Xanthan gum engineering strain construction method based on secondary messenger and metabolic engineering

Abstract

The invention relates to the technical field of bioengineering, in particular to a xanthan gum engineering strain construction method based on secondary messenger and metabolic engineering, and the method comprises the following steps: illuminating a molecular mechanism of c-di-GMP and ppGPP for cooperatively regulating xanthan gum synthesis; a high-yield strain is constructed through secondary messenger module optimization and metabolic engineering modification; and fermentation parameters are optimized by combining a precise fermentation technology. By analyzing a secondary messenger regulation mechanism and optimizing a chassis cell metabolism network, the production performance of xanthan gum is remarkably improved. According to the invention, a c-di-GMP and ppGPP synergistic regulation mechanism is clarified, and secondary messenger module optimization, metabolic engineering transformation and precise fermentation are combined, so that the xanthan gum high-yield strain is constructed, and the yield and sugar conversion rate are improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically, it relates to a method for constructing xanthan gum engineered strains based on secondary messenger and metabolic engineering. Background Technology

[0002] Xanthan gum, an important microbial extracellular polysaccharide, possesses excellent thickening, suspending, emulsifying, and stability properties, and is widely used in food, petroleum, pharmaceutical, and cosmetic industries. Currently, industrial production of xanthan gum mainly relies on microbial fermentation. However, in existing technologies, the complex regulatory network affecting microbial polysaccharide synthesis, particularly the unclear regulatory mechanisms of secondary messenger molecules (such as c-di-GMP and ppGPP), hinders the development of precise synthesis enhancement strategies, thus limiting further improvements in xanthan gum yield and sugar conversion rate. Traditional strain modification methods often focus on enhancing single metabolic pathways, lacking the integration and utilization of global regulatory signals, resulting in limited performance improvements in engineered strains that fail to meet the demands of high yield, high efficiency, and stability for large-scale industrial production. Therefore, elucidating the molecular mechanism of secondary messenger molecules synergistically regulating xanthan gum synthesis, and based on this, constructing efficient engineered strains and optimizing fermentation processes, is crucial to overcoming the current bottlenecks in xanthan gum production.

[0003] Patent CN118460444A discloses an engineered microorganism, its construction method, and a method for producing industrial metabolites using it. In this engineered microorganism, the teaA gene (SEQ ID NO:1) is knocked out, while the teaB gene (SEQ ID NO:2) and teaC gene (SEQ ID NO:3) are overexpressed. This engineered microorganism exhibits increased tetrahydropyrimidine secretion rate, PHA yield, and overall cell dry weight (i.e., growth level), and demonstrates enhanced nitrogen donor utilization in tetrahydropyrimidine synthesis. It can also utilize wool hydrolysate as a feedstock to produce tetrahydropyrimidine, thereby effectively reducing industrial fermentation costs and achieving economical, green, and sustainable fermentation. However, this technical solution mainly focuses on the enhanced production of specific metabolites, without addressing the regulatory role of secondary messenger molecules in xanthan gum synthesis. It fails to fully elucidate the synergistic relationship between secondary messengers and the metabolic network, limiting its application potential in xanthan gum production.

[0004] Patent CN105189740A discloses a *Cyclophorus* strain and related methods and reagents, including engineered regulatory sequences and genes from *Cyclophorus* or the *Cyclophorus* genus, selective markers for engineered microorganisms, methods for mutagenesis of microorganisms, and strains produced by mutagenesis. This technical solution achieves efficient production of specific compounds through genetic engineering; however, in the field of xanthan gum production, research on its regulatory mechanisms of secondary messenger molecules is relatively weak, failing to systematically reveal the relationship between secondary messenger molecules and metabolic engineering, thus limiting its applicability in xanthan gum production. Furthermore, this solution lacks sufficient theoretical support for optimizing fermentation processes, which may affect the efficiency and stability of large-scale production.

[0005] In summary, existing technologies for constructing xanthan gum engineered strains have not fully integrated the regulatory mechanisms of secondary messenger molecules with the overall optimization strategy of metabolic engineering, resulting in limited performance improvement of strains and making it difficult to meet the demands of industrial production for high yield, high efficiency, and stability. Summary of the Invention

[0006] This invention provides a method for constructing xanthan gum engineered strains based on secondary messenger and metabolic engineering, which synergistically regulates the molecular mechanism of xanthan gum synthesis, and optimizes the metabolic network of chassis cells by combining metabolic engineering strategies to construct engineered strains that enhance xanthan gum production.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing xanthan gum engineered strains based on secondary messenger and metabolic engineering, comprising the following steps:

[0009] S10: Elucidating the molecular mechanism by which secondary messenger molecules co-regulate xanthan gum synthesis.

[0010] Using the wild-type xanthan gum production strain NRRL B-1459 as the research object, under high carbon / nitrogen ratio fermentation conditions (carbon-nitrogen ratio not less than 20:1), the molecular mechanism of c-di-GMP and ppGPP synergistic regulation of xanthan gum synthesis was elucidated using molecular biology techniques. The specific mechanisms include the following:

[0011] S10.1: Screening and Validation of Key Regulatory Genes

[0012] Using gene knockout, overexpression, and reverse mutation techniques, mutant strains of the c-di-GMP synthase gene dgcA, the degradative enzyme gene pdeB, the ppGPP synthase gene relA, and the degradative enzyme gene spoT were constructed, respectively. The specific regulatory roles of these genes in xanthan gum synthesis were determined by comparing the xanthan gum yield, synthesis rate, and intracellular secondary messenger concentration between wild-type and mutant strains. For example, knocking out the pdeB gene increased the intracellular c-di-GMP concentration to 1.8 times its original value, and the xanthan gum yield increased from 22 g / L to 28 g / L; overexpression of the dgcA gene further increased the intracellular c-di-GMP concentration, and the xanthan gum yield reached 30 g / L.

[0013] S10.2: Signal Path Interaction Analysis

[0014] Using fluorescent reporter gene fusion expression technology, protein immunoprecipitation technology, and gel migration assays, we investigated whether c-di-GMP and ppGPP synergistically regulate the transcription and expression of the xanthan gum synthesis gene cluster xanA-xanZ by binding to key enzymes or regulatory proteins in the xanthan gum synthesis pathway. For example, through fluorescent reporter gene GFP fusion expression, we found that the transcription level of the xanthan gum synthesis gene cluster xanA-xanZ in the double mutant strain ΔpdeB / ΔspoT was 2.5 times higher than that in the wild-type strain, and the intracellular concentrations of c-di-GMP and ppGPP were increased by 1.8 times and 1.5 times, respectively.

[0015] S10.3: Validation of Metabolic Flux Regulation

[0016] Metabolomics analysis was used to compare the changes in the contents of xanthan gum precursors UDP-glucose, UDP-mannose, and UDP-glucuronic acid in wild-type and mutant strains. Transcriptomics data were combined to analyze how c-di-GMP and ppGPP synergistically promote xanthan gum synthesis by regulating carbon flux allocation (such as the EMP and HMP pathways) and precursor synthesis pathways. For example, the UDP-glucose content in the mutant strain ΔpdeB was 1.6 times higher than that in the wild-type strain, indicating that c-di-GMP promotes precursor synthesis by enhancing EMP pathway activity.

[0017] S20: Construction of a high-yield xanthan gum engineered strain based on secondary messenger and metabolic engineering strategies

[0018] Based on the elucidated synergistic regulatory mechanism, and using NRRL B-1459 as the chassis cell, an engineered strain to enhance xanthan gum production was constructed through a combination of "secondary messenger regulation" and "metabolic engineering modification." Specifically, the strain includes the following:

[0019] S20.1: Optimization of the Second-Level Messenger Module

[0020] The synthesis signal is enhanced by regulating the steady-state levels of c-di-GMP and ppGPP: Overexpression of the c-di-GMP synthase gene dgcA and knockout of the degradation enzyme gene pdeB increase the intracellular c-di-GMP concentration; simultaneously, the expression of the ppGPP synthase gene relA is regulated by a weak promoter to maintain ppGPP levels and avoid excessive accumulation that inhibits cell growth, thus achieving synergistic activation of xanthan gum synthesis by both. The weak promoter is one with a lower strength than the strong constitutive promoter P43, and can maintain the expression level of the ppGPP synthase gene relA at 0.5 to 1.0 times the wild-type relA gene expression level. For example, integrating the dgcA gene into the chromosomal pdeB gene locus (knocking out pdeB while overexpressing dgcA) yields strain S1, whose intracellular c-di-GMP concentration is 2.2 times higher than the wild-type strain, and its xanthan gum yield reaches 30 g / L.

[0021] S20.2: Metabolic Engineering Module Modification

[0022] Optimizing carbon metabolism and precursor supply in chakra cells: Overexpressing key enzyme genes galU and manA in the xanthan gum synthesis precursor pathway enhances the synthesis of precursors such as UDP-glucose and UDP-mannose; knocking out key genes ldh and ackA in the byproduct synthesis pathway reduces the diversion of carbon sources to lactate and acetic acid; and optimizing carbon flow allocation in the glycolysis and pentose phosphate pathways by promoter engineering the central carbon metabolism key enzyme gene pfkA, thereby improving precursor supply efficiency. The promoter engineering involves replacing the natural promoter of the central carbon metabolism key enzyme gene pfkA with an artificially designed tunable promoter containing at least one known regulatory element (e.g., an inducible promoter or a synthetic promoter with a variable promoter sequence) and being able to regulate the expression level of the pfkA gene to between 1.2 and 1.8 times that of the wild type. For example, strain S2 was obtained by overexpressing the galU and manA genes on the basis of strain S1. Its UDP-glucose and UDP-mannose contents were increased by 2.1 times and 1.9 times respectively compared with the wild strain, and the xanthan gum yield reached 29 g / L.

[0023] S20.3: Screening and Validation of Engineered Strains

[0024] The modified modules were introduced into chassis cells via homologous recombination or expression vectors to obtain a recombinant strain library. Initial screening was performed at the shake flask level (250 mL shake flask, 50 mL liquid volume) to detect xanthan gum yield and cell growth rate. High-yielding strains were selected for secondary screening in a 7 L fermenter to verify their production performance under controlled conditions, ultimately obtaining the target engineered strain. For example, strain S3 achieved a xanthan gum yield of 29 g / L in shake flask fermentation, a 31.8% increase compared to the wild-type strain, and the sugar conversion rate increased from 45% to 55%.

[0025] S30: Optimized Precision Fermentation Process

[0026] To achieve the industrial application of engineered strains, fermentation parameters were systematically optimized in fermenters of different sizes using precision fermentation technology. This included the following:

[0027] S30.1: Optimization of Laboratory Size Parameters

[0028] Using the target engineered bacterial strain as the fermentation strain, the effects of initial pH (6.0–8.0), fermentation temperature (28–34℃), dissolved oxygen (10%–50% air saturation), and feeding patterns (batch feeding, constant-rate feeding, and exponential feeding) on ​​xanthan gum yield, sugar conversion rate, and fermentation cycle were investigated in a 7L stirred fermenter (working volume 5L). Based on the experimental data, a mixed model was used for parameter identification to establish a dynamic prediction model for the fermentation process. For example, with an initial pH of 7.0, a fermentation temperature of 30℃, and dissolved oxygen control strategies of 35% dissolved oxygen for 0–24 hours, 20% for 24–72 hours, and 15% for 72–96 hours, using an exponential feeding pattern (feed solution containing 600 g / L glucose, feeding rate 0.5–1.0 g / L per hour), xanthan gum yield reached 33 g / L, and sugar conversion rate reached 58%.

[0029] S30.2: Parameter Verification for Pilot-Scale Operations

[0030] The optimized parameters were validated in a 100L fermenter (working volume 70L), and the feeding rate and dissolved oxygen control strategy were further adjusted to determine the optimal fermentation conditions for pilot-scale production. For example, in the 100L fermenter, the xanthan gum yield reached 34 g / L and the sugar conversion rate reached 59%.

[0031] S30.3: Industrial-scale verification

[0032] Based on the optimized parameters from the pilot-scale test, scale-up production was carried out in a 260kL industrial fermenter (working volume 200kL). The fermentation cycle was controlled at 96 hours. The pH value was adjusted in real time to 6.5-7.0, temperature to 30±1℃, dissolved oxygen (controlled by linkage between stirring speed and aeration rate), and feed rate (dynamically adjusted according to residual sugar concentration to maintain a carbon source concentration of 15-20g / L). For example, in the 260kL fermenter, under the above parameter control, after 96 hours of fermentation, the xanthan gum yield reached 35g / L, and the sugar conversion rate reached 62%.

[0033] In a second aspect, the present invention provides a xanthan gum engineered strain based on secondary messenger and metabolic engineering, which is constructed according to the method described in any embodiment of the first aspect.

[0034] Thirdly, the present invention provides an industrial production method for xanthan gum, comprising the following steps:

[0035] S40: Seed Culture

[0036] The target engineered strain was inoculated into seed culture medium and cultured for 12 to 16 hours at a temperature of 28 to 32°C and a pH of 6.5 to 7.0 to obtain seed culture.

[0037] S40.1: Fermentation Process Monitoring

[0038] The seed culture is inoculated into the fermenter, and the fermentation process is monitored according to the optimized fermentation parameters described in the first aspect. The pH value, temperature, dissolved oxygen and feeding rate are adjusted in real time to ensure stable operation of the fermentation process.

[0039] S40.2: Product Extraction

[0040] After fermentation, xanthan gum was extracted by ethanol precipitation and then dried to obtain the finished product.

[0041] Fourthly, the present invention provides a xanthan gum production line, wherein xanthan gum is continuously and stably produced according to the engineered strain constructed by the method described in the first aspect and the industrial production method described in the third aspect.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention elucidates the molecular mechanism by which secondary messenger molecules synergistically regulate xanthan gum synthesis, optimizes the metabolic network of chassis cells using metabolic engineering strategies, constructs engineered strains to enhance xanthan gum production, and optimizes fermentation parameters through precision fermentation technology, thereby significantly improving the production performance of xanthan gum. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the process flow of the present invention. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0046] like Figure 1 As shown, in a first aspect, this application provides a method for constructing xanthan gum engineered strains based on secondary messenger and metabolic engineering, comprising the following steps:

[0047] S10: Elucidating the molecular mechanism by which secondary messenger molecules co-regulate xanthan gum synthesis.

[0048] Using the wild-type xanthan gum production strain NRRL B-1459 as the research object, under high carbon / nitrogen ratio fermentation conditions (carbon-nitrogen ratio not less than 20:1), the molecular mechanism of c-di-GMP and ppGPP synergistic regulation of xanthan gum synthesis was elucidated using molecular biology techniques. The specific mechanisms include the following:

[0049] S10.1: Screening and Validation of Key Regulatory Genes

[0050] Mutants of the c-di-GMP synthase gene dgcA, the degradative enzyme gene pdeB, the ppGPP synthase gene relA, and the degradative enzyme gene spoT were constructed using gene knockout, overexpression, and reverse mutation techniques. The specific regulatory roles of these genes in xanthan gum synthesis were determined by comparing the xanthan gum yield, synthesis rate, and intracellular secondary messenger concentration between wild-type and mutant strains. For example, knocking out the pdeB gene increased the intracellular c-di-GMP concentration to 1.8 times its original value, and the xanthan gum yield increased from 22 g / L to 28 g / L; overexpression of the dgcA gene further increased the intracellular c-di-GMP concentration, and the xanthan gum yield reached 30 g / L. It should be noted that the combined mutants were constructed using sequential homologous recombination or CRISPR-Cas9 multi-gene editing technology, with specific steps following the single-gene knockout method.

[0051] In the specific implementation process, the upstream and downstream homologous arm sequences of the target gene were first amplified by PCR and cloned into a knockout vector. Subsequently, the recombinant plasmid was introduced into NRRL B-1459 via electroporation, and positive transformants were screened using antibiotics. Single-gene knockout strains were obtained after PCR verification. For overexpression strains, the target gene was cloned into an expression vector controlled by a strong promoter and similarly introduced into NRRL B-1459 via electroporation to obtain overexpression strains. Intracellular c-di-GMP and ppGPP concentrations were detected by high-performance liquid chromatography, and xanthan gum yield was determined using shake-flask fermentation experiments.

[0052] S10.2: Signal Path Interaction Analysis

[0053] Using fluorescent reporter gene fusion expression technology, protein immunoprecipitation technology, and gel migration assays, we investigated whether c-di-GMP and ppGPP synergistically regulate the transcription and expression of the xanthan gum synthesis gene cluster xanA-xanZ by binding to key enzymes or regulatory proteins in the xanthan gum synthesis pathway. For example, through fluorescent reporter gene GFP fusion expression, we found that the transcription level of the xanthan gum synthesis gene cluster xanA-xanZ in the double mutant strain ΔpdeB / ΔspoT was 2.5 times higher than that in the wild-type strain, and the intracellular concentrations of c-di-GMP and ppGPP were increased by 1.8 times and 1.5 times, respectively.

[0054] In the specific implementation process, the fluorescent reporter gene GFP was linked to the promoter region of the xanthan gum synthesis gene cluster xanA-xanZ, and cloned into an expression vector, which was then introduced into the target strain via electroporation. The fluorescence intensity of GFP was observed using fluorescence microscopy to assess the transcriptional activity of the gene cluster. Simultaneously, protein immunoprecipitation was used to detect the binding of c-di-GMP and ppGPP to the target protein, and their effects on DNA binding sites were verified using gel migration assays.

[0055] S10.3: Validation of Metabolic Flux Regulation

[0056] Metabolomics analysis was used to compare the changes in the contents of xanthan gum precursors UDP-glucose, UDP-mannose, and UDP-glucuronic acid in wild-type and mutant strains. Transcriptomics data were combined to analyze how c-di-GMP and ppGPP synergistically promote xanthan gum synthesis by regulating carbon flux allocation (such as the EMP and HMP pathways) and precursor synthesis pathways. For example, the UDP-glucose content in the mutant strain ΔpdeB was 1.6 times higher than that in the wild-type strain, indicating that c-di-GMP promotes precursor synthesis by enhancing EMP pathway activity.

[0057] In the implementation process, fermentation broth samples from different strains were collected, and the content of metabolites was detected using liquid chromatography-mass spectrometry. Simultaneously, total RNA was extracted, and transcriptome sequencing analysis was performed to screen differentially expressed genes and provide functional annotation. Changes in carbon metabolic flux allocation were assessed using KEGG pathway enrichment analysis.

[0058] S20: Construction of a high-yield xanthan gum engineered strain based on secondary messenger and metabolic engineering strategies

[0059] Based on the elucidated synergistic regulatory mechanism, and using NRRL B-1459 as the chassis cell, an engineered strain to enhance xanthan gum production was constructed through a combination of "secondary messenger regulation" and "metabolic engineering modification." Specifically, the strain includes the following:

[0060] S20.1: Optimization of the Second-Level Messenger Module

[0061] The synthesis signal is enhanced by regulating the steady-state levels of c-di-GMP and ppGPP: Overexpression of the c-di-GMP synthase gene dgcA and knockout of the degradation enzyme gene pdeB increase the intracellular c-di-GMP concentration; simultaneously, the expression of the ppGPP synthase gene relA is regulated by a weak promoter to maintain ppGPP levels and avoid excessive accumulation that inhibits cell growth, thus achieving synergistic activation of xanthan gum synthesis by both. The weak promoter is one with a lower strength than the strong constitutive promoter P43, and can maintain the expression level of the ppGPP synthase gene relA at 0.5 to 1.0 times the wild-type relA gene expression level. For example, integrating the dgcA gene into the chromosomal pdeB gene locus (knocking out pdeB while overexpressing dgcA) yields strain S1, whose intracellular c-di-GMP concentration is 2.2 times higher than the wild-type strain, and its xanthan gum yield reaches 30 g / L.

[0062] In the specific implementation process, a knockout fragment with a homologous arm was designed. The dgcA gene was inserted into the pdeB gene site using homologous recombination technology, while the pdeB gene was knocked out. For the ppGPP module, the relA gene was placed under the control of a weak promoter and cloned into a low copy number plasmid, which was then introduced into the target strain via electroporation.

[0063] S20.2: Metabolic Engineering Module Modification

[0064] Optimizing carbon metabolism and precursor supply in basal cells: Overexpression of key enzyme genes galU and manA in the xanthan gum synthesis precursor pathway enhances the synthesis of precursors such as UDP-glucose and UDP-mannose; knockout of key genes ldh and ackA in the byproduct synthesis pathway reduces the diversion of carbon sources to lactate and acetic acid; promoter engineering of the key enzyme gene pfkA in central carbon metabolism optimizes carbon flow allocation in glycolysis and pentose phosphate pathways, improving precursor supply efficiency. For example, overexpression of galU and manA genes in strain S1 yielded strain S2, whose UDP-glucose and UDP-mannose contents were increased by 2.1 times and 1.9 times, respectively, compared to the wild-type strain, with a xanthan gum yield of 29 g / L.

[0065] In the specific implementation process, the galU and manA genes were cloned into expression vectors controlled by strong promoters and introduced into the target strains via electroporation. For the byproduct synthesis pathway, knockout fragments were designed and the ldh and ackA genes were knocked out using homologous recombination technology. For the pfkA gene, a key enzyme in central carbon metabolism, promoters of different strengths were designed to replace its natural promoter, and changes in expression levels were verified by quantitative real-time PCR.

[0066] S20.3: Screening and Validation of Engineered Strains

[0067] The modified modules were introduced into chassis cells via homologous recombination or expression vectors to obtain a recombinant strain library. Initial screening was performed at the shake flask level (250 mL shake flask, 50 mL liquid volume) to detect xanthan gum yield and cell growth rate. High-yielding strains were selected for secondary screening in a 7 L fermenter to verify their production performance under controlled conditions, ultimately obtaining the target engineered strain. For example, strain S3 achieved a xanthan gum yield of 29 g / L in shake flask fermentation, a 31.8% increase compared to the wild-type strain, and the sugar conversion rate increased from 45% to 55%.

[0068] In the specific implementation process, the modification modules were introduced into the chassis cells one by one in a step-by-step manner, and positive transformants were screened using antibiotics. Preliminary screening was conducted at the shake-flask level, and the strain with the highest xanthan gum production was selected for secondary screening in the fermenter. Fermentation conditions were set as follows: temperature 30℃, initial pH 7.0, dissolved oxygen controlled at 35% air saturation, and fermentation cycle 72 hours.

[0069] S30: Optimized Precision Fermentation Process

[0070] To achieve the industrial application of engineered strains, fermentation parameters were systematically optimized in fermenters of different sizes using precision fermentation technology. This included the following:

[0071] S30.1: Optimization of Laboratory Size Parameters

[0072] Using the target engineered bacterial strain as the fermentation strain, the effects of initial pH (6.0–8.0), fermentation temperature (28–34℃), dissolved oxygen (10%–50% air saturation), and feeding patterns (batch feeding, constant-rate feeding, and exponential feeding) on ​​xanthan gum yield, sugar conversion rate, and fermentation cycle were investigated in a 7L stirred fermenter (working volume 5L). Based on the experimental data, a mixed model was used for parameter identification to establish a dynamic prediction model for the fermentation process. For example, with an initial pH of 7.0, a fermentation temperature of 30℃, and dissolved oxygen control strategies of 35% dissolved oxygen for 0–24 hours, 20% for 24–72 hours, and 15% for 72–96 hours, using an exponential feeding pattern (feed solution containing 600 g / L glucose, feeding rate 0.5–1.0 g / L per hour), xanthan gum yield reached 33 g / L, and sugar conversion rate reached 58%.

[0073] In the implementation process, different combinations of fermentation parameters were set, and multiple batches of fermentation experiments were conducted. Changes in parameters such as pH, dissolved oxygen, and temperature during fermentation were recorded using an online monitoring system, and samples were taken periodically to test xanthan gum yield and residual sugar concentration. Statistical analysis methods were used to screen for the optimal parameter combination, and a mathematical model was established for prediction.

[0074] S30.2: Parameter Verification for Pilot-Scale Operations

[0075] The optimized parameters were validated in a 100L fermenter (working volume 70L), and the feeding rate and dissolved oxygen control strategy were further adjusted to determine the optimal fermentation conditions for pilot-scale production. For example, in the 100L fermenter, the xanthan gum yield reached 34 g / L and the sugar conversion rate reached 59%.

[0076] In the specific implementation process, initial fermentation parameters were set based on the laboratory scale optimization results, and the feeding rate and dissolved oxygen control strategy were dynamically adjusted based on real-time monitoring data during fermentation. Stable fermentation conditions were determined through repeated experiments.

[0077] S30.3: Industrial-scale verification

[0078] Based on the optimized parameters from the pilot-scale test, scale-up production was carried out in a 260kL industrial fermenter (working volume 200kL). The fermentation cycle was controlled at 96 hours. The pH value was adjusted in real time to 6.5-7.0, temperature to 30±1℃, dissolved oxygen (controlled by linkage between stirring speed and aeration rate), and feed rate (dynamically adjusted according to residual sugar concentration to maintain a carbon source concentration of 15-20 g / L). For example, in the 260kL fermenter, under the above parameter control, after 96 hours of fermentation, the xanthan gum yield reached 35 g / L, and the sugar conversion rate reached 62%.

[0079] In the implementation process, an automatic control system is used to monitor the fermentation process in real time to ensure that all parameters remain stable within the set range. The fermentation effect is verified by periodically sampling and testing the concentration of xanthan gum and residual sugar in the fermentation broth.

[0080] Secondly, this application provides a xanthan gum engineered strain based on secondary messenger and metabolic engineering, which is constructed according to the method described in any embodiment of the first aspect.

[0081] Thirdly, this application provides an industrial production method for xanthan gum, comprising the following steps:

[0082] S40: Seed Culture

[0083] The target engineered strain was inoculated into seed culture medium and cultured for 12 to 16 hours at a temperature of 28 to 32°C and a pH of 6.5 to 7.0 to obtain seed culture.

[0084] In the specific implementation process, the seed culture medium formula was as follows: glucose 20 g / L, yeast extract 10 g / L, peptone 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, with the initial pH adjusted to 7.0. The seed culture conditions were: temperature 30℃, shaker speed 200 rpm, and culture time 14 hours.

[0085] S40.1: Fermentation Process Monitoring

[0086] The seed culture is inoculated into the fermenter, and the fermentation process is monitored according to the optimized fermentation parameters described in the first aspect. The pH value, temperature, dissolved oxygen and feeding rate are adjusted in real time to ensure stable operation of the fermentation process.

[0087] In practice, the fermentation tank is filled with 70% of its working volume, and the inoculum size is 10%. During fermentation, an online monitoring system records changes in parameters such as pH, dissolved oxygen, and temperature in real time, and dynamically adjusts the feeding rate and dissolved oxygen level according to a preset control strategy.

[0088] S40.2: Product Extraction

[0089] After fermentation, xanthan gum was extracted by ethanol precipitation and then dried to obtain the finished product.

[0090] In the specific implementation process, the fermentation broth is centrifuged, the supernatant is collected, and 2 times the volume of 95% ethanol is added for precipitation. After standing for 2 hours, the precipitate is filtered and collected. The precipitate is washed twice with anhydrous ethanol and then vacuum dried at 60°C to constant weight to obtain the xanthan gum product.

[0091] Fourthly, this application provides a xanthan gum production line, which continuously and stably produces xanthan gum using the engineered strain constructed according to the method described in the first aspect and the industrial production method described in the third aspect.

[0092] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0093] Example 1

[0094] S10: Using NRRL B-1459 as the research object, a ΔpdeB mutant strain was constructed, and the intracellular c-di-GMP concentration and xanthan gum production were detected. The results showed that the intracellular c-di-GMP concentration increased to 1.8 times the original value, and the xanthan gum production increased from 22 g / L to 28 g / L.

[0095] S20: Based on the ΔpdeB mutant strain, strain S1 was obtained by overexpressing the dgcA gene. Detection results showed that the intracellular c-di-GMP concentration increased to 2.2 times the original level, and the xanthan gum yield reached 30 g / L.

[0096] S30: Fermentation experiments were conducted on strain S1 in a 7L fermenter to optimize fermentation parameters. The results showed that the xanthan gum yield reached 33 g / L and the sugar conversion rate was 58%.

[0097] Example 2

[0098] S10: The ΔspoT mutant strain was constructed, and the intracellular ppGPP concentration and xanthan gum production were detected. The results showed that the intracellular ppGPP concentration increased to 1.5 times the original level, and the xanthan gum production increased from 22 g / L to 26 g / L.

[0099] S20: Based on the ΔspoT mutant strain, strain S2 was obtained by weakening the expression of the relA gene. The results showed that the intracellular ppGPP concentration was 1.3 times higher than that of the wild type, and the xanthan gum yield reached 27 g / L.

[0100] S30: Fermentation experiments were conducted on strain S2 in a 7L fermenter to optimize fermentation parameters. The results showed that the xanthan gum yield reached 32 g / L, and the sugar conversion rate was 57%.

[0101] Example 3

[0102] S10: A ΔpdeB / ΔspoT double mutant strain was constructed, and the intracellular concentrations of c-di-GMP and ppGPP, as well as xanthan gum production, were measured. The results showed that the intracellular concentrations of c-di-GMP and ppGPP increased to 1.8-fold and 1.5-fold, respectively, and the xanthan gum production increased from 22 g / L to 30 g / L.

[0103] S20: Based on the ΔpdeB / ΔspoT double mutant strain, strain S3 was obtained by overexpressing the dgcA gene and weakening the relA gene expression. The results showed that the intracellular c-di-GMP and ppGPP concentrations were increased by 2.2 times and 1.3 times, respectively, compared to the wild type, and the xanthan gum yield reached 29 g / L.

[0104] S30: Fermentation experiments were conducted on strain S3 in a 7L fermenter to optimize fermentation parameters. The results showed that the xanthan gum yield reached 34 g / L and the sugar conversion rate was 59%.

[0105] Comparative Example 1

[0106] Fermentation experiments were conducted using NRRL B-1459 as the fermentation strain under the same fermentation conditions. The results showed that the xanthan gum yield was 22 g / L and the sugar conversion rate was 45%.

[0107] Comparative Example 2

[0108] Fermentation experiments were conducted using the ΔpdeB mutant strain under the same fermentation conditions. The results showed that the xanthan gum yield was 28 g / L and the sugar conversion rate was 50%.

[0109] Comparative Example 3

[0110] Fermentation experiments were conducted using the ΔspoT mutant strain under the same fermentation conditions. The results showed that the xanthan gum yield was 26 g / L and the sugar conversion rate was 48%.

[0111] The results of the above embodiments and comparative examples are summarized in the table below:

[0112]

[0113] As shown in the table, the method provided in this application can significantly improve xanthan gum yield and sugar conversion rate. At the same time, by optimizing the secondary messenger module and metabolic engineering module, the efficiency and stability of xanthan gum production are achieved.

[0114] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A method for constructing xanthan gum engineered strains based on secondary messenger and metabolic engineering, characterized in that, Includes the following steps: S10: Using the wild-type xanthan gum production strain NRRL B-1459 as the starting strain, under high carbon / nitrogen ratio fermentation conditions of not less than 20:1, the synergistic regulatory effects of c-di-GMP and ppGPP on xanthan gum synthesis were analyzed to determine their molecular mechanism for regulating xanthan gum synthesis. S20: A high-yield xanthan gum engineered strain was constructed based on a secondary messenger and metabolic engineering strategy. Using NRRL B-1459 as the chassis cell, xanthan gum synthesis was enhanced by regulating the homeostatic levels of c-di-GMP and ppGPP and optimizing the distribution of carbon metabolic flux. S30: Precision fermentation process optimization, combining precision fermentation technology to systematically optimize fermentation parameters in fermenters of different sizes.

2. The method according to claim 1, characterized in that, Step S10 includes the following: S10.1: Screening and validation of key regulatory genes. Using gene knockout, overexpression and reverse mutation techniques, mutant strains of c-di-GMP synthase gene dgcA, degradation enzyme gene pdeB, ppGPP synthase gene relA and degradation enzyme gene spoT were constructed, respectively. S10.2: Signal pathway interaction analysis: Using fluorescent reporter gene fusion expression technology, protein immunoprecipitation technology and gel migration assay, we can detect whether c-di-GMP and ppGPP can synergistically regulate the transcription and expression of the xanthan gum synthesis gene cluster xanA-xanZ by binding to key enzymes or regulatory proteins in the xanthan gum synthesis pathway. S10.3: Metabolic flux regulation validation. Metabolomics analysis was used to compare the changes in the content of xanthan gum precursors UDP-glucose, UDP-mannose, and UDP-glucuronic acid in wild-type and mutant strains. Transcriptomics data were combined to analyze how c-di-GMP and ppGPP promote xanthan gum synthesis by regulating carbon metabolic flux allocation.

3. The method according to claim 1, characterized in that, Step S20 includes the following: S20.1: The secondary messenger module is optimized by overexpressing the c-di-GMP synthase gene dgcA and knocking out the degradation enzyme gene pdeB to increase the intracellular c-di-GMP concentration, while maintaining the ppGPP level by regulating the expression of the ppGPP synthase gene relA through a weak promoter. S20.2: Metabolic engineering module modification, overexpression of key enzyme genes galU and manA in the xanthan gum synthesis precursor synthesis pathway, enhancement of the synthesis of precursor substances such as UDP-glucose and UDP-mannose, knockout of key genes ldh and ackA in the byproduct synthesis pathway, reduction of carbon source diversion to lactate and acetic acid, and optimization of carbon flow allocation in glycolysis and pentose phosphate pathway by promoter engineering modification of the key enzyme gene pfkA in central carbon metabolism; S20.3: Screening and verification of engineered strains. The above-mentioned modified modules are introduced into the chassis cells through homologous recombination or expression vectors to obtain a recombinant strain library, and primary and secondary screening are carried out in shake flasks and 7L fermenters.

4. The method according to claim 1, characterized in that, Step S30 includes the following: S30.1: Laboratory-scale parameter optimization: The effects of initial pH value of 6.0 to 8.0, fermentation temperature of 28 to 34℃, dissolved oxygen of 10% to 50% air saturation and feeding mode on xanthan gum yield were investigated in a 7L stirred fermenter. S30.2: Pilot-scale parameter verification, further adjusting the feeding rate and dissolved oxygen control strategy in a 100L fermenter; S30.3: Industrial-scale scale-up verification: scale-up production was carried out in a 260kL industrial fermenter, with the fermentation cycle controlled at 96 hours.

5. The method according to claim 1, characterized in that, In step S10.1, the mutant strains include ΔpdeB, ΔspoT, ΔdgcA, ΔrelA, and their combinations.

6. The method according to claim 1, characterized in that, In step S20.1, the weak promoter is a promoter with a strength lower than that of the strong constitutive promoter P43, and is able to maintain the expression level of the ppGPP synthase gene relA at 0.5 to 1.0 times that of the wild-type relA gene.

7. The method according to claim 1, characterized in that, In step S20.2, the promoter engineering modification includes replacing the natural promoter of the central carbon metabolism key enzyme gene pfkA with an artificially designed tunable promoter, adjusting its expression level to between 1.2 and 1.8 times that of the wild type.

8. The method according to claim 1, characterized in that, In step S30.1, the feeding mode includes batch feeding, constant rate feeding and exponential feeding, wherein the feeding solution in the exponential feeding mode contains 600 g / L of glucose and the feeding rate is 0.5 to 1.0 g / L per hour.

9. The method according to claim 1, characterized in that, In step S30.3, the online monitoring system controls the pH value to 6.5-7.0, the temperature to 30±1℃, and the dissolved oxygen to be controlled in real time by linking the stirring speed and the aeration rate. The feeding rate is dynamically adjusted according to the residual sugar concentration to maintain the carbon source concentration at 15 to 20 g / L.

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