CN115927143A - Method and strain for improving N-acetylglucosamine yield through multi-path optimization - Google Patents

Abstract

The invention discloses a method and a strain for improving N-acetylglucosamine yield through multi-path optimization, wherein a genome editing system based on CRISPR/Cpf1 is used for constructing a synthetic strain of N-acetylglucosamine from the beginning on the basis of a bacillus subtilis 168 strain, and a time-based CRISPR system based on a stationary phase promoter and dCpf1 is used for carrying out high-throughput combined optimization on a competitive path, so that a more efficient and stable N-acetylglucosamine synthetic strain is obtained. The gene knockout and the promoter replacement of the recombinant bacillus subtilis constructed by the invention are all completed by using a CRISPR/Cpf 1-based genome traceless editing system, and no plasmid is used in the final strain, so that the glucose can be continuously, stably and efficiently used for synthesizing the acetylglucosamine, the yield of the acetylglucosamine on a shake flask can reach 33.7g/L, and the foundation is laid for the industrialization of the recombinant bacillus subtilis.

Description

Method and strain for improving N-acetylglucosamine yield through multi-path optimization

Technical Field

The invention relates to a method and a strain for improving the yield of N-acetylglucosamine through multi-path optimization, belonging to the technical field of metabolic engineering.

Background

N-acetylglucosamine (GlcNAc) is a monosaccharide in organisms and is widely found in bacteria, yeast, molds, plants and animals. In the human body, N-acetylglucosamine is a synthetic precursor of disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of the function of cartilage and joint tissues. Therefore, N-acetylglucosamine is widely used as a pharmaceutical and dietary supplement to treat and repair joint damage. In addition, N-acetylglucosamine has many applications in the cosmetic field. At present, N-acetylglucosamine is mainly produced by acidolysis of chitin in shrimp shells or crab shells, the waste liquid produced by the method has serious environmental pollution, and the obtained product is easy to cause anaphylactic reaction and is not suitable for people with seafood allergy to take.

Bacillus subtilis is a production host widely used as food enzyme preparation and important nutritional chemicals, and the product is certified as "general regulated as safe" (GRAS) level by FDA. Therefore, the construction of the recombinant bacillus subtilis by using a metabolic engineering means is an effective way for producing food safety-grade N-acetylglucosamine. When synthesizing N-acetylglucosamine using glucose as a substrate, glucose entering the cell is competitively utilized by the glycolysis pathway, pentose phosphate pathway, and peptidoglycan synthesis pathway, thereby limiting the efficient synthesis of N-acetylglucosamine. In addition, glucosamine-6-phosphate (GlcN 6P) is a key intermediate metabolite in the synthesis of N-acetylglucosamine, and is also an important precursor for the synthesis of various products such as neuraminic acid and hyaluronic acid, but cannot be accumulated in large amounts in cells due to the metabolic regulation mechanism of the cells themselves. In patent application 201911174644.1, the competition pathway for synthesizing N-acetylglucosamine is weakened dynamically by a CRISPR system coupled with a GlcN6P response element, the expression of GNA1 is regulated and controlled dynamically on a plasmid by a GlcN6P response promoter, the N-acetylglucosamine synthesis pathway is regulated and controlled dynamically, a key gene alsSD of the acetoin synthesis pathway is knocked out, the yield of the obtained recombinant bacillus subtilis on a shake flask can reach 28.0g/L, and the yield of the recombinant bacillus subtilis on a 15L fermentation tank during fed-batch fermentation can reach 131.6g/L. In addition, in patent application 202110050546.8, by combining a GlcN 6P-responsive promoter with T7RNA polymerase, a biosensor which can respond GlcN6P efficiently was constructed, and N-acetylglucosamine yield of a mutant strain which is screened to have an increased concentration of GlcN6P was increased by 31.6%.

The gene knockout or promoter replacement of the recombinant Bacillus subtilis for N-acetylglucosamine synthesis is realized by a Cre/lox-based genome editing system (reference document: yan X, yu HJ, hong Q, li SP.2008.Cre/lox system and PCR-based genome engineering in Bacillus subtilis.appl.environ.Microbiol.74: 5556-5562), a large number of lox72 sites remain on the genome after multiple genome edits, and then unknown rearrangement reaction of the genome can occur, and the key gene GNA1 is expressed by using plasmids, which results in poor stability of the recombinant strain; in addition, the CRISPRi system used to attenuate the competitive pathway is based on dCas9 protein, which is less regulated by dCas9 and more complex when it is regulated with a multi-target combination than dCpf1 protein commonly used in another CRISPRi system. At present, the problems of poor stability of the N-acetylglucosamine synthesis strain and insufficient fine regulation of a competitive pathway still exist.

Disclosure of Invention

In order to solve the technical problems, the invention takes the bacillus subtilis 168 strain as a base, constructs a synthetic strain of N-acetylglucosamine de novo by using a genome editing system based on CRISPR/Cpf1, and performs high-throughput combinatorial optimization on a competitive pathway by using a time-based CRISPR system based on a stationary phase promoter and dCpf1, thereby obtaining the more efficient and stable N-acetylglucosamine synthetic strain.

The first purpose of the invention is to provide a bacterial strain with improved N-acetylglucosamine yield, wherein the bacterial strain takes bacillus subtilis as a host, a glucosamine transporter coding gene nagP, a 6-acetylglucosamine phosphate deacetylase coding gene nagA, a 6-glucosamine phosphate deaminase coding gene nagB, a 6-glucosamine phosphate deaminase coding gene gamA, a lactate dehydrogenase coding gene ldh and an acetate kinase coding gene ackA are knocked out by adopting a CRISPR/Cpf1 genome editing system, and a glucose-induced promoter P is used for inducing the bacterial strain to produce N-acetylglucosamine _lysC Expressing a glucosamine synthetase encoding gene glmS, regulating and controlling a competitive way of N-acetylglucosamine synthesis by adopting a time-type CRISPR system, and integrating and expressing a GNA1 gene and a phosphatase encoding gene yqaB in a genome;

wherein the time-based CRISPR system comprises dCpf1 protein and crRNA, the nucleotide sequence of the crRNA is shown in SEQ ID NO.6, and the dCpf1 protein uses a constitutive promoter P _groES Expression, crRNA uses the stationary phase promoter P _srfA And (4) expressing.

Furthermore, the host takes B.subtilis 168 as an original strain, and integrates a xylose inducible promoter P on a genome by using CRISPR/Cpf1 technology _xylA The controlled transcription factor comK-comS gene is obtained by reverse mutating the trpC gene into a wild-type gene trpC0 with the nucleotide sequence shown as SEQ ID NO. 9.

Furthermore, the GNA1 gene is integrated and expressed at aprE locus and alsSD locus respectively.

Further, the crRNA is expressed at the amyE site.

Further, the dCpf1 protein is expressed integratedly at the lacA site.

In the present invention, the integration sites of crRNA and dCpf1 protein belong to common integration sites, other common integration sites can be selected, and the expression at different integration sites has little influence on the implementation of the present invention.

Further, the nucleotide sequence of the GNA1 coding gene is shown in SEQ ID NO. 10.

Further, the nucleotide sequence of phosphatase encoding gene yqaB is shown in SEQ ID NO. 11.

In the present invention, the GNA 1-encoding gene and the phosphatase-encoding gene yqaB may be selected from sequences commonly used in the art.

In the present invention, a GlcN6P biosensor is also incorporated into the host bacterium for converting intracellular GlcN6P concentration into a fluorescent signal that can be rapidly measured. When a GlcN6P biosensor is integrated, it is also necessary to integrate the gene encoding the repressor LacI at the nprE site.

The second purpose of the invention is to provide a method for improving the yield of N-acetylglucosamine by multi-path optimization, which comprises the following steps: using bacillus subtilis as a host, knocking out nagP, nagA, nagB, gamA, ldh and ackA genes by using a CRISPR/Cpf1 genome editing system, and inducing a promoter P by using glucose _lysC Expressing a glmS gene, regulating and controlling a competitive path of N-acetylglucosamine synthesis by adopting a time-type CRISPR system, and integrating and expressing a GNA1 gene and a phosphatase coding gene yqaB in a genome;

wherein the time-based CRISPR system comprises dCpf1 protein and crRNA, the nucleotide sequence of the crRNA is shown as SEQ ID NO.6, and the dCpf1 protein uses a constitutive promoter P _groES Expression, crRNA uses the stationary phase promoter P _srfA And (4) expressing.

Furthermore, the host takes B.subtilis 168 as an original strain, and integrates a xylose inducible promoter P on a genome by using CRISPR/Cpf1 technology _xylA The controlled transcription factor comK-comS gene is obtained by reverse mutating the trpC gene into a wild-type gene trpC0 with the nucleotide sequence shown as SEQ ID NO. 9.

The third purpose of the invention is to provide the application of the strain in preparing food, medicines, nutritional health products or cosmetics.

The invention has the beneficial effects that:

the gene knockout and the promoter replacement of the recombinant bacillus subtilis constructed by the invention are all completed by using a CRISPR/Cpf 1-based genome traceless editing system, and no plasmid is used in the final strain, so that the glucose can be continuously, stably and efficiently used for synthesizing the acetylglucosamine, the yield of the acetylglucosamine on a shake flask can reach 33.7g/L, and the basis is laid for the industrialization of the recombinant bacillus subtilis.

Drawings

FIG. 1 shows the construction of Bacillus subtilis for the synthesis of N-acetylglucosamine (a) the N-acetylglucosamine synthesis and catabolism-related metabolic pathway in Bacillus subtilis (b) the knock-out of the N-acetylglucosamine catabolism-related pathway and byproduct synthesis pathway (c) the response of the GlcN6P biosensor in different strains (d) the N-acetylglucosamine production by different strains;

FIG. 2 is a graph of feedback inhibition release of glmS for a gene encoding a GlcN6P synthase (a) glucose-inducible promoter characterization (b) GlcN6P biosensor response following glmS promoter replacement (c) N-acetylglucosamine synthesis following feedback inhibition release of glmS;

FIG. 3 is a relationship between the time-phase CRISPR system construction based on stationary phase promoter and dCpf1 (a) application of time-phase CRISPR system construction in N-acetylglucosamine synthesis competition pathway regulation (b) verification of dCpf 1-based CRISPR system function using mCherry (c) replacement of dCpf 1-based promoter construction time-phase CRISPR system (d) replacement of crRNA promoter construction time-phase promoter (e) fusion of zwf, pfkA and glmM with mCherry, respectively, as reporter genes (f) attenuation of different crRNAs to zwf, pfkA and glmM (g) attenuation of zwf, pfkA and glmM and GlcN6P biosensor response;

FIG. 4 is a combinatorial optimization of N-acetylglucosamine synthesis competition pathway based on the time-phased CRISPR system (a) Synthetic-crRNA-Arrays Blend for Boosting and Leading (ScrABBLE) device composed of crRNA array library (b) ScrABBL device Sanger sequencing assay (c) ScrABBLE device high throughput sequencing assay (d) multi-pathway combinatorial optimization high throughput screening procedure (e) flow sorting process (f) sorting strain 96-well plate fluorometric assay;

FIG. 5 shows a GlcN6P biosensor which is subjected to high throughput screening without addition of erythromycin resistance gene (a) integration of a ScrABBLE device into 2.00 BNZR species and then flow sorting (b) sorting of the cells coated on a plate and then picking of single colony (c) sorting of a 96-well plate for fluorometric analysis;

FIG. 6 is a flow analysis of GlcN6P biosensors in recombinant strains;

FIG. 7 shows that the recombinant strain is used for N-acetylglucosamine synthesis (a) different strains integrate GNA1 gene, and crRNA array sequentially comprises a promoter and crRNA numbers targeting zwf, pfkA, mcherry and glmM (b) increases GNA1 copy number and integrates and expresses yqaB gene.

Detailed Description

The present invention is further described below with reference to specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

DNA polymerase was purchased from Takara, restriction enzyme and T4 ligase from NEB, plasmid extraction kit from Biotechnology engineering (Shanghai) GmbH, and PCR product nucleic acid purification kit from Thermo Scientific.

Both shake flask fermentation seeds and general cell culture used LB medium containing (g/L): tryptone 10, yeast powder 5 and NaCl 10. In the cell culture or screening, antibiotics or inducers are added to the culture medium at the following final concentrations as required: kanamycin 50. Mu.g/mL, chloramphenicol 5. Mu.g/mL, spectinomycin 100. Mu.g/mL, bleomycin 30. Mu.g/mL, erythromycin 5. Mu.g/mL, IPTG 1mM.

Shake flask fermentation medium (g/L): tryptone 6, yeast powder 12, urea 6 ₂ HPO ₄ ·3H ₂ O 12.5，KH ₂ PO ₄ 2.5, glucose 90, magnesium sulfate heptahydrate 3 and glycerol 5.

High Performance Liquid Chromatography (HPLC) detection methods of glucose, acetoin and N-acetylglucosamine: agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, calif.), mobile phase: 5mM H ₂ SO ₄ The flow rate is 0.6mL/min, the column temperature is 40 ℃,the injection volume was 10. Mu.L.

Unless otherwise indicated, the experimental methods, detection methods and preparation methods disclosed in the present invention all employ conventional techniques in the art of molecular biology, biochemistry, cell biology, recombinant DNA technology and related fields, which are well described in the literature.

Example 1: n-acetylglucosamine catabolism and byproduct synthesis related gene knockout

In order to convert intracellular GlcN6P concentration into a fluorescence signal that can be measured rapidly, the GlcN6P biosensor containing T7RNA polymerase and RBS4 of patent application 202110050546.8 was integrated into the strain BSZR (Bacillus subtilis 168epr:: P) using a CRISPR/Cpf1 based genome editing system _xylA Com KS, trpC0, the construction method is shown in patent 202111251656.7, B.subtilis 168 is taken as a starting strain, and a xylose inducible promoter P is integrated on a genome by using CRISPR/Cpf1 technology _xylA Controlling transcription factor comK-comS gene, and reverse mutating trpC gene into aprE site of wild gene trpC 0) genome whose nucleotide sequence is shown in SEQ ID NO.9 to obtain strain BNZR0.00. CRISPR/Cpf1 based genome traceless editing systems are disclosed in references: wu, Y, liu, Y, lv, X, li, J, du, G, liu, L, 2020.CAMERS-B CRISPR/Cpf1 associated multiple-genes encoding and modulation system for Bacillus subtilis and Bioengineering 117,1817-1825. As shown in FIG. 1a, the degradation pathway of N-acetylglucosamine exists in Bacillus subtilis, so that extracellular N-acetylglucosamine is transported into cells and utilized as a carbon source. In addition, bacillus subtilis also produces acetic acid, lactic acid and other by-products during fermentation. Thus, based on the strain BNZR0.00, 4 genes (nagP, nagA, nagB, gamA) of the pathways involved in N-acetylglucose catabolism and 2 genes (ldh, ackA) of the pathways involved in the synthesis of acetic acid and lactic acid as by-products were knocked out to obtain the strains BNZR0.10, BNZR0.40 and BNZR0.60 in this order (FIG. 1 b). As shown in FIG. 1c, immediately after measuring the response of GlcN6P biosensor in the four strains BNZR0.00, BNZR0.10, BNZR0.40 and BNZR0.60, it was found thatThe biosensor cannot be activated by adding glucose into the strain, which indicates that intracellular GlcN6P is not accumulated; the biosensors of the four strains can be activated when GlcN is added, and the activation degree is increased along with the increase of the number of the knockout genes, which indicates that the knockout of the genes is beneficial to intracellular GlcN 6P. Finally, we transformed plasmid pP43-GNA1 containing the GNA1 gene of the N-acetylglucosamine synthesis pathway into the above four strains (ref: 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 biosensior-CRISPR genetic circuits for dynamic and autonomous flux-control of metabolic flux in Bacillus subtilis, nucleic Acids Research 48, 996-1009.) and found that only BNR 0.40 and ZR0.60 introduced into the GNA1 gene could synthesize N-acetylglucosamine and BNR 0.60 synthesized with a high yield of N-acetylglucosamine synthesis pathway BNR 0.40 and BNR 0.60 was synthesized with a high yield of N-acetylglucosamine synthesis pathway (see BNR 0.40 and BNR 0.60. Restriction on the presence of N-acetylglucosamine synthesis pathway). Finally, strain BNZR0.60 was selected for subsequent engineering.

Example 2: elimination of feedback inhibition of glmS for the GlcN6P synthase encoding gene

As shown in FIG. 1a, in the N-acetylglucosamine synthesis pathway, the glmS gene encoding GlcN6P synthase is feedback inhibited by the product GlcN6P, which prevents intracellular accumulation of GlcN6P in large amounts and limits N-acetylglucosamine synthesis. Therefore, we further replaced the original promoter of glmS gene in strain BNZR0.60 by means of promoter replacement to release it from feedback regulation. In view of the role of the above feedback regulation in metabolic flux allocation and cell growth, we herein attempted to replace the promoter with one induced by glucose as shown in table 1 to avoid overexpression of glmS resulting in an imbalance in intracellular metabolic flux. As shown in FIG. 2b, the response of the selected promoters to glucose concentration was first measured using fluorescent protein, and it was found that although the different promoters were different in their intensities in the absence of glucose, the expression intensity of most promoters was enhanced in the presence of glucose. When it is attempted to replace the promoter of glmS with these promoters,only constitutive promoter P _veg And glucose inducible promoter P _cggR 、P _cggR′ 、P _lysC 、P _pdhA The substitution into strain BNZR0.60 was successful and yielded strains BNZR1.00, BNZR1.02, BNZR1.03, BNZR1.06 and BNZR1.12 (FIG. 2 b). The above four glucose-inducible promoters also have sufficient strength without the addition of glucose, indicating that the expression of glmS itself, which is the essential gene, cannot be too weak, which may be the reason why other promoters cannot accomplish the replacement. After addition of glucose, the GlcN6P biosensors in both strains BNZR1.00 and BNZR1.06 can be activated, indicating that the accumulation of GlcN6P is greater in both strains, and that the biosensors are also stronger when GlcN is added than when the glmS promoter is replaced (fig. 2 b). The synthesis of N-acetylglucosamine was determined after introduction of the plasmid pP43-GNA1 into the strains BNZR1.00, BNZR1.06 and BNZR1.12, in which the yield of strain BNZR1.06/pP43-GNA1 was up to 12.6g/L, 2.5 times that of BNZR0.60/pP43-GNA 1; in addition, the constitutive promoter P was used _veg The final yield of strain BNZR1.00/pP43-GNA1 expressing glmS was comparable to that of BNZR1.06/pP43-GNA1, but the cell growth was poor, indicating that the use of glucose-inducible promoter was superior to the direct use of constitutive promoter when the feedback control of glmS was released. Therefore, strain BNZR1.06 was finally selected for subsequent engineering.

TABLE 1 glucose inducible promoter

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Example 3: construction of time-based CRISPR system based on stationary phase promoter and dCpf1

As shown in FIG. 3a, there are three degradation pathways in N-acetylglucosamine synthesis, namely, the glycolysis pathway (EMP), the pentose phosphate pathway (HMP), and the Peptidoglycan Synthesis Pathway (PSP), which limit further increases in N-acetylglucosamine production. The three pathways have a crucial effect on the growth of cells, so that the three pathways cannot be completely blocked; by controlling the expression of the key genes zwf, pfkA and glmM in these three competing pathways, the metabolic flux can be regulated. For spontaneous control of these three pathways, promoters for stationary phase expression were selected here, and the dCpf 1-based CRISPR system described in references Wu, Y, liu, Y, lv, X, li, J, du, G, liu, L, 2020.CAMERS-B CRISPR/Cpf1 associated multiple-genes encoding and modulation system for Bacillus subtilis, biotechnology and Bioengineering 117,1817-1825 was modified. On one hand, the modified period-type CRISPI system can be used for performing spontaneous weakening on the target gene from the stable period, so that excessive influence on cell growth is avoided; on the other hand, dCpf1 has a larger regulation range, can realize fine regulation of multiple genes, and can realize simultaneous regulation of multiple genes under the guidance of a single crRNA array. As shown in fig. 3b, the function of the CRISPRi system based on dCpf1 was first verified using the red fluorescent protein mCherry (see table 2 for crRNA used), and it was found that the CRISPRi system produces an attenuation effect on the mCherry, and the control range under guidance of different crRNA is greatly different.

On the basis of the above, two strategies are tried to construct an epoch-type CRISPRi system. As shown in FIG. 3c, the promoter of dCpf1 was replaced with the stationary phase expression promoter P in strategy 1 _srfA 、P _yqfCD Or P _ysdB While crRNA uses a constitutive promoter P _veg When the expression is carried out, the expression of the reporter gene mCherry is not weakened, which indicates that the CRISPR system with the expected function cannot be obtained by the strategy; as shown in FIG. 3d, however, in strategy 2 dCpf1 used a constitutive promoter P _groES Expression, crRNA uses the stationary phase promoter P _srfA 、P _yqfCD Or P _ysdB Expression is carried out, the CRISPR has normal regulation function at the moment, the weakening strength of the mCherry is reflected at the beginning of 10h and is enhanced along with the time extension, which shows that the phasic CRISPR system is successfully constructed at the moment (a plasmid pLCgNo-dCpf1 for integrating dCpf1 into lacA site is shown as SEQ ID NO.1, a plasmid pcrasr for integrating crRNA into amyE site,pcrayq and pcrays are shown in SEQ ID NO.2-4, respectively). As shown in fig. 3e, in order to apply the phase-type CRISPRi system to the expression control of target genes zwf, pfkA and glmM, the mcherry gene was fused to the C-termini of the three genes, and the three genes were subjected to the site-mutation inactivation (H241A, D127A and S100A) to prevent the influence on cell growth after overexpression. 12 crrnas as shown in table 2 were designed for each gene, and the inhibition strength of target genes by different crrnas was verified by fusion reporter gene containing fluorescent protein mCherry, and it was found that the CRISPRi system had inhibition strengths of zwf, pfkA and glmM of 58.0% to 96.7%,18.2% to 94.7% and 5.5% to 76.5% respectively under the guidance of different crrnas (fig. 3 f), which indicates that the CRISPRi system based on dCpf1 indeed has a great control range. On the basis, response conditions of the GlcN6P sensor after attenuation of zwf, pfkA and glmM expression are further analyzed, and the attenuation intensities of the three target genes and the response intensity of the GlcN6P sensor are found to have positive correlation (FIG. 3 g), which indicates that intracellular GlcN6P accumulation can be promoted after inhibition of the competitive pathways.

TABLE 2 crRNA sequences for targeted gene mcherry, zwf, pfkA and glmM regulation

Example 4: combined optimization and fine regulation of N-acetylglucosamine synthesis competitive pathway

For combinatorial optimized regulation of the three competing pathways for N-acetylglucosamine synthesis, references were used: wu, Y, liu, Y, lv, X, li, J, du, G, liu, L, 2020.CAMERS-B CRISPR/Cpf1 associated multiple-genes editing and formatting system for Bacillus subtilis Biotechnology and Bioengineering 117,1817-1825, 12 crRNAs acting on zwf, pfkA and glmM target genes, crRNA-mc5 acting on mCherry and three stationary phase expression promoters P _srfA 、P _yqfCD Or P _ysdB And (4) combining. As shown in FIG. 4a, 5184 kinds of materials are constructed in the above mannerThe library of crRNA array combinations was named for Synthetic-crRNA-Arrays Blend for Boosting and Leading (ScrABBLE) device. The sequencing results in fig. 4b show that the ScrABBLE device has a single peak at all positions except for overlapping peaks at the recognition sequence complementary to different sites of the target gene, indicating that the crRNA array library was successfully constructed and that the ScrABBLE device was consistent with the expected sequence by high throughput sequencing analysis (fig. 4 c). Next, in order to allow the GlcN6P biosensor to be controlled by IPTG after elimination of the gene editing plasmid pHT-XCR6 containing the repressor LacI, a gene encoding the repressor LacI was integrated at the nprE site of the strain BNZR1.06 to obtain the strain BNZR1.06L (where the action of the repressor inhibits the leakage expression of the biosensor and the gene encoding the repressor may not be integrated in the case of preparing the production strain without adding the biosensor), and further a constitutive promoter P was integrated at the lacA site _groES The expressed dCpf1 protein resulted in strain BNZR2.00. The integration and expression of the exogenous genes can avoid the use of plasmid vectors, thereby ensuring the stability of the recombinant strains. In order to prevent recombinant strains containing crRNA array libraries from being eliminated during culture due to growth attenuation, the strain BNZR2.00e was obtained by adding a co-transcribed erythromycin resistance gene after eGFP in the GlcN6P biosensor of strain BNZR2.00, so that the erythromycin resistance dependent on BNZR2.00e increased with the increase of GlcN6P biosensor response.

FIG. 4d is a schematic diagram of the screening process, in which after the ScrABBLE device is introduced into BNZR2.00E, erythromycin is added for culturing, then the cells with stronger fluorescence are sorted by using a flow cytometer, after the obtained cells are coated on a bleomycin resistant plate (the ScrABBLE device contains the resistance marker), single colonies are selected to be placed into a 96-well plate for measuring fluorescence, the crRNA array amplified by the strain with higher fluorescence value is subjected to sequencing analysis, and finally the sequenced crRNA array is integrated into BNZR2.00 to prevent other unknown mutations from occurring in the screening process. As shown in fig. 4e, the fluorescence signal of the GlcN6P biosensor was enhanced by integrating the ScrABBLE device into bnzr2.00e, and we sorted the 0.1% of the highest fluorescence and plated it into bleomycin resistant plates. As shown in FIG. 4f, a single colony on the plate was inoculated into a 96-well plate and cultured, and the distribution of fluorescence was determined to approximately match the normal distribution, and we selected 28 strains with the highest fluorescence for sequencing and integrated the complete crRNA array obtained at this time into BNZR2.00.

As shown in FIG. 5, it was also tried to introduce the ScrABBLE device directly into BNZR2.00 for high-throughput screening, and since the biosensor did not have an erythromycin resistance gene, it was not necessary to add erythromycin during the culture. As shown in FIG. 5a, the cells were collected in the entire Q4 compartment because the overall fluorescence levels of the cells following the integration of the ScrABBLE device into BNZR2.00 were significantly less than the fluorescence levels of the cells following the integration of the ScrABBLE device into BNZR 2.00E. After plating the cells onto bleomycin plates (fig. 5 b), a single colony was picked up and assayed for fluorescence intensity in a 96-well plate, with most strains being non-fluorescent. We still selected the 24 strains with the highest fluorescence and analyzed the crRNA array on their genomes (fig. 5 c). Using 5 and 9 intact crRNA arrays obtained from the screening of BNZR2.00 and BNZR2.00E, respectively, 14 recombinant strains (BNZR2.01R to BNZR2.14R) were obtained after integrating the crRNA arrays into BNZR2.00.

The response of a GlcN6P biosensor in different recombinant strains, which was not activated in both the starting strain BNZR0.00 and the knock-out strain BNZR0.60 but activated in BNZR1.06L, which released the feedback inhibition by glmS, was analyzed by flow cytometry as shown in FIG. 6, and was similar in intensity to that of strain BNZR2.00. In different strains subsequently incorporating crRNA array, glcN6P biosensors were significantly stronger at 12h than BNZR2.00; however, after the incubation time was extended to 24h, the response of the GlcN6P biosensor in many strains fell back, and only the three strains BNZR2.03R, BNZR2.06R and BNZR2.12R remained stable, indicating that GlcN6P in the three strains remained stable at a higher level. In addition, fluorescence of the GlcN6P biosensor falls back when most strains extend from 12h to 24h, which indicates that the attenuation of three pathways of glycolysis, pentose phosphate and peptidoglycan synthesis leads to unstable cell metabolism, thereby causing the fluctuation of intracellular GlcN6P concentration. Finally, strains BNZR2.03R, BNZR2.06R, BNZR2.12R and BNZR2.09R are selected for further modification.

The numbering of the promoter and the crRNA targeting zwf, pfkA, mcherry and glmM in the crRNA array of strains BNZR2.03R, BNZR2.06R, BNZR2.12R and BNZR2.09R is shown in FIG. 7a, and the sequence information of the crRNA array is respectively shown in SEQ ID NO. 5-8. We further integrated the GNA1 gene into the aprE site of the above 4 strains and BNZR2.00 to obtain BNZR2.03R-apGb, BNZR2.06R-apGb, BNZR2.12R-apGb, BNZR2.09R-apGb and BNZR2.00-apGb, and found that the strain BNZR2.06R-apGb produced the highest amount (22.6 g/L) of N-acetylglucosamine than the strain BNZR2.00-apGb without crRNA array by shake flask fermentation, and the dry cell weight and byproduct acetoin of BNZR2.06R-apGb were also reduced compared with BNZR 2.00-apGb. As shown in FIG. 7b, BNZR2.00-apGb and BNZR2.06R-apGb were finally selected for further transformation. After a second copy of GNA1 gene is integrated at a side product acetoin synthesis pathway gene alsSD, the yields of N-acetylglucosamine of the obtained strains BNZR2.00-Gb2 and BNZR2.06R-apGb2 are respectively 7.3g/L and 32.0g/L, and the side product acetoin is not generated any more in both strains; in order to enhance the conversion of intracellular acetylglucosamine 6-phosphate (GlcNAc 6P) to N-acetylglucosamine, a phosphatase-encoding gene yqaB was further integrated into the genome 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 biosensior-CRISPR genetic circuits for dynamic and automatic dual-control of metallic flux in Bacillus subtilis nucleic Acids Research 48, 996-1009) to yield strains BNZR2.00-Gb2y and BNR 2.06R-apGb2y, which yield 7.3g/L and 33.7g/L, respectively, in shake flasks.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A bacterial strain with improved N-acetylglucosamine yield is characterized in that the bacterial strain takes bacillus subtilis as a host, a CRISPR/Cpf1 genome editing system is adopted to knock out a glucosamine transporter coding gene nagP, a 6-acetylglucosamine phosphate deacetylase coding gene nagA, a 6-glucosamine phosphate deaminase coding gene nagB, a 6-glucosamine phosphate deaminase coding gene gamA, a lactate dehydrogenase coding gene ldh and an acetate kinase coding gene ackA, and a glucose induction promoter P is used for inducing the bacterial strain to have high yield and high stability, and the bacterial strain is suitable for the application of the CRISPR/Cpf1 genome editing system in the aspects of improving the yield of the N-acetylglucosamine and the yield of the N-acetylglucosamine _lysC Expressing a glucosamine synthetase encoding gene glmS, regulating and controlling a competitive approach of N-acetylglucosamine synthesis by adopting a time-type CRISPR system, and integrating and expressing a GNA1 gene and a phosphatase encoding gene yqaB in a genome;

wherein, the period type CRISPR system packageComprises dCpf1 protein and crRNA, the nucleotide sequence of the crRNA is shown in SEQ ID NO.6, and the dCpf1 protein uses a constitutive promoter P _groES Expression, crRNA uses the stationary phase promoter P _srfA And (4) expressing.

2. The strain with increased N-acetylglucosamine yield of claim 1, wherein B.subtilis 168 is used as a starting strain for the host, and a xylose inducible promoter P is integrated on the genome by CRISPR/Cpf1 technology _xylA The controlled transcription factor comK-comS gene is obtained by reverse mutating the trpC gene into a wild-type gene trpC0 with the nucleotide sequence shown as SEQ ID NO. 9.

3. The strain with improved N-acetylglucosamine production of claim 1, wherein the GNA1 gene is integrated and expressed at aprE site and alsSD site respectively.

4. The strain with increased N-acetylglucosamine production according to claim 1, wherein the crRNA is expressed by integration at the amyE site.

5. The strain with increased N-acetylglucosamine production according to claim 1, wherein the dCpf1 protein is expressed by integration at the lacA site.

6. The strain with improved N-acetylglucosamine production of claim 1, wherein the nucleotide sequence of the gene encoding GNA1 is shown in SEQ ID NO. 10.

7. The strain with improved N-acetylglucosamine production according to claim 1, wherein the nucleotide sequence of phosphatase encoding gene yqaB is represented by SEQ ID No. 11.

8. A method for improving N-acetylglucosamine yield through multi-path optimization is characterized by comprising the following steps: to dryThe Bacillus subtilis is taken as a host, genes nagP, nagA, nagB, gamA, ldh and ackA are knocked out by adopting a CRISPR/Cpf1 genome editing system, and a glucose-induced promoter P is used _lysC Expressing a glmS gene, regulating and controlling a competitive path of N-acetylglucosamine synthesis by adopting a time-type CRISPR system, and integrating and expressing a GNA1 gene and a phosphatase coding gene yqaB in a genome;

wherein the time-based CRISPR system comprises dCpf1 protein and crRNA, the nucleotide sequence of the crRNA is shown as SEQ ID NO.6, and the dCpf1 protein uses a constitutive promoter P _groES Expression, crRNA uses the stationary phase promoter P _srfA And (4) expression.

9. The method of claim 8, wherein the host is a starting strain of B.subtilis 168, and the CRISPR/Cpf1 technology is used for integrating a xylose-inducible promoter P on a genome _xylA The controlled transcription factor comK-comS gene, and the trpC gene is back mutated into a wild gene trpC0 with the nucleotide sequence shown in SEQ ID NO. 9.

10. Use of a strain according to any one of claims 1 to 7 for the preparation of a food product, a pharmaceutical product, a nutraceutical product or a cosmetic product.

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