Genetically engineered bacterium for producing extracellular water-soluble beta-1, 3-glucan at high yield and construction method of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for highly producing extracellular water-soluble beta-1, 3-glucan and a construction method of the genetically engineered bacterium, and belongs to the field of microbial genetic engineering. According to the invention, rhizobium ZB01-H is used as a host, and gene engineering strains ZB01-zlzF and ZB01-zlzW are obtained by overexpressing genes zlzF and zlzW; further, a negative regulation factor gluA is knocked out, so that genetic engineering strains ZB01-pFDA and ZB01-pWDA are obtained; an inducer IPTG (isopropyl-beta-d-thiogalactoside) is added in the 4th hour in the fermentation process of the strain to start gene expression, so that the yield of beta-1, 3-glucan reaches 11.62 g / L and 11.24 g / L. According to the invention, the problem of yield bottleneck of extracellular soluble beta-1, 3-glucan produced by rhizobium is effectively solved, a method thought is provided for modification of microbial strains, and a foundation is laid for industrial production of beta-1, 3-glucan.

Description

Technical Field

[0001] This invention relates to genetically engineered bacteria that produce high levels of extracellular water-soluble β-1,3-glucan and their construction methods, belonging to the field of microbial genetic engineering. Background Technology

[0002] In recent years, β-1,3-glucan has shown broad application potential in functional foods, pharmaceuticals, and cosmetics due to its diverse biological activities and excellent functional properties. β-1,3-glucan not only possesses various physiological functions such as immunomodulation, anti-tumor activity, hypoglycemia, and cholesterol reduction, but also exhibits practical processing properties such as thickening, emulsification, and gelation, making its application value outstanding. Currently, commercially available β-1,3-glucan is mainly derived from grains and yeast, but it is usually found in the cell wall, resulting in complex extraction processes, low yields, and susceptibility to contamination by impurities such as starch and protein, limiting product purity, quality, and large-scale production.

[0003] Microbial fermentation for the production of extracellular β-1,3-glucan is considered an effective approach to address the aforementioned problems, offering advantages such as simple extraction and low cost. Agrobacterium has been used for the industrial production of β-1,3-glucan (Curdlan), but its poor water solubility at room temperature severely limits its practical application in food processing and drug delivery systems. Although chemical modification can improve solubility, this approach is strictly limited in the food industry from a food safety perspective. Therefore, developing microorganisms capable of producing high yields of water-soluble extracellular β-1,3-glucan has become an important direction for industry development.

[0004] Against this backdrop, rhizobia exhibit unique advantages, possessing the ability to directly synthesize and secrete extracellular water-soluble β-1,3-glucan, effectively simplifying downstream separation and purification processes. For example, patent CN106434495A discloses a type of *Pusa rhizobia* (…). Rhizobium pusense (CGMCC No. 12954) Achieved a β-1,3-glucan yield of 5.5 g / L. Currently, the key gene clusters for polysaccharide synthesis in rhizobia have been preliminarily elucidated. To further increase extracellular polysaccharide yield, the β-1,3-glucan synthesis pathway can be optimized and its yield increased through targeted expression regulation of key genes. For example, patent CN117625507A represents the inventors' preliminary exploration of key genes in rhizobia, achieving a β-1,3-glucan yield of 5.64 g / L. However, the regulatory mechanism for the synthesis of water-soluble β-1,3-glucan in rhizobia is still in its early stages. There is an urgent need to further explore key regulatory proteins, reconstruct and optimize their metabolic pathways to achieve efficient industrial-scale synthesis of extracellular water-soluble β-1,3-glucan. Summary of the Invention

[0005] [Technical scheme] In order to solve the above technical problems, the present application provides a genetically engineered strain of rhizobium, which realizes the efficient synthesis of beta-1,3-glucan by adjusting its metabolic pathway, and the yield in the laboratory reaches 9.82 g / L-11.62 g / L, laying a foundation for the industrial production of beta-1,3-glucan.

[0006] The first object of the present application is to provide a genetically engineered strain for efficiently synthesizing beta-1,3-glucan, which takes rhizobium as a host cell and is modified by at least one of the following (a) and (b): (a) overexpression zlzF of a gene; (b) overexpression zlzW of a gene.

[0007] In an embodiment of the present application, the function of the gluA gene in the genome of the genetically engineered strain is deleted or knocked out.

[0008] In an embodiment of the present application, the nucleotide of the zlzF gene is shown as SEQ ID NO. 4, the nucleotide of the zlzW gene is shown as SEQ ID NO. 5, and the nucleotide of the gluA gene is shown as SEQ ID NO. 6.

[0009] In an embodiment of the present application, the rhizobium is ZB01, ZB01-H.

[0010] In an embodiment of the present application, the rhizobium ZB01-H is recorded in the literature "Synthesis of beta-glucan in rhizobium and its regulation on triglyceride metabolism".

[0011] The second object of the present application is to provide a method for increasing the yield of beta-1,3-glucan, which overexpresses the target gene zlzF and / or zlzW in rhizobium.

[0012] In an embodiment of the present application, the function of the gluA gene in the genome of the rhizobium is deleted or knocked out.

[0013] In an embodiment of the present application, the nucleotide of the zlzF gene is shown as SEQ ID NO. 4, the nucleotide of the zlzW gene is shown as SEQ ID NO. 5, and the nucleotide of the gluA gene is shown as SEQ ID NO. 6.

[0014] In an embodiment of the present application, the rhizobium is ZB01, ZB01-H.

[0015] A third object of the present application is to provide a method for efficiently synthesizing β-1,3-glucan, which is fermentation culture using the genetically engineered strain.

[0016] In an embodiment of the present application, the genetically engineered strain is inoculated into a culture medium, and an inducer is added after fermentation for 4-12 h, and then induced culture is performed for 70-80 h.

[0017] In an embodiment of the present application, seed liquid of the genetically engineered strain is inoculated into a fermentation medium, an inducer is added after fermentation for 4 h, and then induced culture is performed for 72 h.

[0018] In an embodiment of the present application, the inducer is IPTG.

[0019] In an embodiment of the present application, the amount of the inducer added is 0.5-1.5 mM.

[0020] In an embodiment of the present application, the amount of the inducer added is 1 mM.

[0021] In an embodiment of the present application, the seed liquid is prepared by inoculating the genetically engineered strain into a seed culture medium, and then performing oscillation culture at 25-30°C and 180-240 rpm until OD 600 is 0.8-1.0 to obtain the seed liquid.

[0022] A fourth object of the present application is to provide application of the genetically engineered strain or the method in synthesis of β-1,3-glucan or a product containing β-1,3-glucan.

[0023] In an embodiment of the present application, the product includes food, medicine, and cosmetics.

[0024] [Advantages] By overexpressing zlzF , zlzW in the rhizobium ZB01-H, the engineered strains ZB01-pzlzF and ZB01-pzlzW are constructed. On this basis, the negative regulatory factor gluA is further knocked out to obtain the engineered strains ZB01-pFDA and ZB01-pWDA with high yield of soluble β-1,3-glucan. The yield of β-1,3-glucan is increased from 8.47 g / L (ZB01-H) to 9.82 g / L (ZB01-pzlzF) and 10.11 g / L (ZB01-pzlzW), respectively; and further by knocking out the negative regulatory factor of the rhizobiumgluA The knockout of the gene and IPTG induction of gene expression (IPTG was added at the 4th hour during fermentation) ultimately increased the production of β-1,3-glucan to 11.62 g / L (ZB01-pFDA) and 11.24 g / L (ZB01-pWDA), respectively, laying a foundation for the industrial production of microbial fermentation of β-1,3-glucan. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The production of exocellular polysaccharide β-1,3-glucan of the mutagenized engineering strain ZB01-H and each overexpression gene engineering strain was added with 1 mM IPTG inducer after the start of fermentation culture; Figure 2 is the production of exocellular polysaccharide β-1,3-glucan of the engineering strains ZB01-pFDA and ZB01-pWDA added with 1 mM IPTG inducer at different times during fermentation culture; (A) is ZB01-pWDA, and (B) is ZB01-pFDA; Figure 3 The production of exocellular polysaccharide β-1,3-glucan of the mutagenized engineering strain ZB01-H and the engineering strains ZB01-pFDA and ZB01-pWDA was added with 1 mM IPTG inducer at the 4th hour during fermentation. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0027] The reagents and materials involved in the following examples are as follows: 1. Reagents and materials Rhizobium ZB01 is described in Structural characterization of a water-soluble and antimicrobial β-glucan secreted by Rhizobium pusense in the literature.

[0028] Rhizobium ZB01-H is described in the literature entitled “Synthesis of β-glucan of Rhizobium and research on regulation of triglyceride metabolism”.

[0029] Escherichia coli DH5a and Escherichia coli DH5a competent cell (Beijing Zoman Biotech Co., Ltd.); Sucrose, NaCl and anhydrous ethanol were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). PCR product purification kit, plasmid extraction kit, DNA extraction kit and DNA agarose gel recovery kit were purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China); PrimeSTAR Max DNA Polymerase, Premix Taq, DL 10000 DNA Marker, T4 DNA ligase and Restriction endonuclease were purchased from Boshijia Biotech Co., Ltd. (Beijing, China); MultiF Seamless Assembly Mix was purchased from Aibiotek Biotech Co., Ltd.; Nucleic Acid Stain, Loading buffer and various antibiotics were purchased from Sangon Biotech Co., Ltd. (Shanghai, China); Primer synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.).

[0030] 2. Culture medium: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, adjust pH to 7.2, sterilize at 121℃ for 20 min.

[0031] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 15 g / L, pour into sterile plate after sterilization at 121℃ for 20 min, it is antibiotic-free LB solid medium; add antibiotics to the corresponding antibiotic selective LB solid medium, condense and store in the refrigerator at 4℃ for standby.

[0032] Fermentation liquid medium: sucrose 30 g / L, tryptone 1 g / L, Na2HPO4 1 g / L, CaCl2 0.07 g / L, MgCl2 0.2 g / L, adjust pH to 7.5, sterilize at 121℃ for 20 min.

[0033] Antibiotic addition: the final concentration of kanamycin (kana) is 200 μg / mL; the final concentration of nalidixic acid (Naac) is 60 μg / mL.

[0034] 3. Primer Table 1 Primer

[0035] 4. Plasmid Table 2 Plasmids and their carrying strains and types

[0036] 5、Strains Table 3 Engineering strains and their genotypes

[0037] Methods involved in the following examples 1、Basic operations of molecular biology: (1) Extraction of E. coli plasmid, genome extraction, purification of PCR products, and gel recovery were performed according to the corresponding kit instructions, unless otherwise specified.

[0038] (2) PCR technology and overlap extension PCR technology were used for DNA fragment amplification and fragment fusion, respectively, and different reaction systems and programs were used according to different experimental needs. The reaction system was referred to Table 2, and the reaction program was referred to Table 3.

[0039] Table 4 PCR reaction system

[0040] Table 5 PCR reaction program

[0041] (3) Seamless cloning: the reaction system and reaction conditions of seamless cloning were referred to the relevant product instructions or official recommended scheme of ABclonal website (https: / / abclonal.com.cn / ), unless otherwise specified.

[0042] (4) Enzymatic digestion and ligation: the reaction system and reaction conditions of DNA double digestion and T4 ligase were referred to the (Takara) website (https: / / www.takarabiomed.com.cn / ), unless otherwise specified.

[0043] (5) E. coli competent heat shock transformation: first, 5-10 μL of ligation product was added to 50-100 μL of frozen competent cells, and ice bathed for 30 min; then the competent cells were placed in a 42℃ metal bath for heat shock for 50 s, and quickly placed on ice, ice bathed for 2 min; then 1 mL of LB liquid medium was added, and incubated at 37℃ for 60 min; finally, 200 μL of bacterial solution was spread on the corresponding resistant solid LB plate, and incubated at 37℃ overnight.

[0044] Example 1 Construction of overexpression and knockout plasmids (1) Construction of the overexpression vector pBBR1MCS-2-ptac: Using the commercial plasmid pBBR1MCS-2 as a template, the plasmid fragment pBBR1MCS-2-△plac was obtained by reverse PCR amplification using primers pB2Qplac-F / pB2Qplac-R; Using the commercial plasmid pGEX-4T-1 as a template, the lacI gene expression cassette (nucleotide sequence shown in SEQ ID NO.1), the ptac promoter gene fragment (nucleotide sequence shown in SEQ ID NO.2), and the lacI gene expression cassette were obtained by PCR amplification using primers lacI-F / lacI-R, ptac-F / ptac-R, and GST-F / GST-R, respectively. GST Fusion tag gene fragment (nucleotide sequence as shown in SEQ ID NO.3); Using fusion PCR with primers lacI-F / GST-R, lacI Fragment, ptac Fragments and GST Fragment splicing and fusion; The fusion fragment and plasmid fragment pBBR1MCS-2-△plac were ligated with a seamless cloning reagent at 50℃ for 15 min, and then transformed into Escherichia coli (DH5α). After obtaining transformants, the plasmid was extracted to obtain the overexpression vector pBBR1MCS-2-ptac.

[0045] (2) Construction of gene overexpression plasmids: Using the plasmid pBBR1MCS-2-ptac constructed in step (1) as a template, the linear fragment of plasmid pBBR1MCS-2-ptac was obtained by reverse PCR amplification using primers pB2ptac-F / pB2ptac-R. Using the genome of Rhizobium ZB01-H as a template, the target gene fragment was obtained by PCR amplification using primers zlzA-F / zlzA-R. zlzA ; Target gene fragment zlzA After ligating the linear fragment with a seamless cloning reagent at 50°C for 15 min, it was transformed into competent E. coli (DH5α) cells. After bacterial growth, positive strains were screened to obtain carrier cells. zlzA The donor strain of the gene overexpression plasmid pBBR1MCS-2-pzlzA.

[0046] Target gene zlzB~zlzX The construction of the overexpression plasmid was similar, and the primers are shown in Table 1. Simultaneously, donor strains carrying the target gene overexpression plasmids pBBR1MCS-2-pzlzB~pBBR1MCS-2-pzlzX were obtained. Plasmid information is shown in Table 2.

[0047] (3) Construction of gene knockout plasmid: The target gene fragment gluAu (the nucleotide sequence of which is shown as SEQ ID NO. 7) and the target gene fragment gluAd (the nucleotide sequence of which is shown as SEQ ID NO. 8) were obtained by PCR amplification using the primer gluA-up-F / gluA-up-R and gluA-down-F / gluA-down-R, respectively, with the genome of rhizobium ZB01-H as the template. gluAu gluAd The target gene fragment gluAu and the target gene fragment gluAd were fused by fusion PCR using the primer gluA-up-F / gluA-down-R with the target gene fragment gluAu and the target gene fragment gluAd as the templates, and the fusion fragment 5' and 3' end carried XbaI and HindIII enzyme cutting sites, respectively. The fusion fragment and the commercialized vector pK18mobSacB were cut by restriction enzymes XbaI and HindIII for 1 h, and then connected by T4 ligase at 16℃ overnight (12 h), and then transformed into E. coli competent cells (DH5α). After colonies grew, positive strains were screened to obtain the donor strain carrying the gene knockout plasmid pKgluA. gluA

[0048] Example 1: A plurality of E. coli strains containing different overexpression plasmids and knockout plasmids were constructed, as shown in the donor strain in Table 4. The primers used are shown in Table 1.

[0049] Example 2: Construction of genetically engineered strains: The donor strain was inoculated into LB liquid medium containing kanamycin (kana) and cultured at 37℃ with 220 rpm shaking; the recipient strain (the chassis strain) was inoculated into LB liquid medium without resistance and cultured at 28℃ with 220 rpm shaking; the helper strain was inoculated into LB liquid medium with kana and cultured at 37℃ with 220 rpm shaking; the recipient strain, the helper strain and the donor strain were cultured to OD 600 1.0, centrifuged, and the supernatant was removed and the bacterial cells were washed with 0.9% physiological saline three times, then the three bacterial cells were mixed together according to the mass ratio of helper strain: recipient strain: donor strain = 1:2:2, and 1 mL of LB liquid medium without resistance was added to suspend the bacterial cells, to obtain the mixed bacterial liquid; ​​​The nitrocellulose membrane is laid on an antibiotic-free LB plate, and 20 μL of the mixed bacterial solution is coated on the nitrocellulose membrane, and the inverted culture is carried out at 28°C for 48 h; a certain amount of 0.9% physiological saline is used to wash the bacterial body from the nitrocellulose membrane, and 50 μL of the mixed solution is coated on the LB plate containing kana and Naac, and the inverted culture is carried out at 28°C for 48 h, and then single colonies are picked for PCR colony verification.

[0050] Example 2 constructs a plurality of different genetically engineered strains, as shown in Table 5. The auxiliary strains and donor strains used are shown in Table 4.

[0051] Example 3: IPTG induction method of genetically engineered strains for producing β-1,3-glucan (1) Fermentation of genetically engineered strains ZB01-pzlzA~ZB01-pzlzY for preparing β-1,3-glucan Twenty-five genetically engineered strains (ZB01-pzlzA~ZB01-pzlzY) are inoculated into LB liquid medium containing kanamycin resistance (kana), and cultured at 28°C with 220 rpm oscillation until the OD 600 value is 1.0~1.2, and the seed is obtained. The seed liquid is inoculated into fermentation liquid medium containing kanamycin resistance (kana) at an inoculation amount of 10% (v / v), and 1 mM IPTG is directly added. The culture is carried out at 28°C with 220 rpm oscillation for 72 h, and three repeats are set for each group. The results are shown in Table 2. The β-1,3-glucan production of ZB01-pzlzF and ZB01-pzlzW reaches 9.82 g / L and 10.11 g / L, respectively. Figure 1

[0052] (2) Optimization of the addition time of IPTG (isopropyl-β-D-thiogalactoside) inducer: The genetically engineered strains ZB01-pFDA and ZB01-pWDA are inoculated into LB liquid medium containing kanamycin resistance (kana), and cultured at 28°C with 220 rpm oscillation until the OD 600 value is 1.0~1.2, and the seed is obtained. The seed liquid is inoculated into fermentation liquid medium containing kanamycin resistance (kana) at an inoculation amount of 10% (v / v), and 1 mM IPTG is directly added. The culture is carried out at 28°C with 220 rpm oscillation for 72 h, and three repeats are set for each group. The results are shown in Table 2. The β-1,3-glucan production of ZB01-pzlzF and ZB01-pzlzW reaches 9.82 g / L and 10.11 g / L, respectively. 600 ​The IPTG was added at 0 h, 4 h, 8 h, 12 h (the final concentration was 1 mM), and the control was set without adding IPTG, and the fermentation was carried out at 28 DEG C, 220 rpm, until 72 h, and three repeats were set in each group, and the results are shown in FIG. 2 (A) and FIG. 2 (B). The best IPTG addition time was 4 h, and the yield of beta-1, 3-glucan of the genetically engineered strains ZB01-pFDA and ZB01-pWDA was 11.62 g / L and 11.24 g / L, respectively, which was increased by 37.2% and 32.7% compared with the initial strain ZB01-H (FIG. 2 (A) and FIG. 2 (B) ). Figure 3 ).

[0053] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and the protection scope of the present application should be defined by the claims.

Claims

1. A genetically engineered strain for efficient synthesis of β-1,3-glucan, characterized in that, Using rhizobium as the host cell, the cells were modified by at least one of the following (a) and (b): (a) Overexpression zlzF Genes, the ones mentioned zlzF The nucleotides of the gene are shown in SEQ ID NO.4; (b) Overexpression zlzW Genes, the ones mentioned zlzW The nucleotides of the gene are shown in SEQ ID NO.

5.

2. The genetically engineered strain according to claim 1, characterized in that, The nucleotides in the genome of the genetically engineered strain are as shown in SEQ ID NO.

6. gluA The function of the gene is lost or knocked out.

3. The genetically engineered strain according to claim 1 or 2, characterized in that, The rhizobium is ZB01 or ZB01-H.

4. A method for increasing the yield of β-1,3-glucan, characterized in that, The method involves using rhizobia as a host to overexpress the target gene. zlzF and / or zlzW The rhizobium is ZB01 or ZB01-H. The zlzF The nucleotides of the gene are shown in SEQ ID NO.

4. zlzW The nucleotides of the gene are shown in SEQ ID NO.

5.

5. The method according to claim 4, characterized in that, The nucleotides in the genome of the rhizobium are as shown in SEQ ID NO.

6. gluA The function of the gene is lost or knocked out.

6. A method for efficiently synthesizing β-1,3-glucan, characterized in that, The method involves fermentation culture using any of the genetically engineered strains described in claims 1 to 3.

7. The method according to claim 6, characterized in that, The seed culture of any one of the genetically engineered strains described in claims 1 to 3 is inoculated into a fermentation medium, fermented for 4 to 12 h, and then an inducer is added, followed by induction culture for 70 to 80 h; optionally, the inducer is IPTG.

8. The method according to claim 7, characterized in that, The amount of the inducer added is 0.5~1.5 mM.

9. The use of the genetically engineered strain according to any one of claims 1 to 3, or the method according to any one of claims 6 to 8, in the synthesis of β-1,3-glucan or products containing β-1,3-glucan.

10. The application according to claim 9, characterized in that, The products include food, medicine, and cosmetics.

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