that after 48 h of shake flask fermentation, when the concentration of soybean peptone (Wokai) was 22 g / L and the concentration of industrial corn steep liquor was 18 g / L, the AmyS enzyme activity of the recombinant bacteria WS9KA4 was the highest, which was 5899 U·mL -1 , which is higher than the AmyS enzyme activity when soybean protein or corn syrup is used as the single nitrogen source. Therefore, the subsequent optimization will be based on this composite nitrogen source ratio.

[0187] (4) Optimization of carbon source types

[0188] In the optimization of the composite nitrogen source (soybean peptone concentration of 22 g·L -1 The industrial corn syrup concentration is 18 g·L -1 ), fructose, corn starch, glucose, maltose, soluble starch, sucrose, potato starch, corn dextrin, xylose, maltodextrin or dextrin at a concentration of 5 g / L were used to replace the glycerol in the original culture medium for shake flask fermentation of the recombinant bacteria WS9KA4. Other fermentation conditions were consistent with Example 1. The results showed that after 48 hours of shake flask fermentation, the carbon sources used by the obtained AmyS enzyme activity from high to low were: potato starch, corn starch, maltose, soluble starch, and the corresponding AmyS enzyme activities were: 6982 U·mL -1 、6944U·mL -1 、6924U·mL -1 、6744U·mL -1 .

[0189] (5) Optimization of carbon source concentration

[0190] Based on the optimization of the carbon source type mentioned above, the enzyme production of recombinant bacteria WS9KA4 in shake flask fermentation in medium with corn starch, soluble starch, potato starch or maltose at concentrations of 5, 10, 15, 20, 25 and 30 g / L was investigated. The rest of the fermentation conditions were consistent with Example 1. The results showed that after 48 h of shake flask fermentation, when soluble starch at a concentration of 25 g / L was used as the carbon source, the AmyS enzyme activity reached 8163 U·mL -1 When soluble starch at a concentration of 30 or 20 g / L was used as the carbon source, the AmyS enzyme activity could reach 7593 U·mL -1 and 7502U·mL -1When potato starch with a concentration of 10 g / L was used as the carbon source, the AmyS enzyme activity could reach 7156 U·mL -1 Since soluble starch is relatively expensive and the enzyme activity advantage is only significant at high concentrations, the subsequent culture medium optimization will use 10g / L potato starch as the carbon source.

[0191] (6) Optimization of metal ion types

[0192] Based on the optimization of the above carbon source types (10 g·L -1 Potato starch was used as the carbon source, with 22 g·L -1 Soybean peptone 18g·L -1 Industrial corn syrup as nitrogen source) and the recombinant strain WS9KA4 was investigated in the presence of 1.0 mmol·L -1 Ca 2+ 、Al 3+ , Mn 2+ , Fe 3+ , Fe 2+ Mg 2+ 、Zn 2+ 、Co 2+ and Cu 2+ The results showed that after 48 h of fermentation, 1.0 mmol·L -1 Fe 2+ or Ca 2+ Both contribute to AmyS expression, with enzyme activities of 7695 U·mL -1 and 7283U·mL -1 Therefore, the following will explore Fe 2+ and Ca 2+ The optimal amount of addition.

[0193] (7) Optimization of metal ion concentration

[0194] Based on the optimization of the above carbon source types (10 g·L -1 Potato starch was used as the carbon source, with 22 g·L -1 Soybean peptone 18g·L -1 Industrial corn syrup was used as the nitrogen source. The recombinant strain WS9KA4 was investigated in the presence of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mmol·L -1 Ca 2+ or Fe 2+ The results showed that when 3.5 mmol·L -1 Fe2+ Optimal, enzyme activity can reach 8433U·mL -1 , which was 17% higher than that of the control group with deionized water added; Ca 2+ The optimum addition amount is 1mmol·L -1 , enzyme activity is 7695U·mL -1 , which was only 7% higher than the control group with deionized water. 2+ The positive effect was not significant, so only 3.5 mmol·L -1 Fe 2+ .

[0195] (8) Fermentation temperature optimization

[0196] Based on the optimization of the above culture medium components (10 g·L -1 Potato starch was used as the carbon source, with 22 g·L -1 Soybean peptone 18g·L -1 Industrial corn syrup is the nitrogen source and the metal ion content is 3.5mmol·L -1 Fe 2+ ), the production of AmyS by shake flask fermentation of recombinant bacteria WS9KA4 under fermentation conditions of different temperatures (25, 30, 33, 37 and 42°C) was investigated. The remaining fermentation conditions were consistent with Example 1. The results showed that after 48 hours of fermentation, as the fermentation temperature increased, the AmyS enzyme activity of the recombinant bacteria WS9KA4 showed a trend of first increasing and then decreasing. When the fermentation temperature was 25-42°C, the enzyme activity could reach 3133U·mL -1 The fermentation temperature is 37℃, which is the most suitable for AmyS expression, and the enzyme activity can reach 9099U·mL -1 .

[0197] (9) pH optimization

[0198] In the above optimization results (10g·L -1 Potato starch was used as the carbon source, with 22 g·L -1 Soybean peptone 18g·L -1 Industrial corn syrup is the nitrogen source and the metal ion content is 3.5mmol·L -1 Fe 2+ , fermentation temperature was 37°C), and the production of AmyS by recombinant bacteria WS9KA4 in shake flasks in medium with pH values ​​of 5.5 to 8.5 was investigated. The remaining fermentation conditions were consistent with Example 1. The results showed that after 48 hours of fermentation, as the pH value of the medium increased, the AmyS enzyme activity of the recombinant bacteria WS9KA4 showed a trend of first increasing and then decreasing. When the pH value of the medium was 6.5, the AmyS enzyme activity was the highest, at 9582U·mL -1.

[0199] Based on the above optimization results, the optimal fermentation conditions of recombinant bacteria WS9KA4 were as follows: nitrogen source was 22 g / L soy peptone and 18 g / L corn steep liquor; carbon source was 10 g / L potato starch; metal ion was 3.5 mmol·L -1 Fe 2+ The optimal shake flask fermentation temperature and pH were 37°C and 6.5, respectively. The highest enzyme activity obtained after optimization of shake flask fermentation conditions was 9582 U·mL -1 , which is 1.15 times that before optimization.

[0200] Example 12 3-L fermenter fermentation of strain WS9KA4

[0201] α-Amylase (55kDa) was used as the exogenous target protein expression object. In order to further improve the ability of recombinant strain WS9KA4 to produce AmyS and reduce the cost of industrial enzymes, the carbon source type and carbon-nitrogen ratio in the feed medium were optimized at the 3-L tank level. 250g / L maltose and 250g / L glycerol were used as the carbon source of the feed medium, specifically:

[0202] Fermentation medium (g / L): corn steep liquor 18.00, soy peptone 22.00, potato starch 10.00, magnesium sulfate ferrous heptahydrate 0.97, magnesium phosphate heptahydrate 1.00, ammonium citrate 1.00, ammonium sulfate 2.68, sodium sulfite 2.00, dipotassium hydrogen phosphate 14.60, sodium dihydrogen phosphate monohydrate 4.00. 3.00 mL of trace element solution should be added to each liter of basal medium.

[0203] Feed medium (g / L): corn steep liquor 35, soy peptone 35, maltose 250.00 and glycerol 250.00. 40.00 mL of trace element solution should be added to each liter of basic medium.

[0204] Trace element solution (g / L): manganese sulfate monohydrate 0.10, cobalt chloride hexahydrate 0.18, calcium chloride 0.50, copper sulfate pentahydrate 0.16, zinc sulfate heptahydrate 0.18, ferric chloride 8.35, disodium ethylenediaminetetraacetic acid 10.05.

[0205] Using the flame-sealed inoculation method, 100 mL of seed solution was quickly added to a 3-L fermenter containing fermentation medium, the dissolved oxygen was maintained at 30%, the dissolved oxygen coupling was set, the speed and oxygen ventilation were adjusted according to the dissolved oxygen, and the system temperature was 37°C. When the dissolved oxygen showed an upward trend, feeding was started and pH coupling was turned on, the pH value was set to 6.5, and 20% (v·v -1 ) phosphoric acid and pure ammonia water to adjust the pH. When feeding, the feed rate was 0.01 mL min -1Feed medium was added to the fermenter at a flow rate of 100 mL / min. According to the changes in bacterial growth during the fermentation process, the feed flow rate was gradually increased until it reached 0.1 mL / min. -1 , then, keep the feed flow rate constant until the end of fermentation.

[0206] After 112 h of fermentation, the AmyS of recombinant strain WS9KA4 reached 18,715 U·mL -1 .

[0207] Example 13 Optimization of 3-L Fermentation Tank Feed Medium for Strain WS9KA4

[0208] The specific implementation manner is the same as Example 12, except that the carbon source of the feed medium is adjusted to 500 g / L glucose and 500 g / L maltose respectively, and the other fermentation parameters remain unchanged.

[0209] The results showed that when 500 g / L glucose was used as the carbon source of the feed medium, the AmyS of the recombinant strain WS9KA4 reached the highest level at 22,581 U·mL after 104 h of fermentation. -1 ; When 500 g / L maltose was used as the carbon source of the feed medium, the AmyS of the recombinant strain WS9KA4 reached the highest level at 28,227 U·mL after 108 h of fermentation. -1 .

[0210] Example 14 Optimization of the carbon-nitrogen ratio of the feed medium of strain WS9KA4 in a 3-L fermenter

[0211] Specific implementation method The same as Example 12, except that the carbon-nitrogen ratio in the feed medium is adjusted to 4:1, and the feed medium composition is (g / L): corn steep liquor 50.40, soy peptone 61.60, glucose 500, and 40.00 mL of trace element solution is added per liter of basal medium. The other fermentation parameters remain unchanged.

[0212] The results showed that the AmyS enzyme activity increased rapidly after 120 h and reached the highest level at 148 h, reaching 102,983 U·mL -1 , which is the highest level of AmyS expression so far.

[0213] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for increasing the expression of exogenous protein in Bacillus subtilis, characterized in that: The key gene affecting the expression of recombinant proteins is inhibited, and the class A penicillin-binding protein PonA is expressed; the key gene affecting the expression of recombinant proteins is the gene rodZ that determines the cell shape; the class A penicillin-binding protein PonA is expressed through a plasmid, and the key gene affecting the expression of recombinant proteins is inhibited by integrating its targeting sgRNA into the genome to inhibit the expression of the corresponding gene.

2. The method according to claim 1, characterized in that: The targeted sgRNA of the key genes affecting the expression of the recombinant protein was integrated into the lacA, mpr or spoIIAC sites on the Bacillus subtilis genome.

3. The method according to claim 1, characterized in that pAD123 was used as the expression vector and the promoter P glv , P HpaII or P aprE Regulate the expression of the class A penicillin-binding protein gene shown in SEQ ID NO.

1.

4. The method according to claim 2, characterized in that: pAD123 was used as the expression vector and the promoter P glv , P HpaII or P aprE Regulate the expression of the class A penicillin-binding protein gene shown in SEQ ID NO.

1.

5. The method according to any one of claims 1 to 4, characterized in that: The exogenous protein is alpha-amylase, beta-galactosidase, ultra-high temperature amylase or chitinase.

6. A Bacillus subtilis chassis cell with improved recombinant protein expression ability, characterized in that: The Bacillus subtilis chassis cells inhibit key genes that affect the expression of recombinant proteins; inhibit the expression of any gene described in (a) to (c): (a) rodZ and lytS genes; (b) rodZ, lytS, hrcA and ympS genes; (c) rodZ, lytS, hrcA, ympS and ftsZ genes.

7. The chassis cell according to claim 6, characterized in that: The class A penicillin-binding protein PonA from Bacillus subtilis was also expressed.

8. The chassis cell according to claim 7, characterized in that: pAD123 was used as the expression vector and P HpaII Regulate the expression of the class A penicillin-binding protein gene ponA shown in SEQ ID NO.

1.

9. The chassis cell according to any one of claims 6 to 8, characterized in that: Bacillus subtilis 1A976 or WS9 was used as the host cell.

10. Use of the Bacillus subtilis chassis cell according to any one of claims 6 to 9 in expressing foreign proteins.

11. A recombinant Bacillus subtilis expressing a target protein in the Bacillus subtilis chassis cell according to any one of claims 6 to 9; the target protein is α-amylase, β-galactosidase, ultrahigh temperature amylase or chitinase.

12. The recombinant Bacillus subtilis according to claim 11, characterized in that pUB110 was used as the expression vector to express the target protein.

13. The recombinant Bacillus subtilis according to claim 12, characterized in that The α-amylase gene shown in SEQ ID NO.2 was expressed.

14. Use of the recombinant Bacillus subtilis according to any one of claims 11 to 13 in fermentation enzyme production.

15. A method for producing α-amylase by fermentation, characterized in that: The recombinant Bacillus subtilis according to claim 13 is cultured in a culture medium for a period of time and induced to produce enzymes with maltose.

16. The method according to claim 15, characterized in that The fermentation is carried out using one or more of soybean peptone, yeast extract, industrial Angel yeast extract, corn steep liquor, and industrial corn steep liquor as nitrogen sources; and the fermentation is carried out using one or more of potato starch, corn starch, maltose, and soluble starch as carbon sources.

17. The method according to claim 16, characterized in that The medium contains Fe 2+ and / or Ca 2+ .

18. The method according to claim 16, characterized in that Ferment at 25-42°C.

19. The method according to claim 16, characterized in that The fermentation pH is 5.5-8.

5.

20. The method according to claim 16, characterized in that Feeding was carried out during the fermentation process.

21. Use of the method according to any one of claims 1 to 5, the chassis cell according to any one of claims 6 to 9, or the method according to any one of claims 15 to 20 in the fields of food, chemical industry, sugar production or feed.

Images