A colorless gellan gum high-yield engineering strain and a construction method and application thereof

Abstract

The application discloses a colorless gellan gum high-yield engineering strain and a construction method and application thereof, and belongs to the technical field of biotechnology. The technical problem to be solved is to provide a colorless gellan gum high-yield engineering strain, which can effectively remove pigments in a fermentation liquor, improve gellan gum product quality, reduce colloid post-processing difficulty, and effectively improve gellan gum fermentation production efficiency without increasing additional costs. The technical solution is characterized in that the colorless gellan gum high-yield engineering strain is obtained by inactivating crtI a gene in a less mobile sphingomonad and overexpressing vhb a gene and gelB-rmlD a transcription unit, wherein the transcription unit comprises gelB-rmlD a gene, gelB a gene, rmlA a gene, rmlC a gene, rmlB a gene and rmlD a gene.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the fields of microbial and genetic engineering, and specifically relates to a colorless high-yield engineered strain of gellan gum, its construction method, and its application. Background Technology

[0002] For understanding the technical content of this invention: Sphingomonas elodea (Also known as) Pseudomonas paucimobilis Later reclassified as Sphingomonas paucimobilis Sphingosine monocytogenes (SMM) is a non-pathogenic Gram-negative bacterium that produces complex microbial extracellular polysaccharide gellan gum.

[0003] Gellan gum is composed of repeating tetrasaccharide units, each containing two β-1,3-D-glucose, one β-1,4-D-glucuronic acid, and one α-1,4-L-rhamnose, with the monosaccharides linked by glycosidic bonds. As a natural biopolysaccharide, gellan gum has wide applications in food and beverage, pharmaceutical, personal care, and daily chemical industries.

[0004] Early modifications of gellan gum-producing strains primarily relied on mutagenesis breeding (e.g., patent documents CN120758402A and CN109971688A). Since the NCBI database reported the gellan gum biosynthetic gene cluster AY217008, research has gradually shifted towards target-oriented strain modification based on metabolic engineering. Several strategies have been reported in existing technologies, including: optimizing precursor metabolic flux allocation (CN120005791A), blocking the synthesis of byproducts such as fucoxanthin and polyhydroxybutyrate (PHB) (CN119875976A), and introducing exogenous *Vibrio hygroscopicus* hemoglobin. Vitreous hemoglobin (J ApplMicrobiol, Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production, 2011, 110(2): 422-430), to improve the metabolic level and fermentation titer of the producing strain under hypoxic conditions. In addition, by regulating the gellan gum chain length-related gene (CN120555315A) and the acyl modification-related gene (CN118956997A), gellan gum with lower molecular weight or altered gel properties can be obtained, thereby meeting the special needs of different application scenarios.

[0005] Other relevant patent documents retrieved: The country of origin for this publication is China, publication number CN120005791A, publication date February 24, 2025. The document, entitled "A Recombinant Engineered Sphingomonas sphingosine monocytogenes and Its Construction Method and Application," discloses a recombinant engineered strain of *Sphingomonas sphingosine monocytogenes*, its construction method, and its application. This engineered strain was first obtained by knocking out a metabolic bypass gene in *Sphingomonas sphingosine monocytogenes*. The metabolic bypass gene is... crtI Gene or phaC Genes. The ΔphaC knockout strain increased gellan gum production. Furthermore, overexpression of energy metabolism-related genes... yes or clpB or ycaJ Among them, strains overexpressing the pgi gene showed the greatest yield increase. Therefore, overexpression of the pgi gene in ΔphaC knockout strains was recommended. yes Genes, gellan gum production further increased.

[0006] Other relevant non-patent literature retrieved: The journal *Acta Microbiologica Sinica*, the article titled "Metabolic Engineering Modification of *Sphingosine Monomeris* for Gellan Gel Production," by Sun Peng et al., published on April 16, 2025, discloses the construction of a high-gella-producing engineered strain from *Sphingosine Monomeris* using metabolic engineering, and the optimization of the fermentation process, providing theoretical support and technological basis for the efficient synthesis of gellan gum using this bacterium. This paper develops a gene-editing tool suitable for *Sphingosine Monomeris* based on the CRISPR-Cas9 system and utilizes this tool to integrate constitutive promoter-driven gellan gum synthesis gene cluster regulatory protein genes into the genome. come ) and β-1,4-glucuronyltransferase gene ( people ).

[0007] Although the above methods have increased the yield of gellan gum or improved its physicochemical properties to some extent, the prior art represented by the aforementioned literature has at least the following unresolved technical problems or defects: Key transcriptional units in the gellan gum biosynthetic pathway have not been fully explored. In particular, the regulatory target sites related to L-rhamnose metabolism lack systematic research and industrial application. L-rhamnose, as an important monosaccharide component of the gellan gum tetrasaccharide repeating unit (accounting for 1 / 4 of the tetrasaccharide unit), may have metabolic efficiency that is one of the key factors limiting high gellan gum yield. However, current technologies lack technical solutions to increase gellan gum yield by enhancing the rhamnose precursor supply pathway. Meanwhile, while existing technologies have attempted to improve gellan gum yield by knocking out byproduct synthesis genes (such as…),… crtI While methods can be used to remove pigments, these modifications often result in a decrease or stagnation in gellan gum production, making it difficult to increase output while simultaneously improving product quality. Summary of the Invention

[0008] The purpose of this invention is to provide: A colorless, high-yield gellan gum strain, its construction method and application, and related technologies are disclosed to address technical problems such as low gellan gum fermentation yield and difficulty in removing pigments from fermentation broth in existing technologies, or combinations thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of the standard terminology can be found in the references “Molecular Cloning: A Laboratory Manual (Sambrook J, Russell D W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, 2001),” “Microbiology Tutorial (4th Edition), Higher Education Press, author: Zhou Deqing, 2020,” “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19,” and “Genetic Engineering, Higher Education Press, 2013-08-01.”

[0012] Unless otherwise specified, conventional methods within the scope of the art, such as PCR amplification, gene cloning, electroporation, resistance screening, quantitative real-time PCR, and gellan gum yield determination, shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] The term "overexpression" as used in this article refers to the process of using genetic engineering techniques to increase the expression level of a target gene in a host cell to a level higher than its endogenous expression level, such as by using a strong promoter to drive the expression of the target gene or by increasing the copy number of the target gene.

[0016] The term "recombinant engineered bacteria" as used in this article refers to engineered bacterial strains obtained by modifying the genome or plasmids of host bacteria through genetic engineering techniques, including gene knockout, gene overexpression, and combinations thereof.

[0017] The term "used in this article" crtI "Gene" refers to the gene encoding phytoenedesaturase, a key enzyme gene in the biosynthesis of nostoxanthin, responsible for catalyzing the conversion of phytoenxin into lycopene.

[0018] The term "used in this article" vhb The "gene" refers to the gene encoding Vitreoscillahemoglobin (VHb), an oxygen-binding protein that can improve cell growth, oxidative metabolism, protein synthesis, and the production efficiency of metabolites, thereby improving the growth status of aerobic organisms in hypoxic environments.

[0019] The term "in this article" gelB-rmlD "Transcription unit" refers to a multi-gene transcription unit located in the gellan gum biosynthesis gene cluster, containing the gene encoding glucosyl isopentenyl phosphotransferase. come The gene encoding glucose-1-phosphate thymidine transferase rmlA dTDP-6-deoxy-D-glucose-3,5-epimerase encoding gene rmlC dTDP-D-glucose-4,6-dehydratase encoding gene rmlB and the gene encoding dTDP-6-deoxy-L-mannose dehydrogenase rmlD There are a total of 5 structural genes, controlled by a single promoter. Among them... rmlA , rmlB , rmlC and rmlD The four genes are mainly related to the synthesis of L-rhamnose units in the gellan gum molecule.

[0020] The term "in this article" S. elodea"or 'Sphingosine monocytogenes'" refers to strains capable of producing gellan gum, including but not limited to... Sphingomonas elodea strain 2101, also known as Pseudomonas paucimobilis or Sphingomonas paucimobilis, Strain source: ATCC (American Center for Type Culture Collection) Sphingomonas few people ATCC 31461.

[0021] The term "Nostoc xanthophyll" as used in this article refers to a yellow pigment byproduct produced by *Sphingomonas sphingosine monocytogenes* during fermentation, whose biosynthetic pathway uses phytohexene as a precursor, and then... crtI The gene-encoded phytoene dehydrogenase catalyzes the conversion of lycopene into lycopene, which is then further catalyzed by subsequent enzymes to generate novoxanthin.

[0022] In a first aspect, the present invention provides: a colorless, high-yield engineered strain of gellan gum, wherein the colorless, high-yield engineered strain is inactivated in *Sphingosine monocytogenes*. crtI Genes, and overexpression vhb Genes and gelB-rmlD Transcription units are obtained, wherein the gelB-rmlD Transcription units contain come Gene, rmlA Gene, rmlC Gene, rmlB Genes and rmlD Gene.

[0023] According to some embodiments of the present invention, the *Sphingosine monocytogenes* strain is derived from ATCC (American Type Culture Collection). Sphingomonas paucimobilis ATCC 31461, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0024] According to some embodiments of the present invention, the crtI The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0025] According to some embodiments of the present invention, the vhb The nucleotide sequence of a gene, as shown in SEQ ID NO:2, or the nucleotide sequence shown in SEQ ID NO:2, is obtained by substituting one or more bases to obtain a nucleotide sequence encoding a protein with the same function.

[0026] According to some embodiments of the present invention, the gelB-rmlD Transcription units contain come Gene, rmlA Gene, rmlC Gene, rmlB Genes and rmlD The amino acid sequences encoded by the gene are shown in SEQ ID NO:3-7.

[0027] Furthermore, the aforementioned gelB-rmlD The nucleotide sequence of the transcription unit is obtained by substituting one or more bases with SEQ ID NO:8 or the nucleotide sequence shown in SEQ ID NO:8 to obtain a nucleotide sequence encoding the same functional protein.

[0028] According to some embodiments of the present invention, the colorless gellan gum high-yield engineered strain contains a strain carrying... vhb overexpression plasmids and carriers of genes gelB-rmlD Overexpression plasmids of transcription units.

[0029] Among them, the preferred technical feature of "overexpression" is that the expression of the target gene is driven by a strong promoter.

[0030] Furthermore, the aforementioned vhb Genes can be transmitted through strong promoters PTRC Driven overexpression.

[0031] Furthermore, the aforementioned gelB-rmlD Transcription units drive overexpression via their natural or foreign promoters.

[0032] Furthermore, carrying vhb overexpression plasmids and carriers of genes gelB-rmlD The vector for the overexpression plasmid of the transcription unit can be independently selected from commonly used free expression vectors in the Pseudomonas system, such as pBBR1MCS-5 or pBBR-kanR.

[0033] According to some embodiments of the present invention, the deactivation crtI Gene knockout crtI Gene.

[0034] Among them, the technical feature "knockout" crtI "Gene" refers to something that changes through gene mutation. crtI Loss of gene function or activity, wherein the gene mutation includes gene deletion, gene insertion or gene substitution, preferably gene deletion.

[0035] Secondly, this invention provides a method for constructing a colorless, high-yield engineered strain of gellan gum, comprising the following steps: S1, inactivated Sphingosaminoplasma purpureus crtI Genes, Acquisition crtI Gene-inactivating mutants; S2, will vhb Genes and gelB-rmlD Transcription unit import crtI In gene-inactivating mutant strains, vhb Genes and gelB-rmlD Overexpression of transcription units, to obtain crtI Gene inactivation and vhb Genes and gelB-rmlD A high-yield engineered strain of colorless gellan gum overexpressing transcription units.

[0036] According to some embodiments of the present invention, in step S1, the deactivation crtI The gene knockout method is: homologous recombination knockout. crtI Gene.

[0037] Homologous recombination knockout crtI The specific steps involved in gene generation include: (a) Using the genome of Sphingosine monocytogenes as a template, primer pairs were designed to amplify the genome. crtI Upstream and downstream homologous arm fragments of a gene; (b) will crtI The upstream and downstream homologous arm fragments of the gene are fused together; (c) will crtI The fusion product of upstream and downstream homologous arm fragments of the gene is recombined with the knockout plasmid vector fragment to obtain... crtI Gene knockout plasmids; (d) will crtI Gene knockout plasmids were introduced into *Sphingosine monocytogenes* via electroporation or other transformation methods, and after homologous recombination and double crossover were completed, the gene knockout plasmid was obtained. crtI Gene-inactivated mutant strain.

[0038] Among them, the crtI The gene knockout plasmid is pLp12-Δ crtI .

[0039] According to some embodiments of the present invention, in step S2, the... vhb Genes and gelB-rmlD Methods for importing transcription units include: constructing... vhb Overexpression plasmids and gelB-rmlD Overexpression plasmid, the vhb Overexpression plasmids and gelB-rmlD overexpression plasmid introduction crtI Gene-inactivated mutant strain.

[0040] Among them, the construction obtained vhb Methods for overexpressing plasmids include: (a) containing vhb The gene plasmid serves as a template, and the amplification includes the promoter and... vhb Fragments of genes; (b) The fragment is recombined with the expression vector to obtain... vhb Overexpression plasmids; For example, the promoter is the Ptrc promoter. The expression vector is pBBR1MCS-5. vhb The overexpression plasmid is pBBR-ptrc-vhb.

[0041] Among them, the construction obtained gelB-rmlD Methods for overexpressing plasmids include: (a) Using the genome of Sphingosine monocytogenes as a template, amplification gelB-rmlD Gene fragments of transcription units; (b) will gelB-rmlD Gene fragments from transcription units are recombined with expression vectors to obtain... gelB-rmlD Overexpression plasmids.

[0042] For example, the expression vector is pBBR-kanR. gelB-rmlD The overexpression plasmid is pBBR-kan-gelB-rmlD.

[0043] Among them, the aforementioned vhb Overexpression plasmids and gelB-rmlD overexpression plasmid introduction crtI Gene-inactivating mutants can be selected from: (1) First, the gene-inactivating mutants can be selected from: vhb overexpression plasmid introduction crtI Gene inactivation mutants were screened to obtain mutants overexpressing vhb, and then... gelB-rmlD The overexpression plasmid was introduced into a mutant strain overexpressing vhb, and inactivated strains were screened. crtI Genes, and overexpression vhb Genes and gelB-rmlD Engineered strains of transcription units; or (2) first... gelB-rmlD overexpression plasmid introduction crtI Gene inactivation mutants were screened to obtain overexpression strains. gelB-rmlD The mutant strain, and then vhb Overexpression plasmid introduction for overexpression gelB-rmlD From the mutant strains, inactivated strains were screened out. crtI Genes, and overexpression vhb Genes and gelB-rmlD (3) Simultaneously, engineered strains of transcription units; vhb overexpression plasmids and gelB-rmlD overexpression plasmid introduction crtI Gene-inactivating mutants were screened to obtain inactivated mutants. crtI Genes, and overexpression vhb Genes and gelB-rmlD Engineered strains of transcription units.

[0044] Thirdly, the present invention provides a fermentation production method for gellan gum, comprising: fermenting and culturing the colorless gellan gum high-yield engineered strain described in the first aspect or the colorless gellan gum high-yield engineered strain obtained by the construction method described in the second aspect, and collecting gellan gum from the fermentation broth.

[0045] The fermentation culture includes seed culture and fermentation culture.

[0046] The seed culture medium used for seed culture includes: 10-30 g / L sucrose, 3-8 g / L peptone, 1-5 g / L yeast extract, 3-8 g / L sodium chloride, and pH 7.0-7.5.

[0047] The fermentation medium used in the fermentation culture includes: sucrose 5-20 g / L, glucose 10-50 g / L, soybean meal powder 1-8 g / L, potassium dihydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.3-1.5 g / L, calcium carbonate 0.5-2 g / L, and pH 7.0-7.5.

[0048] The fermentation culture is carried out at a temperature of 28-32℃, a rotation speed of 180-250 rpm, and a culture time of 36-72 h.

[0049] Fourthly, the present invention provides the application of the colorless gellan gum high-yield engineered strain described in the first aspect or the colorless gellan gum high-yield engineered strain obtained by the construction method described in the second aspect in gellan gum production.

[0050] Examples 1-8 of this invention at least support the protection scope of "colorless gellan gum high-yield engineered strain and its construction method".

[0051] The term "colorless high-yield engineered strain of gellan gum and its construction method" is summarized from the foregoing explanation and / or the corresponding technical solutions in Examples 1-8. Therefore, those skilled in the art can reasonably infer that "colorless high-yield engineered strain of gellan gum and its construction method," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of "colorless high-yield engineered strain of gellan gum and its construction method."

[0052] The present invention has at least the following beneficial effects: 1. Compared with existing technologies, the colorless gellan gum high-yield engineered strain 2101-Δ constructed by the present invention is a triple-combination modified strain. crtI - vhb - come - rmlD The gellan gum yield reached 19.48 mg / g, which was 31.9% higher than that of the unmodified starting strain *Sphingosine monocytogenes* (14.77 mg / g), and the fermentation broth was colorless and transparent (visible). Figure 6 and Figure 7 ).

[0053] 2. Compared with existing technologies, the colorless gellan gum high-yield engineered strain constructed in this invention exhibits good genetic stability. Engineered strain 2101-Δ crtI - vhb - come - rmlD After five consecutive subcultures, the potency during shake-flask fermentation remained relatively stable, fluctuating between 19.125 mg / g and 19.48 mg / g (see...). Figure 8 It has good prospects for industrial application.

[0054] In summary, the colorless gellan gum high-yield engineered strain provided by this invention can effectively remove pigments from the fermentation broth without increasing additional costs, thereby improving the quality of gellan gum products, reducing the difficulty of post-processing of the colloid, and effectively improving the fermentation production efficiency of gellan gum. Attached Figure Description

[0055] Figure 1 for gelB-rmlD A schematic diagram showing the location of transcription units in the gellan gum biosynthesis gene cluster.

[0056] Figure 2 A map was constructed for the pLp12-ΔcrtI plasmid.

[0057] Figure 3 A map was constructed for the pBBR-ptrc-vhb plasmid.

[0058] Figure 4 Construct a map for the pBBR-kanR plasmid.

[0059] Figure 5 Construct a map for the pBBR-kan-gelB-rmlD plasmid.

[0060] Figure 6 The results of gellan gum fermentation yields for the starting strain and each engineered strain are shown (specifically, for the starting strain). S. elodea 2101 crtI Inactivated mutant strain 2101-ΔcrtI, crtI Inactivated mutant strain superimposed with empty vector control strain 2101-ΔcrtI-pbbr crtI Inactivated mutant strain superimposed with vhb overexpression strain 2101-ΔcrtI-vhb, crtI Inactivated mutants superimposed with overexpression come - rmlD strain 2101-ΔcrtI-gelB-rmlD and crtI Inactivated mutants with dual plasmid overexpression vhb and come - rmlD(Gellan gum yield of strain 2101-ΔcrtI-vhb-gelB-rmlD, determined by fermentation at 30℃ for 48 hours in fermentation medium).

[0061] Figure 7 Image of the gellan gel fermentation shake flask of the starting strain and the final engineered strain (left: starting strain) S. elodea 2101, fermentation broth is yellow; Right: final engineered strain 2101-ΔcrtI-vhb-gelB-rmlD, fermentation broth is white and transparent).

[0062] Figure 8 The results show the passage stability of the high-yield engineered strain 2101-ΔcrtI-vhb-gelB-rmlD. Detailed Implementation

[0063] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0065] All data processing in this invention was performed using OriginPro 2024 software. Analysis between different groups was conducted using independent samples t-tests or one-way ANOVA tests. P <0.05 indicates that the difference is statistically significant.

[0066] The strains and plasmids used in the following examples are shown in Table 1. *Escherichia coli* was cultured in LB liquid medium (1% peptone, 0.5% yeast extract, 1% sodium chloride) or on LB solid plates supplemented with 2% agar at 37°C. The gellan gum-producing strains were cultured in liquid medium (0.1% yeast extract, 0.3% beef extract, 0.5% peptone, 0.5% sodium chloride, 0.5% sucrose, adjusted to pH 7.2), solid medium (1% sucrose, 0.5% peptone, 0.3% yeast extract, 0.2% beef extract, 0.5% sodium chloride, adjusted to pH 7.2), seed medium (2% sucrose, 0.5% peptone, 0.3% yeast extract, 0.5% sodium chloride, adjusted to pH 7.2), and fermentation medium (1% sucrose, 3% glucose, 0.4% soybean meal, 0.1% potassium dihydrogen phosphate, 0.06% magnesium sulfate, 0.1% calcium carbonate, adjusted to pH 7.2) at 30°C and 220 rpm.

[0067] All biological reagents used in the following examples were purchased from reagent companies. General technical procedures for Escherichia coli and Pseudomonas were performed according to standard operating procedures. Gene synthesis, primer synthesis, and DNA sequencing were performed by Suzhou Genewiz Biotechnology Co., Ltd.

[0068] Table 1. Strains and plasmids used in the experiments of this invention.

[0069] SEQ ID NO:11:

[0070] Example 1 crtI Construction of the knockout strain 2101-ΔcrtI: (1) In order to block the biosynthetic pathway of nodular xanthophyll, a major byproduct in the biosynthesis of gellan gum, in order to S. elodea Using the genome as a template, and with crtI-F1 (SEQ ID NO:12: tgagagcttagtacgttagcGCCAGTCGGCAGGCAGATA), crtI-R1 (SEQ ID NO:13: AATGAAGCGTAGCTGTAATGCGCTTTCCCG), crtI-F2 (SEQ ID NO:14: CATTACAGCTACGCTTCATTGCATGCTCCT), and crtI-R2 (SEQ ID NO:15: agaggatctgaagatcagcaAGCGGATCGTCACCAAGTTC) as primers, amplification was performed respectively. crtI Gene( crtI The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the CRTI protein is shown in SEQ ID NO:9. Homologous arm fragments (upstream and downstream) were used. Using pLp12 plasmid as a template, and VpLp12-F (SEQ ID NO:16: gctaacgtactaagctctcatggc) and VpLp12-R (SEQ ID NO:17: tgctgatcttcagatcctctacgc) as primers, the pLp12 vector fragment was amplified using the high-fidelity DNA polymerase system (catalog number P515) from Nanjing Novizan Biotechnology Co., Ltd. The amplification reaction conditions and procedures were performed according to the kit instructions. PCR products were recovered using the microcolumn concentrated DNA gel recovery kit (catalog number: ZPV202) from Beijing Zhuangmeng International Biotechnology Co., Ltd. The specific DNA recovery process was performed according to the kit instructions.

[0071] (2) The amplified and recovered homologous arm fragments of the crtI gene were recombinated with the pLp12 vector. The recombination was performed using the Beijing TransGen Biotech Basic Version Homologous Recombination Seamless Cloning Kit (Catalog No.: CU201). The recombination process was carried out according to the kit instructions to obtain the pLp12-ΔcrtI plasmid (see [link]). Figure 2 ); (3) The pLp12-ΔcrtI plasmid was introduced by electroconversion. S. elodeaIn section 2101, recipient bacteria were electroporated and inoculated into seed medium at 30°C with shaking for 12-16 hours. 1 mL of the bacterial culture was centrifuged, and the precipitate was resuspended twice in pre-cooled sterile water, retaining a final electroporation volume of 100 μL of competent recipient bacteria. Electroporation was performed using a 2 mm electroporator at 2.5 kV. After electroporation, 1 mL of pre-cooled antibiotic-free seed medium was quickly added, mixed, and transferred to sterile EP tubes. The culture was incubated at 30°C with shaking for 2 hours. The bacterial culture was centrifuged, and some supernatant was discarded, retaining approximately 100 μL of resuspended bacteria. The entire bacterial culture was spread onto a 10 mg / L gentamicin-resistant plate and incubated at 30°C upside down for 48 hours to obtain single-exchange transformants (single-exchange strains). The single-exchange strains were inoculated into antibiotic-free seed medium and incubated overnight at 30°C. A small amount of the bacterial culture was diluted and spread onto antibiotic-free solid plates supplemented with 0.4% L-arabinose, and incubated at 30°C for approximately 2 days to complete the double exchange process. When single colonies grow on the plate, single colonies are picked and screened by replication on solid plates with and without antibiotics and with 10 mg / L gentamicin. The strains that have lost their resistance are then verified by PCR: using crtI-F1 and crtI-R2 as identification primers, and the control group as... S. elodea The PCR product of the 2101 genome should have a band of 1088 bp in the positive transformant and a band of 1653 bp in the wild-type control. Based on the fragment size of the PCR product, the mutant strain 2101-ΔcrtI with successful crtI gene knockout was obtained.

[0072] SEQ ID NO:1: .

[0073] SEQ ID NO:9: MKRIAVYCGSASPADPVYIEAARTLGRTLAERGIGVVYGGGRLGLMGAVADAALEAGGEVIGVIPTLLVNAEVAHKGLTSLEVCETMHERKARFTELADGFVNLPGGTGTMDELWEAMSWAQLGYHADPIGLLNIAGYYDKLVEFWEHMGKVGFVRPQHQGLLLVDSTIDGLLTKMAEAQPVVPIAQLRREQL .

[0074] Example 2: Exogenous Genes vhb Construction of the overexpression mutant strain 2101-ΔcrtI-vhb: (1) To improve the gellan gum production level of the engineered bacteria, the vhb gene was overexpressed on the basis of strain 2101-ΔcrtI: using pUC57-ptrc-vhb as a template, where vhb The nucleotide sequence of the gene fragment is shown in SEQ ID NO:2 (the amino acid sequence of the VHB protein is shown in SEQ ID NO:10). The complete ptrc-vhb gene fragment was amplified using ptrc-F (SEQ ID NO:18: TCTAGAACTAGTGGATCCaattcgtgtcgctcaaggcgcactcccgttctggataatgt) and vhb-R (SEQ ID NO:19: TCGAGAGGTATCGATAAGCTTTTACTCCACGGCCTGAGCAT) as primers. The pbbr vector fragment was amplified using pBBR1MCS-5 plasmid as a template and V-pbbr-F (SEQ ID NO:20: AAGCTATCGATACCGTCGACC) and V-pbbr-R (SEQ ID NO:21: GGATCCACTAGTTCTAGAGCGGC) as primers. The amplification was performed using the high-fidelity DNA polymerase system (catalog number P515) from Nanjing Novizan Biotechnology Co., Ltd. The amplification reaction conditions and procedures were performed according to the kit instructions. PCR products were recovered using the microcolumn concentrated DNA gel recovery kit (catalog number: ZPV202) from Beijing Zhuangmeng International Biotechnology Co., Ltd. The specific process of DNA recovery was performed in accordance with the kit instructions. (4) The amplified and recovered ptrc-vhb gene fragment was recombined with the pbbr vector fragment to obtain the pBBR-ptrc-vhb plasmid (see Figure 3 ).

[0075] (5) The constructed pBBR-ptrc-vhb plasmid was introduced into 2101-ΔcrtI by electroporation (the method is the same as that in Example 1, where the pLp12-ΔcrtI plasmid was introduced by electroporation). S. elodea (Electroconversion method in 2101). Transformants grown on 10 mg / L gentamicin resistance solid plates were identified by PCR using the gentamicin resistance gene (GmR). GmR-F (SEQ ID NO:22: ATGTTACGCAGCAGCAACGA) and GmR-R (SEQ ID NO:23: TTAGGTGGCGGTACTTGGGT) were used as the identification primer pairs to obtain... crtI Gene deletion combination vhb The gene-overexpressing strain was named 2101-ΔcrtI-vhb.

[0076] SEQ ID NO:2: ATGCTGGACCAGCAGACCATTAATATTATTAAGGCCACCGTGCCCGTCCTTAAGGAGCACGGCGTGACCATTACCACCACCTTCTACAAGAACCTTTTCGCCAAGCACCCCGAGGTGCGCCCTCTTTTCGACATGGGCCGCCAGGAGTCTCTTGAGCAGCCTAAGGCCCTGGCAATGACTGTTCTTGCCGCCGCCCAGAATATTGAGAATCTTCCTGCCAT TCTTCCTGCCGTGAAGAAGATTGCCGTGAAGCATTGTCAGGCCGGCGTTGCAGCCGCCCACTATCCCATTGTGGGCCAGGAGCTTCTTGGTGCAATCAAGGAGGTGCTTGGCGACGCCGCCACTGATGATATTCTGGACGCCTGGGGGAAGGCCTATGGCGTGATTGCCGACGTGTTCATTCAGGTGGAGGCCGATTTGTATGCTCAGGCCGTGGAGTAA.

[0077] SEQ ID NO:10: MLDQQTINIIKATVPVLKEHGVTITTTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE .

[0078] Example 3: Construction of the control strain 2101-ΔcrtI-pbbr overexpressing the empty vector: pBBR1MCS-5 was introduced into 2101-ΔcrtI via electroporation. Transformants grown on 10 mg / L gentamicin resistance solid plates were identified by PCR using the gentamicin resistance gene (GmR) as the target. GmR-F and GmR-R were used as identification primer pairs. The control mutant strain expressing the empty vector with the deletion of the crtI gene was named 2101-ΔcrtI-pbbr.

[0079] Example 4 come - rmlD Analysis of relevant information of gene fragments come - rmlD Transcription unit ( gelB-rmlDThe nucleotide sequence of the transcription unit (as shown in SEQ ID NO:8) contains the gene encoding glucosyl isopentenyl phosphotransferase. come (The amino acid sequence encoded by this gene is shown in SEQ ID NO:3), Glucose-1-phosphate thymidine transferase encoding gene rmlA (The amino acid sequence encoded by this gene is shown in SEQ ID NO:4), dTDP-6-deoxy-D-glucose-3,5-epimerase encoding gene rmlC (The amino acid sequence encoded by this gene is shown in SEQ ID NO:5), dTDP-D-glucose-4,6-dehydratase encoding gene rmlB (The amino acid sequence encoded by this gene is shown in SEQ ID NO:6) and the gene encoding dTDP-6-deoxy-L-mannose dehydrogenase. rmlD (The amino acid sequence encoded by this gene is shown in SEQ ID NO:7) There are a total of 5 structural genes, controlled by a single promoter, all of which are key genes in the gellan gum biosynthesis pathway (such as...). Figure 1 (As shown).

[0080] SEQ ID NO:3: MNAFEAQRAFEEQLRAYAAPKQSAMPAFRRSTVRMILYTELLLIDSIAILASFYFAACMRDSNWLSLAGVNVGIFLLPITLGTAIASGTYSLESLRHPISGVKNILSALFFSILIVL LGSYLLTTELPLSRIQLGAGALMTVVLLMAGRLVFRRHVRAMTGDKLLDELVIIDGVSLDLASDAVALDARIINLSPNPRDPQMLHRLGTTVIGFDRVVVACTEEHRAVWALLLKGMN IKGEILVPQFNALGAIGVDAYDGKDTLVVSQGPLSMPNRAKKRALDLLITVPAVLALAPLMILVAIAIKLESPGPVLFAQDRVGRGNRLFKILKFRSMRQALCDANGNVSASRDDDR ITKVGRFIRKTSIDELPQLLNVLRGDMSVVGPRPHALGSRAADHYFWEIDERYWHRHTLKPGMTGLAQVRGFRGATDRRIDLTNRLQANMEYIDGWDIWRDITILFKTLRVIVHSNAF .

[0081] SEQ ID NO:4: MKGIILAGGSGTRLYPATLISKQLLPVYDKPMIFYPLSVLMLTGIRDVLIISTPRDLPMFQALLGDGSAFGMNLSYAEQPSPNGLAEAFIIGADFVGNNPSALILGDNIYHGEKMGERCKAAAEQASQGGANVFAYHVDDPERYGVVAFDPETGVATSVEEKPAVPKSNWAITGLYFYDKDVVDIAKSIQPSARGELEITDVNRTYLERGDLHITRLGRGYAWLDTGTHDSLHDAGSFVRTLEHRTGVKIACPEEIAFENGWLGAEDLLKRARGLGKTGYAAYLRKLVAAA 。

[0082] SEQ ID NO:5: MTKVHHHELSGVIEFTPKPGDHRGFFSEVFKQSQLDAEGVEARWVQDNQSFSAAAGTIRGLHLQAPAFAQAKLVRVLPGAIYDVAVDIRRGSPTYGRWVGVELSAEKWNQLLVPAGYAHGFMTLVPDCEILYKVSAKYSKDSEMAIRWDDPDLAITWPDIGVEPVLSEKDAVTPFVEFNTPFFYQG 。

[0083] SEQ ID NO:6: MQQQTFLVTGGGFIGSAVVRHLVRQGARVINLDKLTYAGNPASLTAIENAPNYRFVHADIADTATILPLLREEQVDVVMHLAAESHVDRSIDGPGEFIETNVVGTFKLLQSALQYWRELEGEKREGFRFHHISTDEVFGDLPFDSGIFTEETPYDPSSPYSASKAASDHLVRAWGH TYGLPVVLNCSNNYGPHFPEKLIPLTILNALEGKPLPVYGKGENIRDWLYVDDHAKALATIATTGKVGQSYNVGGRNERTNLQVVETICDLLDQRIPLADGRKRRELITFVTDRPGHDRRYAIDATKLETELGWKAEENFDTGIAATIDWYLENEWWWGPIRSGKYAGERLGQTA 。

[0084] SEQ ID NO:7: MRILVTGHDGQVAQSLGEQAQGHELIFTSYPEFDLSRPETIEAAVAKIQPELIVSAAAYTAVDKSESEPELAMAINGDGPGVLARAGAKIGAPIIQLSTDYVFDGSLDRPWREDDPTGPLGVYGATKLAGEQAVQASGATNAVIRLAWVYSPFGNNFVKTMLRLAETRDALNVVEDQQGCPSSALDIASAILAVVERWQRDGAFSGVYHFAGSGETNWADFARAIFAESAKRGGPTATVTGIPSSDYPTPAKRPANSRLDCSRFADTFGYRAPAWQDSLAVVMGRLLG 。

[0085] SEQ ID NO:8:

[0086] Based on the sequence alignment results analysis come The encoded glucosyl isopentenyl phosphotransferase is involved in the assembly of the starting unit during gellan gum production and is mainly responsible for assembling UDP-glucose onto the lipid carriers of the inner membrane intracellularly. rmlA , rmlB , rmlC and rmlD Four genes are mainly related to the synthesis of L-rhamnose units in the gellan gum molecule, among which rmlA Encoding glucose-1-phosphate thymidine transferase, responsible for catalyzing the conversion of glucose-1-phosphate to dTDP-D-glucose. rmlB Encoding dTDP-D-glucose-4,6-dehydratase, responsible for catalyzing the conversion of dTDP-D-glucose to dTDP-4-keto-6-glucose. rmlC Encoding dTDP-6-deoxy-D-glucose-3,5-epimerase, responsible for catalyzing the conversion of dTDP-4-keto-6-glucose to d-TDP-4-keto-L-rhamnose. rmlD It encodes dTDP-6-deoxy-L-mannose dehydrogenase, which is responsible for catalyzing the conversion of d-TDP-4-keto-L-rhamnose to dTDP-L-rhamnose.

[0087] Example 5 come - rmlD Construction of mutant strains 2101-ΔcrtI-gelB-rmlD and 2101-ΔcrtI-vhb-gelB-rmlD with increased transcription unit copy number: (1) In order to improve the gellan gum production level of engineered bacteria, the gelB-rmlD transcription unit gene fragment was further overexpressed on the basis of strain 2101-ΔcrtI-vhb: using pET28a plasmid as template, and kanR-F (SEQ ID NO:24: TTGTTATGGAGCAGCAACGATGAGCCATATTCAACGGGAAACGT) and kanR-R (SEQ ID NO:25: TCTCGGCTTGAACGAATTGGTGGCACTTTTCGGGGAAATGT) as primers, the complete kanamycin resistance gene kanR fragment was amplified; using pBBR1MCS-5 plasmid as template, and pbbr-kanR-F (SEQ ID NO:26: CAATTCGTTCAAGCCGAGATCGG) and pbbr-kanR-R (SEQ ID NO:27: CATCGTTGCTGCTCCATAACATCAAACAT) as primers, the pbbr vector fragment was amplified; (2) The amplified and recovered kanR gene fragment was recombined with the pbbr vector fragment to obtain the pBBR-kanR plasmid (see Figure 4 ).

[0088] (3) with S. elodea Using the genome as a template, and gelB-F (SEQ ID NO:28: TCGACGGTATCGATAAGCTTgtcgggtgtcattccggtac) and rmlD-R (SEQ ID NO:29: GCTCTAGAACTAGTGGATCCcaacgccgacatggacgaca) as primers, the complete genome was amplified. come - rmlD Gene fragment; using pBBR-kanR plasmid as template and Vpbbr-F and Vpbbr-R as primers, amplify the pBBR-kanR vector fragment (the amplification and recovery methods refer to the method for amplifying the pbbr vector fragment in Example 1). (4) The amplified and recovered gelB-rmlD gene fragment was recombined with the pBBR-kanR vector fragment to obtain the pbbr-kanR-gelB-rmlD plasmid (see Figure 5 ); (5) The constructed pBBR-kanR-gelB-rmlD plasmid was introduced into 2101-ΔcrtI and 2101-ΔcrtI-vhb by electroporation (the method is the same as that in Example 1, where pLp12-ΔcrtI plasmid was introduced by electroporation). S. elodea (Electroconversion method in 2101). PCR identification was performed using the kanamycin resistance gene kanR as the target, with kanR-F and kanR-R as the identification primer pair, to obtain... come - rmlD The overexpressing strains with increased gene fragment copy number were named 2101-ΔcrtI-gelB-rmlD and 2101-ΔcrtI-vhb-gelB-rmlD, respectively, with 2101-ΔcrtI-vhb-gelB-rmlD being the colorless gellan gum high-yield engineered strain of the present invention.

[0089] Test Example 1: Determination of gellan gum yield from shake-flask fermentation products: The transformants (starting strains from Examples 1-6) S. elodea 2101、(2) crtI Inactivated mutant strain 2101-ΔcrtI, (3) crtI The inactivated mutant strain was superimposed with the empty vector control strain 2101-ΔcrtI-pbbr, (4) crtIThe inactivated mutant strain superimposed with the vhb overexpression strain 2101-ΔcrtI-vhb, (5) crtI Inactivated mutants superimposed with overexpression come - rmlD Plant 2101-ΔcrtI-gelB-rmlD and (6) crtI Inactivated mutants with dual plasmid overexpression vhb and come - rmlD Strain 2101-ΔcrtI-vhb-gelB-rmlD was transferred to fresh solid plates for enrichment and cultured at 30℃ for 48 h to obtain bacterial colonies. A 1 cm sample was scraped off. 2 The mycelial growth was inoculated into seed culture medium and cultured at 30℃ with shaking at 220 rpm for 48 h. Then, it was transferred to fermentation medium and cultured at 30℃ with shaking at 220 rpm for another 48 h. After fermentation, 30 g of the fermentation broth was accurately weighed, and three times its volume of 95% ethanol was added to precipitate gellan gum. The obtained gellan gum was washed 1-2 times with 95% ethanol, dried to constant weight, and then weighed. The gellan gum yield was calculated (the formula is as follows: yield = crude gellan gum mass / fermentation broth mass × 100%). All results were repeated three times, and the average value was taken.

[0090] The results of gellan gum fermentation yield of the above strains are shown in [the table below]. Figure 6 And Table 2.

[0091] Table 2

[0092] After shake-flask fermentation and gellan gum yield determination, the gellan gum yield of the 2101-ΔcrtI-vhb-gelB-rmlD strain of this invention reached 19.48 mg / g, which is higher than that of the original strain. S. elodea 2101 (14.77 mg / g) increased by 31.9%, while 2101-ΔcrtI, 2101-ΔcrtI-pbbr, 2101-ΔcrtI-vhb, and 2101-ΔcrtI-gelB-rmlD increased by only 3.66%, 2.84%, 11.37%, and 22.00%, respectively.

[0093] Test Example 2: The effect of 2101-ΔcrtI-vhb-gelB-rmlD on removing pigments from fermentation broth: Comparison of pigments in the fermentation broth of engineered bacteria and control strains: Compared to the starting strain S. elodea 2101, the yellow pigment in the fermentation broth of the final engineered strain 2101-ΔcrtI-vhb-gelB-rmlD was almost completely removed (see...). Figure 7 This engineered strain can be used to produce colorless, high-quality gellan gum, reducing the cost of subsequent industrial processing.

[0094] Test Example 3: Observation of the genetic stability of 2101-ΔcrtI-vhb-gelB-rmlD: To determine the genetic stability of the high-yielding engineered strain, the engineered strain 2101-ΔcrtI-vhb-gelB-rmlD was continuously passaged to investigate the changes in gel production levels with increasing passage number. The bacterial culture of engineered strain 2101-ΔcrtI-vhb-gelB-rmlD was spread onto solid plates containing 50 mg / L kanamycin and 50 mg / L gentamicin, and incubated at 30°C for 48 h. The culture was then transferred to shake flasks containing seed culture medium and incubated at 30°C with shaking at 220 rpm for 48 h. The culture was then transferred to fermentation shake flasks and incubated at 30°C with shaking at 220 rpm for 48 h. The shake-flask fermentation level of gellan gum was measured. Simultaneously, bacterial culture from the seed shake flasks was spread onto solid plates containing 50 mg / L kanamycin and 50 mg / L gentamicin for continuous passage.

[0095] The results of the passaging stability experiment are as follows Figure 8 As shown, the results indicate that after five consecutive subcultures, the shake-flask fermentation titer of the colorless high-yield gellan gum engineered strain remained relatively stable, and the gellan gum yield remained stable between 19.125 mg / g and 19.48 mg / g, demonstrating good genetic stability and potential for industrial application.

[0096] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A colorless, high-yield engineered bacterial strain for gellan gum production, characterized in that, The colorless, high-yield engineered strain of gellan gum was inactivated by *Sphingosine monocytogenes*. crtI Genes, and overexpression vhb Genes and gelB-rmlD Transcription units are obtained, wherein the gelB-rmlD Transcription units contain gelB Gene, rmlA Gene, rmlC Gene, rmlB Genes and rmlD Gene; The *Sphingomonas paucimobilis* strain is ATCC 31461. The crtI The nucleotide sequence of the gene is shown in SEQ ID NO:1; The vhb The nucleotide sequence of the gene is shown in SEQ ID NO:2; The gelB-rmlD Transcription units contain gelB Gene, rmlA Gene, rmlC Gene, rmlB Genes and rmlD The amino acid sequences encoded by the gene are shown in SEQ ID NO:3-7.

2. The colorless gellan gum high-yield engineered strain according to claim 1, characterized in that, The gelB-rmlD The nucleotide sequence of the transcription unit is obtained by substituting one or more bases with SEQ ID NO:8 or the nucleotide sequence shown in SEQ ID NO:8 to obtain a nucleotide sequence encoding the same functional protein.

3. The colorless gellan gum high-yield engineered strain according to claim 1, characterized in that, The colorless gellan gum high-yield engineered strain contains [a specific strain]. vhb overexpression plasmids and carriers of genes gelB-rmlD Overexpression plasmids of transcription units.

4. The colorless gellan gum high-yield engineered strain according to claim 1, characterized in that, The deactivation crtI Gene knockout crtI Gene.

5. The method for constructing the colorless gellan gum high-yield engineered strain according to any one of claims 1-4, characterized in that, Includes the following steps: S1, inactivated Sphingosaminoplasma purpureus crtI Genes, Acquisition crtI Gene-inactivating mutants; S2, will vhb Genes and gelB-rmlD Transcription unit import crtI In gene-inactivating mutant strains, vhb Genes and gelB-rmlD Overexpression of transcription units, to obtain crtI Gene inactivation and vhb Genes and gelB-rmlD A high-yield engineered strain of colorless gellan gum overexpressing transcription units.

6. The construction method according to claim 5, characterized in that, In step S1, the deactivation crtI The gene knockout method is: homologous recombination knockout. crtI Gene.

7. The construction method according to claim 5, characterized in that, In step S2, the vhb Genes and gelB-rmlD Methods for importing transcription units include: constructing... vhb Overexpression plasmids and gelB-rmlD Overexpression plasmid, the vhb Overexpression plasmids and gelB-rmlD overexpression plasmid introduction crtI Gene-inactivated mutant strain.

8. A fermentation production method for gellan gum, characterized in that, include: The colorless gellan gum high-yield engineered strain according to any one of claims 1-4 or the colorless gellan gum high-yield engineered strain obtained by the construction method according to any one of claims 5-7 is fermented and cultured, and the gellan gum in the fermentation broth is collected.

9. The application of the colorless gellan gum high-yield engineered strain according to any one of claims 1-4 or the colorless gellan gum high-yield engineered strain obtained by the construction method according to any one of claims 5-7 in gellan gum production.

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