Genetically engineered bacteria for synthesizing natural cyclic diguanosine monophosphate in large quantities and application thereof

Abstract

This invention discloses a genetically engineered bacterium capable of synthesizing large quantities of natural cyclic diguanylate (c-di-GMP) and its applications. It involves knocking out the major phosphodiesterase gene chr1_233 as described in claim 1 within the sphingobium xenophagum C2 strain. This invention rationally designs and modifies the c-di-GMP synthesis and degradation pathways and the inhibitory site of major diguanylate cyclase activity in heterologous sphingobium-consuming bacteria, constructing a series of genetically engineered bacteria capable of synthesizing large quantities of natural c-di-GMP. This provides the industry with a green, simple, economical, and efficient method for producing natural c-di-GMP, and offers a series of high-quality and inexpensive raw materials for the preparation of STING agonists or immune adjuvants, or for the preparation of drugs for treating diseases related to STING protein function.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and bioengineering, and specifically relates to a genetically engineered bacterium that synthesizes large quantities of natural cyclic diguanosine monophosphate and its applications. Background Technology

[0002] Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger molecule in bacteria, playing a crucial role in bacterial motility and colonization, cell differentiation, biofilm formation, pathogenic factor production, and intercellular communication. Studies have found that c-di-GMP possesses potent immunostimulatory properties, producing excellent immunomodulatory effects when acting on eukaryotic cells. c-di-GMP can bind to the innate immune-related protein STING (interferon gene stimulator) and induce conformational changes and spatial shifts, thereby activating downstream signaling pathways to induce the expression of type I interferon and pro-inflammatory cytokines, thus activating the innate immune response. Therefore, c-di-GMP can be used to prepare STING agonists or immunoadjuvants, or to develop drugs for treating diseases related to STING protein function. With the rapid development of immunotherapy, using cyclic dinucleotide STING agonists to modulate the STING signaling pathway has become a novel approach in immunotherapy, and the synthesis of natural cyclic dinucleotides and their analogues has become a research hotspot in recent years due to their significant application value.

[0003] Currently, c-di-GMP is mainly synthesized through chemical and enzymatic methods. Chemical synthesis suffers from drawbacks such as long processing time, numerous steps, and environmental unfriendliness, while enzymatic synthesis requires expensive GTP substrates and pure enzymes. Therefore, commercially available c-di-GMP remains very expensive, costing 3000-4000 RMB / mg (based on Merck's sales price). Utilizing the bacterial c-di-GMP synthesis and degradation pathways is another option for producing natural c-di-GMP. The synthesis and degradation metabolism of c-di-GMP in bacteria are regulated by guanylate cyclase and phosphodiesterase, respectively. The active site of guanylate cyclase is the GGDEF domain, catalyzing the synthesis of one molecule of c-di-GMP from two molecules of GTP. The active site of phosphodiesterase is the EAL or HD-GYP domain, which degrades c-di-GMP into pGpG or GMP, respectively. However, the concentration of c-di-GMP within bacteria is controlled by a complex and delicate regulatory network, governed by interactions between synthases and degradative enzymes, as well as interactions with other proteins. Under the action of major phosphodiesterase, intracellular c-di-GMP is maintained at an extremely low level. Furthermore, the synthesis of c-di-GMP is also subject to feedback inhibition by c-di-GMP itself, with the inhibitory site RXXD in the GGDEF domain binding to c-di-GMP, resulting in an upper limit to the concentration of intracellular c-di-GMP synthesis. Therefore, using traditional gene expression methods, it is often impossible to achieve high yields of natural c-di-GMP in wild-type bacteria.

[0004] This invention aims to construct a genetically engineered bacterium capable of synthesizing natural c-di-GMP in large quantities by rationally designing and modifying the c-di-GMP synthesis and degradation pathway and the inhibitory site of guanylate cyclase activity in sphingosine monophosphate bacteria. This provides the industry with a green, simple, economical, and efficient method for producing natural c-di-GMP, and provides a high-quality and inexpensive raw material for the preparation of STING agonists or immune adjuvants, or for the preparation of drugs for treating diseases related to STING protein function. Summary of the Invention

[0005] The first objective of this invention is to provide the major phosphodiesterase gene chr1_233, which plays a decisive role in the degradation of c-di-GMP within C2 strain cells.

[0006] The nucleotide sequence of the main phosphodiesterase gene chr1_233 is shown in SEQ ID NO.1.

[0007] A second objective of this invention is to provide the application of knocking out the major phosphodiesterase gene chr1_233 in the sphingobium xenophagum strain to enhance intracellular synthesis of c-di-GMP.

[0008] A third objective of this invention is to provide the guanylate cyclase gene chr1_457 or a mutant thereof, the nucleotide sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of which is shown in SEQ ID NO.6.

[0009] The fourth objective of this invention is to utilize the overexpression of the major diguanylate cyclase gene chr1_457 or its mutant in Sphingobium xenophagum to enhance the intracellular synthesis of c-di-GMP in the strain.

[0010] A fifth objective of this invention is to provide a promoter tf962, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0011] The sixth objective of this invention is to provide the application of overexpressing the guanylate cyclase gene chr1_457 or its mutants in Sphingobium xenophagum using the promoter tf962 to improve the intracellular synthesis of c-di-GMP in the strain.

[0012] The seventh objective of this invention is to provide a c-di-GMP-producing sphingobium xenophagum, which is obtained by knocking out the major phosphodiesterase gene chr1_233 in sphingobium xenophagum.

[0013] The eighth objective of this invention is to provide a c-di-GMP-producing sphingobium xenophagum, which is a sphingobium xenophagum strain that overexpresses the major diguanylate cyclase gene chr1_457 or a mutant thereof.

[0014] Preferably, the sphingobium xenophagum is sphingobium xenophagum C2.

[0015] This invention obtained the major phosphodiesterase gene chr1_233, which plays a decisive role in the intracellular degradation of c-di-GMP, by knocking out the EAL domain of 10 c-di-GMP synthases with GGDEF / EAL double domains in the genome of the heterotrophic sphingobium xenophagum C2 strain. Knocking out the major phosphodiesterase gene chr1_233 increased the intracellular concentration of c-di-GMP synthesized by S. xenophagum C2 strain by 4.83 times. By analyzing the expression changes of 16 genes related to c-di-GMP synthesis and degradation in the chr1_233 mutant strain *S. xenophagum* C2 (Δ233), the major guanylate cyclase gene *chr1_457*, which plays a decisive role in c-di-GMP synthesis in the C2 strain, was obtained. An inducible expression plasmid pCMT_457 for the major guanylate cyclase gene *chr1_457* was constructed using the 4-isopropylbenzoic acid-induced CMT operon sequence. Under the induction of 50 µM 4-isopropylbenzoic acid, the concentration of c-di-GMP synthesized intracellularly in *S. xenophagum* C2 strain increased by 6.58-fold. A constitutive expression plasmid pTF962_457 for the major guanylate cyclase gene *chr1_457* was constructed using the strong promoter tf962 sequence in the *S. xenophagum* C2 genome. Under the action of the strong promoter tf962, the synthesis of c-di-GMP in *S. xenophagum* C2 strain increased by 6.58-fold. The concentration of c-di-GMP synthesized intracellularly in *S. xenophagum* C2 strain increased by 9.15-fold. The inhibitory site R187 of the guanylate cyclase Chr1_457 was modified to P187 using an in vitro point mutagenesis method. A constitutive expression plasmid pTF962_457-R187P, representing the modified guanylate cyclase gene chr1_457-R187P, was constructed using the strong promoter tf962 sequence. Under the influence of the strong promoter tf962, the concentration of c-di-GMP synthesized intracellularly in *S. xenophagum* C2 strain increased by 16.21-fold. By rationally designing and modifying the c-di-GMP synthesis and degradation pathways and the inhibitory site of guanylate cyclase in bacteria that feed on heterologous sphingosine, a series of genetically engineered bacteria capable of synthesizing natural c-di-GMP in large quantities were constructed. This provides the industry with a green, simple, economical, and efficient method for producing natural c-di-GMP, and offers a series of high-quality and inexpensive raw materials for the preparation of STING agonists or immune adjuvants, or for the preparation of drugs for treating diseases related to STING protein function. Attached Figure Description

[0016] Figure 1Structural characteristics and homology analysis of c-di-GMP synthesis and degradation enzymes in the genome of S. xenophagum C2, a heterologous sphingosine-eating bacterium;

[0017] Figure 2 Characteristics of c-di-GMP synthesis in different phosphodiesterase EAL domain gene knockout strains in the C2 genome of heterologous sphingosine-consuming bacteria S. xenophagum;

[0018] Figure 3 Changes in the expression of genes related to c-di-GMP synthesis and degradation in the chr1_233 knockout strain of the major phosphodiesterase gene and the characteristics of c-di-GMP synthesis in the induced expression of the chr1_457 major diguanylate cyclase gene.

[0019] Figure 4 Characteristics of c-di-GMP synthesis in constitutively expressed c-di-GMP gene chr1_457 in the major phosphodiesterase chr1_233 gene knockout strain;

[0020] Figure 5 The characteristics of c-di-GMP synthesis in the constitutive expression of the major diguanylate cyclase gene chr1_457-R187P, in the chr1_233 gene knockout strain where the active inhibitory site R187 was modified to P187. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1: c-di-GMP synthesis and degradation gene elements in the genome of the heterologous sphingosine-eating bacterium S. xenophagum C2

[0023] Sphingosine-eating bacteria *S. xenophagum* C2 strain (= CCTCC AB 2015427 = KCTC52051) were inoculated into liquid LB medium (containing 10.0 g peptone, 5.0 g yeast extract, and 5.0 g NaCl per liter, with water as the solvent; prepared by dissolving all components in water and sterilizing). The medium was then incubated at 30°C in a shaker at 200 rpm / min until the late logarithmic growth phase. The bacterial OD... 600≈1.0. The bacterial culture was centrifuged at 9,000×g for 5 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were sent to Annoroad Gene Technology (Beijing) Co., Ltd. for whole genome extraction and sequencing analysis. Whole-genome sequencing analysis revealed that the C2 strain chromosomal genome contained a total of 16 gene elements related to c-di-GMP synthesis and degradation. Among them, 6 were guanylate cyclase genes containing only the GGDEF domain, namely chr1_457, chr1_510, chr1_734, chr1_1644, chr1_2598, and chr1_2629; the other 10 were dual-domain enzyme (guanylate cyclase / phosphodiesterase) genes containing both the GGDEF and EAL domains, namely chr1_233, chr1_838, chr1_1712, chr1_1793, chr1_1808, chr1_2048, chr1_2373, chr1_2787, chr1_2836, and chr2_385. Homology alignment analysis of the protein sequences of these 16 enzymes related to c-di-GMP synthesis and degradation revealed that six guanylate cyclases with a single GGDEF domain clustered in one branch, while ten guanylate cyclases / phosphodiesterases with a GGDEF / EAL double domain clustered in another branch. Among the double-domain enzymes, Chr1_2048 stood alone in one branch, showing low similarity to the other nine double-domain enzymes; Chr1_838, Chr1_1808, Chr1_233, Chr1_1793, and Chr1_2836 showed closer similarity to each other, clustering in one branch; while Chr1_2787, Chr2_385, Chr1_1712, and Chr1_2373 clustered in another branch. Figure 1 ).

[0024] Example 2: Construction of gene knockout strains with different phosphodiesterase EAL domains and their c-di-GMP synthesis characteristics

[0025] Of the 10 bidomain enzymes of *S. xenophagum* C2, chr1_1793, chr1_2787, and chr1_2048 are located downstream of the operon structure, while the remaining bidomain enzymes are single, independent genes. Figure 2A). Ten gene knockout strains of the EAL domain in two-domain enzymes were constructed using pAK405 suicide plasmid-mediated homologous recombination. First, genomic DNA was extracted from strain C2 using a bacterial genomic DNA extraction kit from Nanjing Novizan Biotechnology Co., Ltd., and then inducing DNA with the following primers: 233_5O and 233_5I, 838_5O and 838_5I, 1712_5O and 1712_5I, 1793_5O and 1793_5I, 1808_5O and 1808_5I, 2048_5O and 2048_5I, 2373_5O and 2373_5I, 2787_5O and 2787_5I, 2836_5O and 2836_5I, and 385_5O and 385_5I. The upstream fragment of the corresponding dual-domain gene EAL domain was amplified using primers 233_3I and 233_3O, 838_3I and 838_3O, 1712_3I and 1712_3O, 1793_3I and 1793_3O, 1808_3I and 1808_3O, 2048_3I and 2048_3O, 2373_3I and 2373_3O, 2787_3I and 2787_3O, 2836_3I and 2836_3O, and 385_3I and 385_3O. Next, DNA was extracted from the pAK405 plasmid using a plasmid extraction kit from Nanjing Novizan Biotechnology Co., Ltd., and the FastDigest restriction endonuclease EcoR from Thermo Fisher Scientific (China) Co., Ltd. was used. and Hind The pAK405 plasmid was digested with enzymes in FastDigest buffer at 37°C for 15 minutes. Then, upstream and downstream fragments of different dual-domain genes' EAL domains, as well as pAK405 plasmid fragments digested with restriction enzymes, were purified using a product purification kit from Nanjing Novizan Biotechnology Co., Ltd. The pAK405 plasmid was then ligated with the corresponding upstream and downstream fragments of different dual-domain genes' EAL domains using homologous recombinase from Beijing TransGen Biotech Co., Ltd., and transformed into *E. coli* competent cells. The cells were then subjected to 50... After screening for kanamycin resistance at μg / mL and sequencing verification, recombinant plasmids pAK405_233, pAK405_838, pAK405_1712, pAK405_1793, pAK405_1808, pAK405_2048, pAK405_2373, pAK405_2787, pAK405_2836, and pAK405_385 were successfully ligated for gene knockout. These plasmids represent deletions of 279bp, 216bp, 201bp, 336bp, 210bp, 258bp, 309bp, 339bp, 261bp, and 285bp within the EAL domain of the corresponding genes, respectively. Then, the 10 recombinant plasmids used for gene knockout were transformed into competent C2 cells via electroporation (25 µF, 200 Ω, 2000 V). The transformed bacterial cultures were plated on LB agar plates containing 25 μg / mL kanamycin for the first recombination exchange and incubated at 30°C for 72 h. Single colonies growing on the 25 μg / mL kanamycin-resistant plates were streaked onto LB agar plates containing 50 μg / mL kanamycin and incubated at 30°C for 24 h. Single colonies growing on the 50 μg / mL kanamycin-resistant plates were streaked onto LB agar plates containing 120–240 μg / mL streptomycin for the second recombination exchange and incubated at 30°C for 24 h. Single clones grown on LB agar plates containing 120–240 μg / mL streptomycin were picked and streaked onto LB agar plates containing 120–240 μg / mL streptomycin and 50 μg / mL kanamycin, and incubated at 30°C for 24 h.Finally, colonies sensitive to kanamycin (50 μg / mL) were selected, and genomic DNA was extracted using a bacterial genomic DNA extraction kit. Using primers 233_LF and 233_LR, 838_LF and 838_LR, 1712_LF and 1712_LR, 1793_LF and 1793_LR, 1808_LF and 1808_LR, 2048_LF and 2048_LR, 2373_LF and 2373_LR, 2787_LF and 2787_LR, 2836_LF and 2836_LR, and 385_LF and 385_LR, the EAL domain fragments of the corresponding dual-domain genes were amplified. Electrophoresis results were used to determine if the gene fragment size was correctly missing. The gene products with missing fragments were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Finally, mutant strains with the correct sequences, having knocked out 279bp, 216bp, 201bp, 336bp, 210bp, 258bp, 309bp, 339bp, 261bp, and 285bp of the EAL domain, were obtained: C2 (Δ233), C2 (Δ838), C2 (Δ1713), C2 (Δ1793), C2 (Δ1808), C2 (Δ2048), C2 (Δ2373), C2 (Δ2787), C2 (Δ2836), and C2 (Δ385).

[0026] Wild-type strain C2 and 10 mutant strains were inoculated into liquid LB medium and cultured at 30°C in a shaker at 200 rpm / min until the late logarithmic growth phase. The bacterial cell OD... 600 ≈1.0. Wild-type strain C2 and 10 mutant strains were inoculated into LB medium at a volume ratio of 2%, and cultured at 30°C for 5–6 h in a shaker at 200 rpm / min until the bacterial cell OD reached 1.0. 600 The bacterial cells were centrifuged at 9,000 × g for 5 min, the supernatant was discarded, and the cells were collected. The cells were washed twice with an inorganic salt buffer (containing 2.0 g of Na₂HPO₄·12H₂O, 0.7 g of KH₂PO₄, 0.5 g of NH₄Cl, 0.3 g of NaCl, 0.1 g of MgSO₄·7H₂O, 0.05 g of CaSO₄·2H₂O, 0.2 mg of FeCl₃·6H₂O, 0.2 mg of NaMoO₄, 0.2 mg of MnCl₂·4H₂O, 0.2 mg of CuCl₂·2H₂O, 0.2 mg of ZnSO₄, 0.3 mg of H₃BO₃, and 0.4 mg of CoCl₂·6H₂O per liter, with water as the solvent; prepared by dissolving all components in water and sterilizing). Finally, the cells were resuspended in the inorganic salt buffer to an OD₂ value of 0.3. 600 ≈1.0. Take 1 mL of OD600 Add a bacterial suspension of approximately 1.0 μL to a glass test tube, add 3 mL of inorganic salt buffer, and measure the OD at this point. 600 Value, denoted as OD 600 1. Add 800 µL of n-hexadecane, vortex for 30 s, let stand for 45 min, and measure the OD at this time. 600 Value, denoted as OD 600 2. The hydrophobicity of bacterial cell surface is calculated using the following formula: CSH % = [OD 600 1 - OD 600 2] / OD 600 1×100%. The results showed that the cell surface hydrophobicity of wild-type C2 and mutant strains C2 (Δ233), C2 (Δ838), C2 (Δ1713), C2 (Δ1793), C2 (Δ1808), C2 (Δ2048), C2 (Δ2373), C2 (Δ2787), C2 (Δ2836), and C2 (Δ385), measured by this method, were 15.98%, 57.05%, 13.11%, 16.67%, 11.02%, 12.35%, 23.70%, 22.14%, 26.65%, 9.46%, and 14.28%, respectively. Figure 2 (B) Only the C2 (Δ233) mutant strain, which knocks out the chr1_233 gene, exhibited high cell surface hydrophobicity; the cell surface hydrophobicity of the other mutant strains was not significantly different from that of the wild-type strain. Additionally, 1 mL of OD... 600 The bacterial suspension (approximately 1.0 g) was transferred to a centrifuge tube, and 4 mL of extraction buffer (methanol:acetonitrile = 1:1, volume ratio) was added. The tube was then frozen at -80°C for 12 h; incubated in a 95°C water bath for 10 min; frozen at -80°C for 12 h; centrifuged at 12,000 × g for 10 min; the supernatant was collected and transferred to a new centrifuge tube, rapidly frozen in liquid nitrogen, and then freeze-dried in a freeze dryer. Finally, the liquid was dissolved in 1 mL of sterile water, and the content of c-di-GMP extracted from the bacterial cells was determined by LC-MS. The results showed that the c-di-GMP contents in wild-type strain C2 and mutant strains C2 (Δ233), C2 (Δ838), C2 (Δ1713), C2 (Δ1793), C2 (Δ1808), C2 (Δ2048), C2 (Δ2373), C2 (Δ2787), C2 (Δ2836), and C2 (Δ385) were 8.99, 43.44, 14.17, 15.83, 10.13, 6.46, 9.34, 14.35, 4.60, 12.34, and 5.54 µg / L, respectively. Figure 2(B) Only the C2 (Δ233) mutant strain, which had the chr1_233 gene knocked out, showed a significant increase in c-di-GMP content; the c-di-GMP content of the other mutant strains did not differ significantly from that of the wild-type strain. These results indicate that the phosphodiesterase encoded by the chr1_233 gene (sequence shown in SEQ ID NO.1, and the encoded amino acid sequence shown in SEQ ID NO.2) is the main phosphodiesterase in strain C2, possessing a strong c-di-GMP degradation capacity and responsible for maintaining a low level of c-di-GMP within the strain. Knocking out the EAL domain of the main phosphodiesterase chr1_233 significantly increased the c-di-GMP production within the strain by 4.83 times.

[0027] Example 3: Inducible expression of primary diguanylate cyclase and characteristics of c-di-GMP synthesis

[0028] In most cases, c-di-GMP synthesis and degradation-related bidomain enzymes exhibit only one activity—either synthesis or degradation—and form linear interaction pairs with one or a few other degradation or synthesis enzymes, regulating c-di-GMP levels within the bacteria. Therefore, after identifying the main phosphodiesterase gene chr1_233, analyzing the expression activities of c-di-GMP synthesis and degradation-related genes regulated by it can help determine the main diguanylate cyclase gene within the bacteria. Wild-type S. xenophagum C2 and mutant S. xenophagum C2 (Δ233) were inoculated into LB broth and cultured at 30°C in a shaker at 200 rpm / min until the late logarithmic growth phase. The bacterial OD... 60≈ 1.0. Centrifuge at 9,000 × g for 5 min, discard the supernatant, and collect the bacterial cells. Wash the bacterial cells twice with inorganic salt buffer, then resuspend the bacterial cells in a certain volume of inorganic salt buffer to adjust the OD of the bacterial cells. 600≈1.0. C2 bacterial suspension and C2 (Δ233) bacterial suspension were inoculated separately into inorganic salt medium at a volume fraction of 1% into the medium (each liter of medium contained 2.0 g Na2HPO4·12H2O, 0.7 g KH2PO4, 0.5 g NH4Cl, 0.3 g NaCl, 0.1 g MgSO4·7H2O, 0.05 g CaSO4·2H2O, 0.2 mg FeCl3·6H2O, 0.2 mg NaMoO4, 0.2 mg MnCl2·4H2O, 0.2 mg CuCl2·2H2O, 0.2 mg ZnSO4, 0.3 mg H3BO3, 0.4 mg CoCl2·6H2O, 0.2 g peptone, 1.0 g yeast extract, and 5.0 g glucose). g (solvent: water; preparation method: dissolve all components in water and sterilize). Incubate at 30°C for 9 h in a shaker at 200 rpm / min. Centrifuge at 9,000 × g for 5 min, discard the supernatant, collect the bacterial cells, flash-freeze in liquid nitrogen, and then send to Guangzhou Gediao Biotechnology Co., Ltd. for sequencing analysis of bacterial transcriptome mRNA expression levels.

[0029] Based on the RPKM (Reads Per kb per Million reads) value of gene expression, and using the formula FDR (false discovery rate) < 0.05 and |Log2 C2 (Δ233) / C2 |>1 Analysis of the expression differences of 16 genes related to c-di-GMP synthesis and degradation in wild-type strain C2 and mutant strain C2 (Δ233) revealed that in the C2 (Δ233) mutant strain where the chr1_233 gene was knocked out, only the expression of the chr1_457 gene (sequence shown in SEQ ID NO.3, encoding amino acid sequence shown in SEQ ID NO.4) was significantly different from that of the wild-type strain, decreasing by 3.32-fold, Log2 C2 (Δ233) / C2 =-1.73, FDR<0.001. The remaining c-di-GMP synthesis and degradation-related genes | Log2 C2 (Δ233) / C2 All are less than 1 ( Figure 3 A) The expression level in the mutant strain did not change significantly compared to the wild-type strain. These results indicate that the guanylate cyclase encoded by the chr1_457 gene is the main guanylate cyclase in strain C2. In the C2 (Δ233) mutant strain with the chr1_233 gene knocked out, because Chr1_233 cannot degrade c-di-GMP, the c-di-GMP concentration in the bacterial cell is maintained at a high level. The high concentration of c-di-GMP inhibits the gene expression of the main guanylate cyclase chr1_457, which interacts linearly with the main phosphodiesterase chr1_233, and its c-di-GMP synthesis activity.

[0030] Therefore, in the C2 (Δ233) mutant strain, the expression of the guanylate cyclase chr1_457 gene was induced by introducing an inducible expression vector to achieve a higher level of c-di-GMP synthesis. Referring to the 4-isopropylbenzoic acid (cumate)-induced CMT operon sequence used for sphingosine monophosphate expression in Appl Environ Microbiol. 2013, 79(21): 6795-802, this sequence was synthesized using the gene synthesis method of Sangon Biotech (Shanghai) Co., Ltd. Using the genomic DNA of strain C2 as a template, primer 457-PU (5′-GCTCTAGAACTAGT) was used to synthesize the cytosolic acid operon. GGATCC TCAGCGCTTGAACTTGGC-3′) and 457-PD (5′-AAGCTTGATATC GAATTC The chr1_457 gene was obtained by amplification using the formula TCAGTGGTGGTGGTGGTGGTGGAGCATCTGCACGACCGGCC-3′. DNA was extracted from the pBBR1MCS-5 broad-host plasmid using a plasmid extraction kit and then processed using the FastDigest restriction endonuclease BamH2O. and EcoR The pBBR1MCS-5 plasmid was digested with enzymes in FastDigest buffer at 37°C for 15 minutes. Then, the CMT operon sequence fragment, the chr1_457 gene fragment, and the restriction enzyme-digested pBBR1MCS-5 plasmid fragment were purified using a product purification kit. The pBBR1MCS-5 plasmid was ligated to the corresponding gene fragment using recombinase and transformed into E. coli competent cells. After screening with 15 μg / mL gentamicin sulfate and sequencing verification, the successfully ligated recombinant plasmid pCMT_457 for inducible expression of the chr1_457 gene was finally obtained.

[0031] Then, the recombinant plasmid pCMT_457 was transformed into competent cells of the C2 (Δ233) mutant strain via electroporation (25 µF, 200 Ω, 2000 V). The transformed bacterial culture was plated on LB agar plates containing 15 μg / mL gentamicin sulfate and incubated at 30°C for 24 h. Single clones of C2 (Δ233, pCMT_457) that grew on the resistant plates were picked and inoculated into LB liquid medium and cultured at 30°C on a shaker at 200 rpm / min until the late logarithmic growth phase. 600≈1.0. C2 (Δ233, pCMT_457) bacterial suspension was inoculated into fresh LB medium at a volume fraction of 2%. Induction was performed by adding 0, 25, 50, 100, and 200 µM of 4-isopropylbenzoic acid, respectively. The medium was then incubated at 30°C for 5–6 h in a shaker at 200 rpm / min until the bacterial cell OD reached 1.00. 600 The value reached 0.3. The cell surface hydrophobicity and intracellular c-di-GMP content of strains induced by different concentrations of 4-isopropylbenzoic acid were determined according to the method described in Example 2 above. The results showed that the cell surface hydrophobicity of strain C2 (Δ233, pCMT_457) induced by different concentrations of 4-isopropylbenzoic acid decreased, possibly because the addition of 4-isopropylbenzoic acid affected the surface properties of the bacterial cells; however, with the increase of the induced concentration of 4-isopropylbenzoic acid, the intracellular c-di-GMP concentration showed a trend of first increasing and then decreasing, reaching its highest level of 59.16 µg / L under the induction of 50 µM 4-isopropylbenzoic acid. Figure 3 B), which showed a 1.36-fold increase in expression compared to strains without induced expression of the major guanylate cyclase gene chr1_457, and a 6.58-fold increase compared to strains without knockout of the major phosphodiesterase gene chr1_233.

[0032] Example 4: Constitutive expression of primary diguanylate cyclase and characteristics of c-di-GMP synthesis

[0033] Considering the cost of exogenous inducers for 4-isopropylbenzoic acid induction and their impact on bacterial cell surface traits, constitutive expression of the chr1_457 gene was performed using a strong promoter from S. xenophagum C2 to further enhance the synthesis level of c-di-GMP within the bacteria. Based on the RPKM of gene expression in wild-type strain C2 and mutant strain C2 (Δ233) in Example 3, the top 5 constitutive genes with the highest expression levels were screened: chr1_235, chr1_59, chr1_2569, chr1_236, and chr1_962. The promoter sequence prediction tool provided by Softberry (http: / / www.softberry.com / ) was used to predict the promoter sequence of the upstream 1000 bp of these genes. Only chr1_962 had a predicted promoter sequence, ATCACGTCGGACACATTGTTCCTCTACAAT, located 382 bp upstream of the chr1_962 gene. Using the genomic DNA of strain C2 as a template, primer tf962 U1 (5′-TGGGTCGC) was used. GGATCCThe promoter fragment tf962 of the chr1_962 gene was amplified using primers AAATCGTCTCTCCATCACGTCGG-3′ and tf962 D (5′-CATGGTCCAGTCCCTTGCTCT-3′) (sequence shown in SEQ ID NO.5); the promoter fragment tf962 of the chr1_962 gene was obtained using primers 962-457 U (5′-CAGAGCAAGGGACTGGACCATGCATTTCTATCTCGCGACG-3′) and 962-457 D1 (5′-TG CTCGAG The chr1_457 gene fragment was obtained by amplification using the primer tf962 (5′-ACAGCAAATGGGTCGC). Using the aforementioned promoter sequence tf962 and the chr1_457 gene fragment as templates, primer tf962 U2 (5′-ACAGCAAATGGGTCGC) was used. GGATCC AAATCGTCTCTC-3′) and 962-457 D2 (5′-TCAGTGGTGGTGGTGGTGGTG CTCGAG The tf962-457 gene fragment was obtained by C-3′ fusion. DNA from the pET-24a plasmid was extracted using a plasmid extraction kit and analyzed with the FastDigest restriction endonuclease BamH2. and Xho The pET-24a plasmid was digested with enzymes in FastDigest buffer at 37°C for 15 minutes. Then, the tf962-457 gene fragment and the pET-24a plasmid fragment digested with restriction enzymes were purified using a product purification kit. The pET-24a plasmid and the tf962-457 gene fragment were ligated using homologous recombinase and transformed into E. coli competent cells. After screening for resistance with 50 μg / mL kanamycin and sequencing verification, the successfully ligated recombinant plasmid pTF962_457 for the chr1_457 genome expression was finally obtained.

[0034] The recombinant plasmid pTF962_457 was transformed into competent cells of the C2 (Δ233) mutant strain via electroporation (25 µF, 200 Ω, 2000 V). The transformed bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 30°C for 48 h. Single colonies of C2 (Δ233, pTF962_457) that grew on the resistant plates were picked and inoculated into LB liquid medium and cultured at 30°C on a shaker at 200 rpm / min until the late logarithmic growth phase. 600 ≈1.0. Inoculate the C2 (Δ233, pTF962_457) bacterial suspension into fresh LB medium at a volume fraction of 2%, and incubate at 30°C for 5–6 h in a shaker at 200 rpm / min until the bacterial cell OD reaches 1.0.600 The value reached 0.3. The cell surface hydrophobicity and intracellular c-di-GMP content of strain C2 (Δ233, pTF962_457) were determined according to the method described in Example 2 above. The results showed that expressing the chr1_457 gene in C2 (Δ233, pTF962_457) cells using the strong promoter tf962 significantly increased the cell surface hydrophobicity and intracellular c-di-GMP content. The cell surface hydrophobicity of C2 (Δ233, pTF962_457) increased to 64.00%, while the in vivo synthesized c-di-GMP content reached 82.26 µg / L (…). Figure 4 The expression rate of the guanylate cyclase gene chr1_457 was 1.39 times higher than that of the strain that did not express the guanylate cyclase gene chr1_457, and 9.15 times higher than that of the strain that did not knock out the guanylate cyclase gene chr1_233.

[0035] Example 5: Mutation at the GGDEF domain activity inhibition site of major diguanylate cyclase and characteristics of c-di-GMP synthesis

[0036] Because the synthesis of guanylate cyclase is subject to feedback inhibition by c-di-GMP itself, specifically by the RXXD inhibitory site in the GGDEF domain binding to c-di-GMP, there is a certain upper limit to the concentration of intracellular c-di-GMP synthesis. Therefore, after addressing the expression level of the guanylate cyclase gene at the gene level, the inhibitory site of the guanylate cyclase Chr1_457 (the mutated nucleotide sequence is shown in SEQ ID NO.6, and its encoded amino acid sequence is shown in SEQ ID NO.7) was modified to further increase the level of c-di-GMP synthesis in bacteria at the protein level. Using the tf962-457 gene fragment from Example 4 as a template, primer tf962 U2 (5′-ACAGCAAATGGGTCGC) was used. GGATCC AAATCGTCTCTC-3′) and primer 457-R187P-D (5′-GACCAGATCGACGCG) GGG CAACAGC-3′ [underlined is the point mutation site from amino acid R to P], amplified the upstream fragment of tf962-457-R187P; using primer 457-R187P-U (5′-GCGCTGTTG) CCC CGCGTCGATCTGG-3′) [underlined indicates the point mutation site from amino acid R to P] and 962-457 D2 (5′-TCAGTGGTGGTGGTGGTGGTGGTG) CTCGAG(C-3′), amplify the downstream fragment of tf962-457-R187P; extract DNA from pET-24a plasmid using a plasmid extraction kit, and use FastDigest restriction endonuclease BamH2O. and Xho The pET-24a plasmid was digested with enzymes in FastDigest buffer at 37°C for 15 minutes. Then, the upstream and downstream fragments of tf962-457-R187P, as well as the pET-24a plasmid fragment digested with restriction enzymes, were purified using a product purification kit. The pET-24a plasmid was ligated to the corresponding gene fragment using homologous recombinase and transformed into competent E. coli cells. After screening for resistance with 50 μg / mL kanamycin and sequencing verification, the recombinant plasmid pTF962_457-R187P, with its ligation of the active inhibitory site R187 modified to P187, was finally obtained and expressed in a genome-complete manner.

[0037] The recombinant plasmid pTF962_457-R187P was transformed into competent cells of the C2 (Δ233) mutant strain via electroporation (25 µF, 200 Ω, 2000 V). The transformed bacterial suspension was plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 30°C for 48 h. Single colonies of C2 (Δ233, pTF962_457-R187P) that grew on the resistant plates were picked and inoculated into LB liquid medium and cultured at 30°C in a shaker at 200 rpm / min until late logarithmic growth was reached, with a cell OD600 ≈ 1.0. A 2% (v / v) inoculum of the C2 (Δ233, pTF962_457-R187P) bacterial suspension was inoculated into fresh LB agar and cultured at 30°C in a shaker at 200 rpm / min for 5–6 h until the cell OD600 reached 1.0. 600 The value reached 0.3. The cell surface hydrophobicity and intracellular c-di-GMP content of strain C2 (Δ233, pTF962_457-R187P) were determined according to the method described in Example 2 above. The results showed that constitutive expression of the chr1_457-R187P gene, which modifies the active inhibitory site R187 to P187, in C2 (Δ233, pTF962_457-R187P) significantly increased the cell surface hydrophobicity and intracellular c-di-GMP content. The cell surface hydrophobicity of C2 (Δ233, pTF962_457-R187P) increased to 71.28%, while the in vivo c-di-GMP content reached 145.70 µg / L (…). Figure 5The expression rate was 1.77 times higher than that of wild-type guanylate cyclase gene chr1_457 constitutively expressed strains without modified active inhibition sites; 2.46 times higher than that of 50 µM 4-isopropylbenzoic acid-induced guanylate cyclase gene chr1_457 expression strains; 3.35 times higher than that of non-expressing guanylate cyclase gene chr1_457 strains; and 16.21 times higher than that of chr1_233 strains without knockout of guanylate cyclase gene.

[0038] Analysis using methanol / acetonitrile extraction combined with LC-MS revealed that OD200 of LB medium cultured to the logarithmic growth phase... 600 The recombinant strain with an OD value of approximately 0.3 can synthesize ~0.15 mg / L of c-di-GMP, and after culturing to the stationary phase... 600 The recombinant strain with a growth rate of ≈3.0 can synthesize ~1.5 mg / L of c-di-GMP; by optimizing the extraction method, culture medium formulation, and fermentation method, higher yields of natural c-di-GMP synthesis can be obtained.

[0039] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0040] SEQ ID NO.1

[0041] >chr1_233

[0042]

[0043] SEQ ID NO.2

[0044] >chr1_233

[0045] MAQRTGQEADVRQSLVESLYASPASLTIGAATGTAVGIAISSLSDFAAIHAVVIAVCVVAALRVVSAIFFHRQLVRGTAVASRNWELAYELGAWAYAGLLGLMGFVTLIGSGNPVIHMLSVSMATGYAGGISGRNAGRVQIAIGQVCLALLPTATGLWMEGSTGYRVLSIAMVAMVLGLAEISSTTHRIVVQALIGKRDKSLLAEKFERLARFDSLTGLENRMAMQMRLRDIFETSQKRNDAVAILWMDVDRFKEINDSLGHMVGDQLLRAVAERLDEVVQGRGRIARFGGDEFVIICPDTDRVTAAAIAQEIITYFGHGFDLSDNHLQVTASIGIAVAPQDGRDMDELMQHADLALYEAKREGRNRASSFNWSLKERFNRVHEVETGLRRALETNELKLLFQPIVDLESGHIDACEALLRWDHPVLGPISPGEFIPIAESMGMIEAMTGWVLREACIAAASWPGEVRIAINISPASLKSHELPGNVIAALLETGLPARRLELEVTESIFLDDSGQTNTILRELQRIGLRLALDDFGTGYSSLSYLRSYRFDTLKVDQSFMAGVSSNAEDRAIVRAIGNLARDLAMDTVAEGIETPDQLLHAREAGFTNVQGYLFSRPVTSERVAEMIAAGPLEGAERPDPVIRKRPQRQA

[0046] SEQ ID NO.3

[0047] >chr1_457

[0048] atgcatttctatctcgcgacgtcattcatttttccgcgctcgctgcgcctgcgcctcttcaccctctgtttcatcgccacccatttgccgctgctgggctattgtggttgggggctaatgaccggacggatcgcgctgaccgaatttgtcctgctgacgctgatgacgctgtttggcacagcgatcgcgctgattggcatgggggcgttgctcaaccccattcatgcgctggcggagacgttcaacggcaaggcggatgccgcgcttccggaagtcggcgacgtgatccagacgctctatgccggggtccatcgcgccgccagcacgacgcgggcgcagatcgacgacctgcatgtcgccgcgcatgaagacccgctgacgggggttgccaaccggcgcggctttctggcgcagctcgacgccctgccccacgaccagcgccgcggctgtgtcgcgatcatcgacattgatcatttcaagcaggtcaacgaccagctgggccatgatgagggcgaccgggtgctggccgcctttgccgaccgcctgtccgcgctgttg cgc cgcgtc gat ctggtcgcgcgctggggtggcgaggaatttgtggttttctttcatggcgcggcagaggatgaagcctgctggtcgctggcgcgtatcgccaaccagatgcggctggacccgatcggccggattcatggccgcccgatcagcttttccgccggtctgtcccgctgggccggagatgccgtggacggcgcattgagcgcggcggacacggcgctctatgacgccaaacagtccgggcgcgaccgcatctgtcgcgcggggccggtcgtgcagatggtgtga

[0049] SEQ ID NO.4

[0050] >chr1_457

[0051] MHFYLATSFIFPRSLRLRLFTLCFIATHLPLLGYCGWGLMTGRIALTEFVLLTLMTLFGTAIALIGMGALLNPIHALAETFNGKADAALPEVGDVIQTLYAGVHRAASTTRAQIDDLHVAAHEDPLTGVANRRGFLAQLDALPHDQRRGCVAIIDIDHFKQVNDQLGHDEGDRVLAAFADRLSALL R RV D LVARWGGEEFVVFFHGAAEDEACWSLARIANQMRLDPIGRIHGRPISFSAGLSRWAGDAVDGALSAADTALYDAKQSGRDRICRAGPVVQMV

[0052] SEQ ID NO.5

[0053] >tf962

[0054] aaatcgtctctcc atcacgtcggacacattgttcctctacaat gcacccgccagaggcaaggtcaatacgcgcctgcaaacgccatccaaacatgagcaacgccacaatagggctgccttgtttggtcgcgcggcatgattgctgcatggcgcaggaccgccgtgcaactatattgctaaccacatgtaaaacagtcatgccacgttcatgttgcatttatgatacaggggcgttcaaaagcggtgaatgcccttttccccggctttacagcgccgttcgccccgcgcagtttccccctatccatatggggggatcgctgtcgggcaccatgtccccctgtaggtgcattagagtggcaggccatctgccaacctccagagcaagggactggacc

[0055] SEQ ID NO.6

[0056] >chr1_457 (R187P)

[0057] atgcatttctatctcgcgacgtcattcatttttccgcgctcgctgcgcctgcgcctcttcaccctctgtttcatcgccacccatttgccgctgctgggctattgtggttgggggctaatgaccggacggatcgcgctgaccgaatttgtcctgctgacgctgatgacgctgtttggcacagcgatcgcgctgattggcatgggggcgttgctcaaccccattcatgcgctggcggagacgttcaacggcaaggcggatgccgcgcttccggaagtcggcgacgtgatccagacgctctatgccggggtccatcgcgccgccagcacgacgcgggcgcagatcgacgacctgcatgtcgccgcgcatgaagacccgctgacgggggttgccaaccggcgcggctttctggcgcagctcgacgccctgccccacgaccagcgccgcggctgtgtcgcgatcatcgacattgatcatttcaagcaggtcaacgaccagctgggccatgatgagggcgaccgggtgctggccgcctttgccgaccgcctgtccgcgctgttg ccc cgcgtc gat ctggtcgcgcgctggggtggcgaggaatttgtggttttctttcatggcgcggcagaggatgaagcctgctggtcgctggcgcgtatcgccaaccagatgcggctggacccgatcggccggattcatggccgcccgatcagcttttccgccggtctgtcccgctgggccggagatgccgtggacggcgcattgagcgcggcggacacggcgctctatgacgccaaacagtccgggcgcgaccgcatctgtcgcgcggggccggtcgtgcagatggtgtga

[0058] SEQ ID NO.7

[0059] >chr1_457 (R187P)

[0060] MHFYLATSFIFPRSLRLRLFTLCFIATHLPLLGYCGWGLMTGRIALTEFVLLTLMTLFGTAIALIGMGALLNPIHALAETFNGKADAALPEVGDVIQTLYAGVHRAASTTRAQIDDLHVAAHEDPLTGVANRRGFLAQLDALPHDQRRGCVAIIDIDHFKQVNDQLGHDEGDRVLAAFADRLSALL P RV D LVARWGGEEFVVFFHGAAEDEACWSLARIANQMRLDPIGRIHGRPISFSAGLSRWAGDAVDGALSAADTALYDAKQSGRDRICRAGPVVQMV。

Claims

1. The use of knocking out the major phosphodiesterase gene chr1_233 in Sphingobium xenophagum to improve the intracellular synthesis of c-di-GMP in Sphingobium strains, characterized in that: The nucleotide sequence of the main phosphodiesterase gene chr1_233 is shown in SEQ ID NO.

1.

2. The use of overexpression of the main diguanylate cyclase gene chr1_457 or mutants of the main diguanylate cyclase gene chr1_457 in the Sphingobium xenophagum strain in which the main phosphodiesterase gene chr1_233 is knocked out to improve the intracellular synthesis of c-di-GMP, characterized in that: The nucleotide sequence of the main guanylate cyclase gene chr1_457 is shown in SEQ ID NO.3, the nucleotide sequence of the mutant of the main guanylate cyclase gene chr1_457 is shown in SEQ ID NO.6, and the nucleotide sequence of the main phosphodiesterase gene chr1_233 is shown in SEQ ID NO.

1.

3. The use of the promoter tf962 to overexpress the master diguanylate cyclase gene chr1_457 or a mutant of the master diguanylate cyclase gene chr1_457 in a Sphingobium xenophagum strain in which the master phosphodiesterase gene chr1_233 has been knocked out for increasing intracellular synthesis of c-di-GMP, characterized in that: The nucleotide sequence of the promoter tf962 is shown in SEQ ID NO.5, the nucleotide sequence of the main guanylate cyclase gene chr1_457 is shown in SEQ ID NO.3, the nucleotide sequence of the mutant of the main guanylate cyclase gene chr1_457 is shown in SEQ ID NO.6, and the nucleotide sequence of the main phosphodiesterase gene chr1_233 is shown in SEQ ID NO.

1.

4. A c-di-GMP high-yield producing Sphingobium xenophagum, characterized by: The main phosphodiesterase gene chr1_233 was knocked out in Sphingobium xenophagum, and the nucleotide sequence of the main phosphodiesterase gene chr1_233 is shown in SEQ ID NO.

1.

5. A sphingobium xenophagum bacterium that produces high levels of c-di-GMP, characterized in that: It is a strain of Sphingobium xenophagum that overexpresses the major guanylate cyclase gene chr1_457 or a mutant of the major guanylate cyclase gene chr1_457, in which the major phosphodiesterase gene chr1_233 is knocked out; the nucleotide sequence of the major guanylate cyclase gene chr1_457 is shown in SEQ ID NO.3, the nucleotide sequence of the mutant of the major guanylate cyclase gene chr1_457 is shown in SEQ ID NO.6, and the nucleotide sequence of the major phosphodiesterase gene chr1_233 is shown in SEQ ID NO.

1.

6. The sphingobium xenophagum according to claim 4 or 5, characterized in that: The sphingobium xenophagum mentioned is Sphingobium xenophagum C2.

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