Multi-pathway optimization method and strain for increasing N-acetylglucosamine production

Abstract

The present invention discloses a method and strain for optimizing the production of N-acetylglucosamine using multiple pathways. The present invention is based on Bacillus subtilis 168 strain, and uses a genome editing system based on CRISPR / Cpf1 to construct a synthetic strain of N-acetylglucosamine from scratch. The competitive pathway is optimized by a high-throughput combination of a period-type CRISPRi system based on a stable phase promoter and dCpf1, thereby obtaining a more efficient and stable N-acetylglucosamine synthetic strain. The gene knockout and promoter replacement of the recombinant Bacillus subtilis constructed by the present invention are both completed using a genome scarless editing system based on CRISPR / Cpf1, and no plasmid is used in the final strain. Therefore, glucose can be sustainably, stably and efficiently synthesized into acetylglucosamine, and its yield on a shake flask can reach 33.7g / L, laying the foundation for its industrialization.

Description

Technical Field

[0001] The present invention relates to a method and a strain for improving N-acetylglucosamine production through multi-pathway optimization, belonging to the technical field of metabolic engineering. Background Art

[0002] N-acetylglucosamine (GlcNAc) is a monosaccharide found in organisms, widely found in bacteria, yeast, mold, plants, and animals. In the human body, N-acetylglucosamine is a precursor to the synthesis of disaccharide units of glycosaminoglycans, which play an important role in repairing and maintaining the function of cartilage and joint tissue. Therefore, N-acetylglucosamine is widely used as a drug to treat and repair joint injuries. Currently, N-acetylglucosamine is mainly produced by acid-hydrolyzing chitin from shrimp or crab shells. The waste liquid generated by this method is relatively polluting to the environment, and the resulting product is prone to allergic reactions, making it unsuitable for people with seafood allergies.

[0003] When synthesizing N-acetylglucosamine using glucose as a substrate, the glycolytic pathway, the pentose phosphate pathway, and the peptidoglycan synthesis pathway competitively utilize the glucose entering the cell, thereby limiting the efficient synthesis of N-acetylglucosamine. In addition, glucosamine 6-phosphate (GlcN6P) is a key intermediate metabolite in the synthesis of N-acetylglucosamine and an important precursor for the synthesis of various products such as neuraminic acid and hyaluronic acid. However, due to the cell's own metabolic regulation mechanism, it cannot accumulate in large quantities in the cell. In patent application 201911174644.1, we used the CRISPRi system coupled with a GlcN6P-responsive element to dynamically weaken the competitive pathway for N-acetylglucosamine synthesis mentioned above. At the same time, we used the GlcN6P-responsive promoter to regulate the expression of GNA1 on the plasmid to dynamically regulate the N-acetylglucosamine synthesis pathway, and knocked out the key gene alsSD in the acetoin synthesis pathway. The resulting recombinant Bacillus subtilis can produce 28.0 g / L in shake flasks and 131.6 g / L in fed-batch fermentation in 15L fermentors. In addition, in patent application 202110050546.8, we constructed a biosensor that can efficiently respond to GlcN6P by combining the GlcN6P-responsive promoter with T7 RNA polymerase. Based on this, the mutant strain with increased GlcN6P concentration was screened, and the N-acetylglucosamine production was increased by 31.6%.

[0004] The gene knockout or promoter replacement of the recombinant Bacillus subtilis for N-acetylglucosamine synthesis mentioned above is achieved through the Cre / lox-based genome editing system (reference: Yan X, Yu HJ, Hong Q, LiSP. 2008. Cre / lox system and PCR-based genome engineering in Bacillus subtilis. Appl. Environ. Microbiol. 74: 5556-5562). After multiple genome editing, a large number of lox72 sites will remain on the genome, which will lead to unknown genomic rearrangement reactions. In addition, the key gene GNA1 is also expressed using a plasmid, which leads to poor stability of the recombinant strain. In addition, the CRISPRi system used to weaken the competition pathway is based on the dCas9 protein. Compared with the dCpf1 protein commonly used in another CRISPRi system, dCas9 has a smaller regulatory range and is more complicated when performing multi-target combination regulation. At present, there are still problems with the poor stability of N-acetylglucosamine synthesis strains and the lack of fine regulation of the competition pathway. Summary of the Invention

[0005] To solve the above technical problems, the present invention is based on Bacillus subtilis 168 strain, uses the CRISPR / Cpf1-based genome editing system to construct an N-acetylglucosamine synthesis strain from scratch, and uses a period-type CRISPRi system based on a stable phase promoter and dCpf1 to perform high-throughput combinatorial optimization of the competing pathway, thereby obtaining a more efficient and stable N-acetylglucosamine synthesis strain.

[0006] The first object of the present invention is to provide a strain with improved N-acetylglucosamine production, wherein the strain uses Bacillus subtilis as a host, uses the CRISPR / Cpf1 genome editing system to knock out the glucosamine transporter encoding gene nagP, the 6-phosphate acetylglucosamine deacetylase encoding gene nagA, the 6-phosphate glucosamine deaminase encoding gene nagB, the 6-phosphate glucosamine deaminase encoding gene gamA, the lactate dehydrogenase encoding gene ldh and the acetate kinase encoding gene ackA, and uses the glucose-inducible promoter P lysC The glucosamine synthase-encoding gene glmS was expressed, and a period-dependent CRISPRi system was used to regulate the competitive pathway of N-acetylglucosamine synthesis. The GNA1 gene and the phosphatase-encoding gene yqaB were integrated and expressed in the genome.

[0007] The CRISPRi system includes dCpf1 protein and crRNA, the nucleotide sequence of crRNA is shown in SEQ ID NO.6, and dCpf1 protein uses constitutive promoter P groES Expression, crRNA uses the stable phase promoter P srfA Express.

[0008] Furthermore, the host is based on B. subtilis 168 as the starting strain, and the xylose-inducible promoter P is integrated into the genome using CRISPR / Cpf1 technology. xylA The transcription factor comK-comS gene is controlled, and the trpC gene is reversely mutated to the wild-type gene trpC0 with a nucleotide sequence as shown in SEQ ID NO.9.

[0009] Furthermore, the GNA1 gene is integrated and expressed at the aprE site and the alsSD site, respectively.

[0010] Furthermore, the crRNA is integrated and expressed at the amyE site.

[0011] Furthermore, the dCpf1 protein is integrated and expressed at the lacA site.

[0012] In the present invention, the integration sites of crRNA and dCpf1 protein are commonly used integration sites. Other commonly used integration sites can be selected. Expression at different integration sites has little effect on the implementation of the present invention.

[0013] Furthermore, the nucleotide sequence of the GNA1 encoding gene is shown in SEQ ID NO.10.

[0014] Furthermore, the nucleotide sequence of the phosphatase encoding gene yqaB is shown in SEQ ID NO.11.

[0015] In the present invention, the GNA1 encoding gene and the phosphatase encoding gene yqaB can be selected from commonly used sequences in the art.

[0016] In the present invention, a GlcN6P biosensor is also integrated into the host bacteria to convert the intracellular GlcN6P concentration into a fluorescent signal that can be rapidly measured. When the GlcN6P biosensor is integrated, the gene encoding the repressor protein LacI is also integrated at the nprE site.

[0017] The second object of the present invention is to provide a multi-pathway optimization method for increasing the yield of N-acetylglucosamine, comprising the following steps: using Bacillus subtilis as a host, using the CRISPR / Cpf1 genome editing system to knock out the nagP, nagA, nagB, gamA, ldh and ackA genes, and using the glucose-inducible promoter P lysC The glmS gene was expressed, and the competitive pathway for N-acetylglucosamine synthesis was regulated using a period-type CRISPRi system. The GNA1 gene and the phosphatase-encoding gene yqaB were integrated and expressed in the genome.

[0018] The CRISPRi system includes dCpf1 protein and crRNA, the nucleotide sequence of crRNA is shown in SEQ ID NO.6, and dCpf1 protein uses constitutive promoter P groES Expression, crRNA uses the stable phase promoter P srfA Express.

[0019] Furthermore, the host is based on B. subtilis 168 as the starting strain, and the xylose-inducible promoter P is integrated into the genome using CRISPR / Cpf1 technology. xylA The transcription factor comK-comS gene is controlled, and the trpC gene is reversely mutated to the wild-type gene trpC0 with a nucleotide sequence as shown in SEQ ID NO.9.

[0020] The third object of the present invention is to provide the use of the strain in preparing medicines.

[0021] The beneficial effects of the present invention are:

[0022] The gene knockout and promoter replacement of the recombinant Bacillus subtilis constructed in the present invention are both accomplished using a CRISPR / Cpf1-based scarless genome editing system, and no plasmids are used in the final strain. Therefore, glucose can be sustainably, stably, and efficiently utilized to synthesize acetylglucosamine, with a yield of up to 33.7 g / L in shake flasks, laying the foundation for its industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Construction of Bacillus subtilis for the synthesis of N-acetylglucosamine (a) Metabolic pathways related to the synthesis and decomposition of N-acetylglucosamine in Bacillus subtilis (b) Knockout of pathways related to the decomposition of N-acetylglucosamine and the synthesis of byproducts (c) Response of the GlcN6P biosensor in different strains (d) N-acetylglucosamine production in different strains;

[0024] Figure 2Figure 3. Relief of feedback inhibition of the GlcN6P synthase encoding gene glmS. (a) Characterization of the glucose-inducible promoter. (b) Response of the GlcN6P biosensor after replacement of the glmS promoter. (c) N-acetylglucosamine synthesis after relief of feedback inhibition of glmS.

[0025] Figure 3 Construction of a period-type CRISPRi system based on a stationary phase promoter and dCpf1 (a) Application of the period-type CRISPRi system in regulating the competitive pathway of N-acetylglucosamine synthesis (b) Verification of the function of the dCpf1-based CRISPRi system using mCherry (c) Replacing the dCpf1 promoter to construct a period-type CRISPRi system (d) Replacing the crRNA promoter to construct a period-type promoter (e) Fusing zwf, pfkA, and glmM with mCherry as reporter genes (f) Attenuation strength of different crRNAs on zwf, pfkA, and glmM (g) Relationship between the attenuation strength of zwf, pfkA, and glmM and the response of the GlcN6P biosensor;

[0026] Figure 4 Combinatorial optimization of competing pathways for N-acetylglucosamine synthesis based on a period-based CRISPRi system. (a) Synthetic-crRNA-Arrays Blend for Boosting and Leading (ScrABBLE) device composed of a crRNA array library. (b) Sanger sequencing analysis of the ScrABBL device. (c) High-throughput sequencing analysis of the ScrABBLE device. (d) High-throughput screening process for combinatorial optimization of multiple pathways. (e) Flow cytometry sorting process. (f) Fluorescence analysis of sorted strains in 96-well plates.

[0027] Figure 5 High-throughput screening of GlcN6P biosensors without the addition of an erythromycin resistance gene. (a) Flow cytometry sorting after integration of the ScrABBLE device into BNZR2.00 strains. (b) Sorted cells were plated and single colonies were picked. (c) Fluorescence analysis of sorted strains in 96-well plates.

[0028] Figure 6 Flow cytometric analysis of GlcN6P biosensor in recombinant strains;

[0029] Figure 7 (a) The GNA1 gene was integrated into different strains for the recombinant strains used for N-acetylglucosamine synthesis. The crRNA array shown in the figure is numbered in sequence for the promoter and crRNA targeting zwf, pfkA, mcherry, and glmM. (b) The GNA1 copy number was increased and the yqaB gene was integrated and expressed. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0031] DNA polymerase was purchased from Takara, restriction endonucleases and T4 ligase were purchased from New England Biolabs, plasmid extraction kits were purchased from Shanghai Biotech Co., Ltd., and PCR product nucleic acid purification kits were purchased from Thermo Scientific.

[0032] For shake flask fermentation seeds and general cell culture, LB medium containing (g / L): tryptone 10, yeast extract 5, and NaCl 10. For cell culture or screening, antibiotics or inducers were added to the medium as needed at the following final concentrations: kanamycin 50 μg / mL, chloramphenicol 5 μg / mL, spectinomycin 100 μg / mL, bleomycin 30 μg / mL, erythromycin 5 μg / mL, and IPTG 1 mM.

[0033] Shake flask fermentation medium (g / L): tryptone 6%, yeast powder 12%, urea 6%, K2HPO4·3H2O 12.5%, KH2PO4 2.5%, glucose 90%, magnesium sulfate heptahydrate 3%, glycerol 5%.

[0034] High-performance liquid chromatography (HPLC) detection of glucose, acetoin, and N-acetylglucosamine: Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5 mM H2SO4, flow rate 0.6 mL / min, column temperature 40°C, injection volume 10 μL.

[0035] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell biology, recombinant DNA technology and related fields in the field of this technology, which have been fully described in existing literature.

[0036] Example 1: Knockout of genes related to N-acetylglucosamine catabolism and by-product synthesis

[0037] In order to convert the intracellular GlcN6P concentration into a fluorescent signal that can be rapidly measured, the GlcN6P biosensor containing T7 RNA polymerase and RBS4 described in patent application 202110050546.8 was integrated into the strain BSZR (Bacillus subtilis 168epr::P xylA-comKS, trpC0, the construction method is as shown in patent 202111251656.7, using B. subtilis 168 as the starting strain, and integrating the xylose-inducible promoter P into the genome using CRISPR / Cpf1 technology. xylA The transcription factor comK-comS gene controlled by the strain was mutated, and the trpC gene was restored to the wild-type gene trpC0 with a nucleotide sequence as shown in SEQ ID NO.9 at the aprE site of the genome to obtain strain BNZR0.00. Based on the CRISPR / Cpf1 genome scarless editing system, see reference: Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L., 2020.CAMERS-B: CRISPR / Cpf1 assisted multiple-genesediting and regulation system for Bacillus subtilis. Biotechnology and Bioengineering 117, 1817–1825. Figure 1 As shown in a, there is a degradation pathway for N-acetylglucosamine in Bacillus subtilis, which causes extracellular N-acetylglucosamine to be transported into the cell and utilized as a carbon source. In addition, Bacillus subtilis also produces by-products such as acetic acid and lactic acid during fermentation. Therefore, based on the strain BNZR0.00, four genes related to the N-acetylglucosamine decomposition pathway (nagP, nagA, nagB, gamA) and two genes in the by-product acetic acid and lactic acid synthesis pathway (ldh, ackA) were knocked out, and strains BNZR0.10, BNZR0.40 and BNZR0.60 ( Figure 1 b). Figure 1As shown in Figure 3, the responses of the GlcN6P biosensor in the four strains BNZR0.00, BNZR0.10, BNZR0.40 and BNZR0.60 were then measured. It was found that adding glucose to the above strains could not activate the biosensor, indicating that GlcN6P had not accumulated in the cell at this time; however, when GlcN was added, the biosensor in the above four strains could be activated, and the degree of activation increased with the increase in the number of knockout genes, indicating that knocking out the above genes is beneficial to intracellular GlcN6P. Finally, we transformed the plasmid pP43-GNA1 containing the GNA1 gene of the N-acetylglucosamine synthesis pathway into the above four strains (reference: Wu, Y., Chen, T., Liu, Y., Tian, ​​R., Lv, X., Li, J., Du, G., Chen, J., Ledesma-Amaro, R., Liu, L., 2020. Design of a programmable biosensor-CRISPRigenetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis. Nucleic Acids Research 48, 996–1009.), and found that only BNZR0.40 and BNZR0.60 could synthesize N-acetylglucosamine after the introduction of the GNA1 gene, and the N-acetylglucosamine production of BNZR0.60 was higher than that of BNZR0.40, indicating that the existence of the catabolism and by-product synthesis pathways limits the synthesis of N-acetylglucosamine ( Figure 1 d). Finally, strain BNZR0.60 was selected for subsequent transformation.

[0038] Example 2: Release of feedback inhibition of the GlcN6P synthase encoding gene glmS

[0039] like Figure 1 As shown in Figure a, in the N-acetylglucosamine synthesis pathway, the gene glmS encoding the GlcN6P synthase is subject to feedback inhibition by the product GlcN6P, which prevents the intracellular GlcN6P from accumulating in large quantities and limits the synthesis of N-acetylglucosamine. Therefore, we further replaced the original promoter of the glmS gene in strain BNZR0.60 by promoter replacement to relieve the feedback regulation. Considering the role of the above-mentioned feedback regulation in metabolic flux distribution and cell growth, we tried to use a glucose-induced promoter as shown in Table 1 for replacement to avoid excessive expression of glmS and cause intracellular metabolic flux imbalance. Figure 2As shown in b, we first used fluorescent proteins to measure the response of the selected promoters to glucose concentrations. We found that although the expression strength of different promoters varied in the absence of glucose, the expression strength of most promoters was enhanced in the presence of glucose. When we tried to use these promoters to replace the promoter of glmS, only the constitutive promoter P veg and glucose-inducible promoter P cggR 、P cggR′ 、P lysC 、P pdhA It can be successfully replaced into strain BNZR0.60, and strains BNZR1.00, BNZR1.02, BNZR1.03, BNZR1.06 and BNZR1.12 ( Figure 2 b) The four glucose-inducible promoters above all maintain sufficient expression even without glucose, indicating that the expression of the essential gene glmS itself cannot be too weak. This may be the reason why the other promoters were unable to complete the replacement. After adding glucose, the GlcN6P biosensor in strains BNZR1.00 and BNZR1.06 was activated, indicating that these two strains accumulate more GlcN6P, and the biosensor strength is also stronger when GlcN is added than when the glmS promoter is replaced ( Figure 2 b) After plasmid pP43-GNA1 was introduced into strains BNZR1.00, BNZR1.06, and BNZR1.12, the production of N-acetylglucosamine was determined. The yield of strain BNZR1.06 / pP43-GNA1 was 2.5 times that of BNZR0.60 / pP43-GNA1, reaching 12.6 g / L. In addition, the production of N-acetylglucosamine by strain BNZR1.06 / pP43-GNA1 was 2.5 times that of strain BNZR0.60 / pP43-GNA1, reaching 12.6 g / L. veg The final yield of strain BNZR1.00 / pP43-GNA1 expressing glmS was comparable to that of BNZR1.06 / pP43-GNA1, but cell growth was poorer, indicating that using a glucose-inducible promoter is superior to directly using a constitutive promoter when deregulating the feedback regulation of glmS. Therefore, strain BNZR1.06 was ultimately selected for subsequent transformation.

[0040] Table 1 Glucose-inducible promoters

[0041]

[0042]

[0043] Example 3: Construction of a phase-dependent CRISPRi system based on a stationary phase promoter and dCpf1

[0044] like Figure 3As shown in a, there are three degradation pathways in N-acetylglucosamine synthesis: the glycolytic pathway (EMP), the pentose phosphate pathway (HMP), and the peptidoglycan synthesis pathway (PSP). This will limit further increases in N-acetylglucosamine production. Furthermore, these three pathways are crucial for cell growth and therefore cannot be completely blocked. However, by controlling the expression of the key genes zwf, pfkA, and glmM in these three competing pathways, their metabolic flux can be regulated. To achieve spontaneous regulation of these three pathways, a promoter expressing in the stable phase was selected, and the dCpf1-based CRISPRi system described in reference Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L., 2020. CAMERS-B: CRISPR / Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering 117, 1817–1825 was modified. On the one hand, the modified period-type CRISPRi system can spontaneously weaken the target gene starting from the stable period to avoid excessive impact on cell growth; on the other hand, dCpf1 has a larger regulatory range, which can achieve fine regulation of multiple genes and can achieve simultaneous regulation of multiple genes under the guidance of a single crRNA array. Figure 3 As shown in Figure 2, the function of the dCpf1-based CRISPRi system was first verified using the red fluorescent protein mCherry (see Table 2 for the crRNA used). It was found that the CRISPRi system weakened mCherry, and the regulatory range under the guidance of different crRNAs was very different.

[0045] On this basis, two strategies were tried to construct a period-type CRISPRi system. Figure 3 As shown in c, in strategy 1, the promoter of dCpf1 was replaced with the stable expression promoter P srfA 、P yqfCD or P ysdB , while crRNA uses the constitutive promoter P veg When the expression of the reporter gene mCherry was performed, the expression of the reporter gene mCherry was not weakened, indicating that this strategy could not obtain a CRISPRi system with the expected function; Figure 3 As shown in d, in strategy 2, dCpf1 uses the constitutive promoter P groES Expression, crRNA uses the stable phase promoter P srfA 、P yqfCD or P ysdBAt this time, CRISPRi has normal regulatory function, and the weakening intensity of mCherry begins to appear at 10h and increases with time, indicating that the period-type CRISPRi system is successfully constructed (the plasmid pLCgNo-dCpf1 used to integrate dCpf1 into the lacA site is shown in SEQ ID NO.1, and the plasmids pcrasr, pcrayq and pcrays used to integrate crRNA into the amyE site are shown in SEQ ID NO.2-4 respectively). Figure 3 As shown in Figure 5, in order to apply this period-type CRISPRi system to the expression regulation of the target genes zwf, pfkA and glmM, the mcherry gene was first fused to the C-terminus of the above three genes, and the three genes were point-inactivated (H241A, D127A and S100A) to prevent overexpression from affecting cell growth. Twelve crRNAs were designed for each gene as shown in Table 2. The inhibitory strength of different crRNAs on the target genes was verified by a fusion reporter gene containing the fluorescent protein mCherry. It was found that the inhibitory strength of the CRISPRi system on zwf, pfkA and glmM under the guidance of different crRNAs was 58.0% to 96.7%, 18.2% to 94.7% and 5.5% to 76.5%, respectively. Figure 3 f), which shows that the CRISPRi system based on dCpf1 does have a wide range of regulation. On this basis, we further analyzed the response of the GlcN6P sensor after weakening the expression of zwf, pfkA and glmM, and found that the weakening intensity of these three target genes was positively correlated with the response intensity of the GlcN6P sensor ( Figure 3 g), indicating that inhibition of these competing pathways can promote the accumulation of intracellular GlcN6P.

[0046] Table 2 crRNA sequences used for regulation of target genes mcherry, zwf, pfkA and glmM

[0047]

[0048]

[0049] Example 4: Combinatorial Optimization and Fine Control of Competing Pathways for N-acetylglucosamine Synthesis

[0050] In order to optimize the combination of the three competing pathways of N-acetylglucosamine synthesis, the crRNA array construction method described in the reference: Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L., 2020. CAMERS-B: CRISPR / Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering 117, 1817–1825 was used to construct the 12 crRNAs targeting the three target genes zwf, pfkA, and glmM, the crRNA-mc5 targeting mCherry, and the three stable expression promoters P srfA 、P yqfCD or P ysdB For example Figure 4 As shown in a, through the above method, a library with 5184 crRNA array combinations was constructed and named the Synthetic-crRNA-Arrays Blend for Boosting and Leading (ScrABBLE) device. Figure 4 The sequencing results of b showed that except for the overlapping peaks at the recognition sequences complementary to different sites of the target gene, the other positions in the ScrABBLE device were single peaks, indicating that the crRNA array library was successfully constructed. High-throughput sequencing analysis also proved that the ScrABBLE device was consistent with the expected sequence ( Figure 4 c). Next, in order to ensure that the GlcN6P biosensor can still be regulated by IPTG after the gene editing plasmid pHT-XCR6 containing the repressor protein LacI is eliminated, the gene encoding the repressor protein LacI is integrated into the nprE site of the strain BNZR1.06 to obtain the strain BNZR1.06L (the role of the repressor protein here inhibits the leaky expression of the biosensor. When preparing the production strain without adding the biosensor, the gene encoding the repressor protein can be omitted). Furthermore, the constitutive promoter P was integrated into the lacA site. groESThe strain BNZR2.00 was obtained by expressing the dCpf1 protein. The integrated expression of the above-mentioned exogenous genes can avoid the use of plasmid vectors, thereby ensuring the stability of the recombinant strain. After weakening, the three target genes of the CRISPRi system will affect the normal growth of cells. To prevent the recombinant strain containing the crRNA array library from being eliminated during the culture process due to weakened growth, a co-transcribed erythromycin resistance gene was added after the eGFP in the GlcN6P biosensor of strain BNZR2.00 to obtain strain BNZR2.00E. In this way, the erythromycin resistance dependent on BNZR2.00E will be enhanced as the response of the GlcN6P biosensor is enhanced.

[0051] Figure 4 d is a schematic diagram of the screening process. After the ScrABBLE device is introduced into BNZR2.00E, erythromycin is first added for culture. Then, cells with stronger fluorescence are sorted using a flow cytometer. The obtained cells are spread onto a bleomycin resistance plate (the ScrABBLE device contains this resistance marker). Single colonies are selected and placed on a 96-well plate to measure fluorescence. For strains with higher fluorescence values, crRNA arrays are amplified for sequencing analysis. Finally, the sequenced crRNA array is integrated into BNZR2.00 to prevent other unknown mutations from occurring during the screening process. Figure 4 As shown in e, the fluorescence signal of the GlcN6P biosensor was enhanced after integrating the ScrABBLE device into BNZR2.00E. We sorted the 0.1% with the highest fluorescence and spread it onto the bleomycin resistance plate. Figure 4 As shown in Figure f, the single colonies on the plate were inoculated into a 96-well plate for culture. After measuring the fluorescence, it was found that the distribution of fluorescence roughly conformed to the normal distribution. We selected the 28 strains with the highest fluorescence for sequencing and integrated the complete crRNA array obtained at this time into BNZR2.00.

[0052] like Figure 5 As shown, we also tried to directly introduce the ScrABBLE device into BNZR2.00 for high-throughput screening. Since there is no erythromycin resistance gene in the biosensor, there is no need to add erythromycin during the culture process. Figure 5 As shown in a, the overall fluorescence level of cells after the ScrABBLE device was integrated into BNZR2.00 was significantly weaker than that when the ScrABBLE device was integrated into BNZR2.00E, so the cells in the entire Q4 partition were collected. After the above cells were spread on the bleomycin plate ( Figure 5b) We picked a single colony and placed it in a 96-well plate to measure the fluorescence intensity. Most of the strains had no fluorescence. We still selected the 24 strains with the highest fluorescence and sequenced and analyzed the crRNA array on their genome ( Figure 5 c) 5 and 9 complete crRNA arrays were screened using BNZR2.00 and BNZR2.00E, respectively. After integrating the above crRNA arrays into BNZR2.00, 14 recombinant strains (BNZR2.01R to BNZR2.14R) were obtained.

[0053] like Figure 6 The results show that flow cytometry was used to analyze the response of the GlcN6P biosensor in different recombinant strains. The biosensor was not activated in the development strain BNZR0.00 and the gene knockout strain BNZR0.60, but was activated in BNZR1.06L, which had the feedback inhibition of glmS removed, and the intensity was similar to that in strain BNZR2.00. In different strains that subsequently integrated the crRNA array, the intensity of the GlcN6P biosensor was significantly stronger than that of BNZR2.00 at 12 hours; however, when the culture time was extended to 24 hours, the response of the GlcN6P biosensor in many strains would decline. Only in the three strains of BNZR2.03R, BNZR2.06R and BNZR2.12R could it remain stable, indicating that GlcN6P in these three strains can be stably maintained at a high level. Furthermore, the fluorescence of the GlcN6P biosensor decreased in most strains from 12 to 24 hours, indicating that weakening of the glycolysis, pentose phosphate, and peptidoglycan synthesis pathways can lead to unstable cellular metabolism and thus fluctuate intracellular GlcN6P concentrations. Ultimately, strains BNZR2.03R, BNZR2.06R, BNZR2.12R, and BNZR2.09R were selected for further modification.

[0054] The promoters and crRNAs targeting zwf, pfkA, mcherry, and glmM in the crRNA arrays of strains BNZR2.03R, BNZR2.06R, BNZR2.12R, and BNZR2.09R are numbered as follows: Figure 7As shown in a, the sequence information of the crRNA array is shown in SEQID NO.5-8 respectively. We further integrated the GNA1 gene into the aprE site of the above four strains and BNZR2.00 to obtain BNZR2.03R-apGb, BNZR2.06R-apGb, BNZR2.12R-apGb, BNZR2.09R-apGb and BNZR2.00-apGb. Through shake flask fermentation, it was found that the strain BNZR2.06R-apGb had the highest N-acetylglucosamine production (22.6g / L), which was 3.4 times that of the strain BNZR2.00-apGb without crRNA array, and the cell dry weight and by-product acetoin of BNZR2.06R-apGb were also lower than those of BNZR2.00-apGb. Figure 7 As shown in Figure b, we finally chose BNZR2.00-apGb and BNZR2.06R-apGb for the next step of transformation. After integrating a second copy of the GNA1 gene at the alsSD gene in the acetoin byproduct biosynthesis pathway, strains BNZR2.00-Gb2 and BNZR2.06R-apGb2 produced N-acetylglucosamine at 7.3 g / L and 32.0 g / L, respectively, and both strains no longer produced the byproduct acetoin. To enhance the conversion of intracellular 6-acetylglucosamine phosphate (GlcNAc6P) to N-acetylglucosamine, the phosphatase encoding gene yqaB was further integrated into the genomes of BNZR2.00-Gb2 and BNZR2.06R-apGb2 (Reference: Wu, Y., Chen, T., Liu, Y., Tian, ​​R., Lv, X., Li, J., Du, G., Chen, J., Ledesma-Amaro, R., Liu, L., 2020. Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis. Nucleic Acids Research 48, 996–1009.), and obtained strains BNZR2.00-Gb2y and BNZR2.06R-apGb2y, with the yields of the above strains in shake flasks being 7.3 g / L and 33.7 g / L, respectively.

[0055] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A strain with improved N-acetylglucosamine production, characterized in that The strain uses Bacillus subtilis as a host and uses the CRISPR / Cpf1 genome editing system to knock out the glucosamine transporter encoding gene nagP , 6-phosphate acetylglucosamine deacetylase encoding gene nagA , 6-phosphate glucosamine deaminase encoding gene nagB , 6-phosphate glucosamine deaminase encoding gene gamA , lactate dehydrogenase encoding gene ldh and acetate kinase encoding genes ackA and using the glucose-inducible promoter P lysC Expression of glucosamine synthase encoding gene glmS , and used a period-type CRISPRi system to target key genes in the competitive pathway of N-acetylglucosamine synthesis zwf 、 pfq and glmM Spontaneous weakening regulation is carried out from the stable phase to avoid the weakening of the competition pathway affecting cell growth, and two genes are integrated and expressed in the genome. GNA1 genes encoding phosphatase yqB ; The CRISPRi system includes dCpf1 protein and crRNA, the nucleotide sequence of crRNA is shown in SEQ ID NO.6, and dCpf1 protein uses constitutive promoter P groES Expression, crRNA uses the stable phase promoter P srfA Express; The host is B. subtilis 168 was the starting strain, and the xylose-inducible promoter P was integrated into the genome using CRISPR / Cpf1 technology. xylA Controlled transcription factors comK-comS genes, and trpC The gene is reverted to the wild type gene with the nucleotide sequence shown in SEQ ID NO.9 trpC0 get; The GNA1 The genes are aprE Site and alsSD The site is integrated and expressed; The crRNA is amyE Site-integrated expression; The dCpf1 protein is lacA Site-integrated expression.

2. The strain with improved N-acetylglucosamine production according to claim 1, characterized in that GNA1 The nucleotide sequence encoding the gene is shown in SEQ ID NO.

10.

3. The strain with improved N-acetylglucosamine production according to claim 1, characterized in that Phosphatase-encoding genes yqB The nucleotide sequence is shown in SEQ ID NO.

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4. A method for optimizing the production of N-acetylglucosamine by multiple pathways, characterized in that: The steps include: using Bacillus subtilis as the host and using the CRISPR / Cpf1 genome editing system to knock out nagP 、 nagA 、 nagB 、 gamA 、 ldh and ackA gene, and uses the glucose-inducible promoter P lysC Express glmS Genes were detected by CRISPRi system, and key genes in the competitive pathway of N-acetylglucosamine synthesis were detected. zwf 、 pfq and glmM Spontaneous weakening regulation is carried out from the stable phase to avoid the weakening of the competition pathway affecting cell growth, and two genes are integrated and expressed in the genome. GNA1 genes encoding phosphatase yqB ; The CRISPRi system includes dCpf1 protein and crRNA, the nucleotide sequence of crRNA is shown in SEQ ID NO.6, and dCpf1 protein uses constitutive promoter P groES Expression, crRNA uses the stable phase promoter P srfA Express; The host is B. subtilis 168 was the starting strain, and the xylose-inducible promoter P was integrated into the genome using CRISPR / Cpf1 technology. xylA Controlled transcription factors comK-comS genes, and trpC The gene is reverted to the wild type gene with the nucleotide sequence shown in SEQ ID NO.9 trpC0 get; The GNA1 The genes are aprE Site and alsSD The site is integrated and expressed; The crRNA is amyE Site-integrated expression; The dCpf1 protein is lacA Site-integrated expression.

5. Use of the strain according to any one of claims 1 to 3 in the preparation of a medicine.

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