Method for efficiently and green synthesizing L-carnosine and application of L-carnosine

Abstract

The invention relates to the technical field of biology, in particular to a method for efficiently synthesizing L-carnosine in a green mode and application of the L-carnosine. The invention provides the L-carnosine synthetase and the strain, and also provides a method for synthesizing the L-carnosine through an enzyme catalysis method, a green and sustainable biological catalysis process is adopted to improve the synthesis efficiency of the L-carnosine and overcome the problems of low yield, low conversion rate, substrate inhibition and the like in the prior art, and substrate accumulation is optimized through transport protein engineering, so that the yield of the L-carnosine is improved. The production capacity of the product is further improved, and a feasible solution is provided for large-scale industrial production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for efficiently and greenly synthesizing L-carnosine and application thereof. Background Art

[0002] L-Carnosine (L-Car) is a naturally occurring dipeptide synthesized from β-alanine (β-Ala) and L-histidine (L-His) by enzymatic reaction. L-Carnosine is widely distributed in mammalian muscle, brain, heart, gastrointestinal tract and other tissues, and has been shown to have biological functions such as antioxidant, anti-inflammatory and neuroprotective. Therefore, L-Carnosine is considered to be a natural antioxidant and bioactive molecule with broad application prospects.

[0003] There are two main methods for synthesizing L-carnosine: traditional chemical synthesis and biocatalytic synthesis. Existing chemical synthesis methods mostly use phthalic anhydride method or other chemical reaction methods, but such methods have problems such as long reaction time, high energy consumption, and serious environmental pollution, and therefore do not conform to the principles of green chemistry. In addition, the yield of L-carnosine synthesized by chemical methods is low, and it is difficult to meet the needs of large-scale production.

[0004] With the development of biotechnology, biocatalysis, especially enzymatic synthesis, has become an effective way to synthesize L-carnosine. Biocatalysis not only has high efficiency, specificity and mild reaction conditions, but also reduces environmental pollution, which meets the requirements of green and sustainable development. In the human body, the synthesis of L-carnosine is catalyzed by carnosine synthase (EC 6.3.2.11), which uses ATP to connect β-Ala and L-His to form L-carnosine. In recent years, researchers have also tried to synthesize L-carnosine through microbial or enzyme catalysis, and have made some progress.

[0005] Existing studies have shown that certain microbial aminopeptidases (such as SmpepD from Serratia marcescens) can effectively catalyze the synthesis of L-carnosine. Through genetic engineering technology, the activity of these enzymes has been significantly improved, making them potential candidate enzymes for the biosynthesis of L-carnosine. In addition, the optimization of enzyme reaction conditions (such as pH, temperature, metal ion concentration) and substrate concentration also plays a key role in improving the synthesis efficiency. However, although the existing enzyme-catalyzed method can achieve the synthesis of L-carnosine, the existing methods generally have problems such as low yield, poor conversion rate, and product inhibition, which makes it difficult to meet the industrial needs of large-scale production.

[0006] In order to further improve the synthesis efficiency of L-carnosine, researchers have also tried to combine a variety of strategies, such as transporter engineering, protein engineering, etc. The application of these strategies can increase the intracellular accumulation of substrates, improve the catalytic efficiency of enzymes, and solve the inhibition problem caused by the accumulation of substrates or products. However, the current research on L-carnosine synthesis is still in the laboratory stage and has not yet been able to achieve large-scale, efficient industrial production.

[0007] The closest existing technology is the biocatalytic method based on enzymatic synthesis of L-carnosine. For example, Kino et al. (2018) successfully synthesized L-carnosine by using L-amino acid ligase in Bacillus subtilis, achieving a high yield of 91.4%, and the reaction required the participation of an equal amount of ATP. This study provided valuable experimental data for the enzymatic synthesis of L-carnosine and provided a theoretical basis for the synthesis method using enzymes as catalysts. However, the ATP consumption of this method is high, which not only increases the energy consumption of the reaction, but also limits its industrial application.

[0008] In addition, Yin et al. (2019) found in their study that through genome mining technology, they screened out a highly active aminopeptidase (SmpepD) from Seratiamarcescens. The enzyme was able to catalyze the synthesis of L-carnosine without using protected β-alanine or its active derivatives, and achieved a high yield of 15.2 g / L under optimized reaction conditions. Although this method has achieved good results in the laboratory, there is still a problem of relatively low yield, and further optimization of enzyme activity, reaction conditions and substrate conversion rate is needed.

[0009] In addition, existing studies have also shown that transporter engineering plays an important role in enhancing the synthesis of L-carnosine. For example, Kutukova et al. (2005) found that the YeaS protein belongs to the RhtB transporter family and can increase the synthesis yield of L-carnosine by enhancing the extracellular transport of L-histidine. This discovery provides a new direction for transporter engineering and opens up a new way to improve the production efficiency of L-carnosine. However, although transporter engineering has improved the synthesis efficiency to a certain extent, existing related research still faces the limitations of transport efficiency and substrate accumulation, which makes it difficult to meet the requirements of industrial production.

[0010] In summary, although the existing L-carnosine synthesis technology has made some progress, there are still certain bottlenecks in production efficiency, cost, substrate utilization, etc. Therefore, finding an efficient, green and large-scale production method for L-carnosine synthesis is still a current research hotspot and challenge.

[0011] Although the existing L-carnosine synthesis methods, especially the enzyme-catalyzed biosynthesis methods, have made some progress in laboratory research, they still have the following major disadvantages, which limit their application in large-scale industrial production:

[0012] 1) Low yield and conversion rate: Although the use of enzyme catalysis to synthesize L-carnosine can improve the yield to a certain extent, the yield of existing enzyme catalysis methods is generally low. For example, Kino et al. (2018) used L-amino acid ligase to achieve a high yield of 91.4%, but the reaction required an equal amount of ATP to participate, which not only increased the energy consumption of the reaction, but also led to an increase in production costs. In addition, even under the most optimized reaction conditions, the synthesis conversion rate of L-carnosine is still far lower than the efficiency required for industrial production.

[0013] 2) Substrate inhibition effect: The synthesis of L-carnosine requires β-alanine and L-histidine as substrates. However, too high a substrate concentration will have an inhibitory effect on the cells, thereby affecting the synthesis process. This is because high concentrations of substrates may cause a metabolic burden on the cells, inhibit enzyme activity, and lead to reduced efficiency in product accumulation. In addition, the balance between substrates and products will also lead to a decrease in conversion rate, affecting the final yield.

[0014] 3) Enzyme stability and tolerance: Another challenge of enzyme catalysis is the stability and tolerance of the enzyme. During a long reaction, the activity of the enzyme may decrease, especially under conditions of high substrate concentration and high reaction temperature, the activity and stability of the enzyme may drop sharply, which directly leads to a decrease in yield. Therefore, it is necessary to improve the stability and tolerance of the enzyme through protein engineering and optimization of reaction conditions.

[0015] 4) Limitation of transporters: The role of transporters is to promote the intracellular accumulation of substrates, thereby improving the catalytic efficiency of enzymes. However, although the existing transporter engineering methods have improved the intracellular accumulation of substrates to a certain extent, the problems of substrate transport efficiency and cell tolerance often still exist due to the selective permeability of the cell membrane. As a result, the effect of transporter engineering in improving the production efficiency of L-carnosine is not as good as expected.

[0016] 5) Lack of efficient and green synthesis pathways: Existing L-carnosine production methods, especially chemical synthesis methods, have problems such as long reaction time, high energy consumption, and high pollution, which seriously do not meet the requirements of green chemistry and sustainable development. Even in biocatalytic synthesis, although green catalysts such as enzymes are used, there are still problems of low yield and high energy consumption. Therefore, how to develop an efficient and environmentally friendly L-carnosine synthesis method has become a challenge that needs to be solved urgently. Summary of the invention

[0017] In view of this, the object of the present invention is to provide a method for synthesizing L-carnosine that is efficient, green and suitable for large-scale industrial production. Specific objectives include:

[0018] 1) Improve the yield and conversion rate: By screening and optimizing highly active enzymes, such as the SmpepD enzyme from Serratia marcescens, and combining protein engineering to transform the enzyme, the synthesis efficiency of L-carnosine can be improved, thereby improving the yield and conversion rate. The realization of this goal can significantly improve the production capacity of L-carnosine and meet the needs of industrial production.

[0019] 2) Reduce substrate inhibition effect: Optimize the intracellular accumulation of substrates through transporter engineering, reduce the inhibitory effect of substrates on cells, and improve substrate conversion efficiency by adjusting reaction conditions (such as substrate concentration, pH, temperature, etc.). This will help reduce the inhibitory effect caused by substrate accumulation and further improve synthesis efficiency.

[0020] 3) Improve enzyme stability and tolerance: Through protein engineering, the structure of the enzyme is modified to enhance its stability and tolerance under reaction conditions such as high substrate concentration and high temperature, prolong the enzyme's catalytic time, and maintain the enzyme's high activity. Improving enzyme stability and tolerance will reduce energy consumption and resource waste during the reaction process, thereby reducing production costs.

[0021] 4) Achieve green and sustainable synthesis: The present invention adopts green catalysts (enzymes) and biocatalysis to avoid harmful chemical reagents and high energy consumption reactions in traditional chemical synthesis, which conforms to the principles of green chemistry. It not only reduces environmental pollution during the production process, but also makes the production process of L-carnosine more environmentally friendly and sustainable.

[0022] 5) Application in large-scale industrial production: By optimizing the whole-cell conversion system and fermentation process, the L-carnosine synthesis method of the present invention can be stably operated in a large-scale bioreactor to achieve the production goals of high yield, low cost and low energy consumption, and has broad industrial application prospects.

[0023] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0024] The invention provides application of an enzyme with NCBI accession number WP_034652116 in synthesizing L-carnosine or improving the synthesis rate of L-carnosine.

[0025] The present invention also provides a genetically engineered bacterium comprising at least one of the following:

[0026] (A1), adding the gene of the enzyme with NCBI accession number WP_034652116;

[0027] (A2), hisG with Δ209-281 mutation;

[0028] (A3), adding the gene of the enzyme with UniProt accession number Q5JJ82;

[0029] (A4), adding yeaS gene and cycA gene.

[0030] In some specific embodiments of the present invention, the genetically engineered bacteria comprises:

[0031] (B1), an expression vector having a gene for an enzyme with NCBI accession number WP_034652116; and / or

[0032] (B2) An expression vector having a gene for an enzyme with UniProt accession number Q5JJ82.

[0033] In some specific embodiments of the present invention, in the above-mentioned application or genetically engineered bacteria, the version number of the enzyme with NCBI accession number WP_034652116 is WP_034652116.1.

[0034] In some specific embodiments of the present invention, the chassis of the genetically engineered bacteria is Escherichia coli.

[0035] In some specific embodiments of the present invention, the adding of the yeaS gene and the cycA gene to the genetically engineered bacteria comprises: integrating the yeaS gene and the cycA gene into the flgG site.

[0036] The invention also provides application of genetic engineering bacteria in synthesizing L-carnosine.

[0037] The present invention also provides a method for synthesizing L-carnosine, comprising: subjecting the genetic engineering bacteria to fed-batch fermentation.

[0038] In some specific embodiments of the present invention, the above-mentioned synthesis method comprises the following steps:

[0039] S1. Inoculate the seed solution of the above genetically engineered bacteria into the culture medium and culture at a temperature of 37°C, a rotation speed of 300 rpm, and a ventilation volume of 1.5 vvm;

[0040] S2. When the glucose in the culture medium is exhausted, feed at a rate of 10 mL / h and continue culturing for 6 to 12 hours;

[0041] S3, change the feed rate to 20 mL / h and continue culturing for 12-24 hours;

[0042] S4. When OD 600 When the concentration reaches above 150, lactose is induced and fermented to obtain L-carnosine.

[0043] In some specific embodiments of the present invention, the culture medium of the above synthesis method comprises: 13.3 g / L KH2PO4, 4 g / L (NH4)2HPO4, 1.2 g / L MgSO4·7H2O, 20 g / L glucose and 5 g / L nicotinamide (NAM).

[0044] In some specific embodiments of the present invention, the feed solution used in the above-mentioned synthesis method comprises: 600 g / L glucose, 60 g / L yeast extract and 60 g / L tryptone.

[0045] The invention also provides a method for increasing the yield of L-carnosine, comprising: synthesizing L-carnosine based on an enzyme with NCBI accession number NZ_CP058353, thereby increasing the yield of L-carnosine.

[0046] In some specific embodiments of the present invention, the above method further comprises: overexpressing the hisG gene in the strain producing L-carnosine to achieve an increase in the yield of L-carnosine;

[0047] The base of the strain producing L-carnosine is Escherichia coli, and the enzyme with NCBI accession number WP_034652116 is expressed.

[0048] In some specific embodiments of the present invention, the method further comprises: introducing S143F mutation, E217K mutation or Δ209-281 mutation into the hisG gene in the strain producing L-carnosine to increase the yield of L-carnosine;

[0049] The base of the strain producing L-carnosine is Escherichia coli, and the enzyme with NCBI accession number WP_034652116 is expressed.

[0050] In some specific embodiments of the present invention, the method further comprises: expressing multiple copies of the his operon in the strain producing L-carnosine to increase the yield of L-carnosine;

[0051] The base of the strain producing L-carnosine is Escherichia coli, and the enzyme with NCBI accession number WP_034652116 is expressed.

[0052] In some specific embodiments of the present invention, the method further comprises: introducing a gene of an enzyme having UniProt accession number A8F5K4, B5Y862, Q60358, Q5JJ82, A0B9M9, O27188 or Q171S0 into a strain producing L-carnosine to increase the yield of L-carnosine;

[0053] The chassis of the strain producing L-carnosine is Escherichia coli, the enzyme with NCBI accession number WP_034652116 is expressed, and the hisG gene has a mutation of Δ209-281.

[0054] In some specific embodiments of the present invention, the method further comprises: adding the yeaS gene and the cycA gene to the strain producing L-carnosine to increase the yield of L-carnosine;

[0055] The chassis of the strain producing L-carnosine is Escherichia coli, the enzyme with NCBI accession number WP_034652116 is expressed, the hisG gene has a mutation of Δ209-281, and the gene of the enzyme with UniProt accession number Q5JJ82 is contained.

[0056] In some specific embodiments of the present invention, the above method also includes optimizing the microbial metabolic pathway, including using other microorganisms such as yeast (Saccharomyces cerevisiae) or lactic acid bacteria (Lactobacillus). These microorganisms naturally have different metabolic pathways, and under appropriate genetic modification, they can become effective L-carnosine production hosts, especially in terms of fermentation control and substrate utilization.

[0057] In some specific embodiments of the present invention, the above method also includes adopting a metabolic flux regulation strategy, including using metabolic flux analysis (MFA) to re-arrange the metabolic pathway of the host bacteria, reduce the generation of by-products and improve the utilization of substrates, so that the production of L-carnosine can be increased by changing the metabolic flux distribution without relying on specific enzymes or genes.

[0058] In some specific embodiments of the present invention, the above method also includes plant or animal cell culture. Although the relative cost is high, L-carnosine can be produced by using plant cell culture or insect cell culture. The advantage of these systems is that the safety of the production process is higher.

[0059] In some specific embodiments of the present invention, the above method also includes a combination of chemical synthesis and biocatalysis, specifically including:

[0060] (a) Chemical and biological combined synthesis method: The precursor molecule of L-carnosine can be synthesized by chemical means first, and then the final product conversion can be completed by biocatalysis. This method can greatly reduce the harsh requirements for fermentation conditions and reduce the environmental impact caused by chemical methods;

[0061] (b) Cell-free synthesis system: Using a cell-free biosynthesis system, L-carnosine is produced by isolating and purifying the relevant enzymes and performing enzymatic reactions in vitro. This method can circumvent the challenges associated with cell growth during fermentation and improve the conversion rate of substrate to product;

[0062] (c) Combination of enzyme engineering and reactor technology: Using immobilized enzyme reactors, L-carnosine is produced by catalyzing immobilized high-efficiency L-carnosine synthase. Enzyme immobilization can improve its stability, prolong the catalytic reaction time, and can be reused, reducing production costs.

[0063] In some specific embodiments of the present invention, the above method also includes these alternative production strategies that may achieve industrial production of L-carnosine, and which strategy to choose depends on actual application requirements and cost considerations. For example, the use of a cell-free synthesis system may be suitable for small-scale production of high-value products, while a fermentation strategy that optimizes metabolic flow is more suitable for large-scale industrial production.

[0064] The present invention solves the technical bottleneck in the existing L-carnosine synthesis method by various means, and the specific advantages are as follows:

[0065] Achievement of high yield and conversion rate: By screening high-efficiency enzymes, the present invention significantly improves the synthesis efficiency of L-carnosine. The optimized enzymes show good activity, so that the conversion rate of the product reaches the level required for industrial production. This greatly reduces production time and cost in practice and improves the economic value of the product.

[0066] Reduce substrate inhibition effect and improve cell utilization of substrates: By optimizing the substrate transport system, including the integration and overexpression of yeaS and cycA transporters, the present invention greatly increases the effective concentration of substrates (such as L-histidine and β-alanine) in cells and reduces the inhibitory effect caused by substrate accumulation.

[0067] By optimizing the substrate transport system, cells can use substrates more efficiently, thereby increasing the final yield of L-carnosine, making the synthesis process of L-carnosine more efficient and reducing the waste of raw materials.

[0068] Green and sustainable synthesis process: The present invention adopts a green biocatalytic process, avoiding the high pollution and high energy consumption problems in traditional chemical synthesis, and conforms to the principles of green chemistry.

[0069] By modifying the metabolic pathway, the host bacteria can independently synthesize necessary substrates (such as β-alanine), reducing dependence on exogenous chemicals, further reducing pollution and costs in the production process, and making the production of L-carnosine more environmentally friendly and sustainable.

[0070] Feasibility of being suitable for large-scale industrial production: The L-carnosine synthesis method of the present invention has been optimized through the fed-batch fermentation process and can be stably operated in a large-scale fermentation tank, achieving the goals of high yield, low cost and low energy consumption. Through the scale-up experiment verification of a 5 L fermentation tank, an L-carnosine yield of up to 21.2 g / L was successfully achieved, showing the industrial application potential of the technology of the present invention. This means that the method is not only suitable for small-scale production under laboratory conditions, but can also be promoted and applied to large-scale industrial production.

[0071] Reduce production costs and improve economic benefits: By enhancing the biosynthetic pathway of substrates (such as the synthesis of histidine and β-alanine), the present invention reduces the need for supplementation of exogenous substrates and reduces the cost of raw materials. At the same time, the application of the fed-batch fermentation strategy effectively increases the unit yield of L-carnosine, thereby reducing the comprehensive cost of producing unit quality products and significantly improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0073] Figure 1 The effect of the construction of β-Ala biosynthesis pathway on the production of L-carnosine is shown;

[0074] Figure 2 The effect of optimizing substrate transport system on L-carnosine production is shown;

[0075] Figure 3 The results show the production of L-carnosine by fed-batch fermentation. DETAILED DESCRIPTION

[0076] The invention discloses a method for efficiently and greenly synthesizing L-carnosine and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0077] The present invention provides a method for synthesizing L-carnosine by enzyme catalysis, adopts a green and sustainable biocatalytic process to improve the synthesis efficiency of L-carnosine, overcomes the problems of low yield, low conversion rate and substrate inhibition in the prior art, optimizes substrate accumulation by transporter protein engineering, further improves the production capacity of the product, and provides a feasible solution for large-scale industrial production.

[0078] The technical solution of the present invention mainly includes the following key steps:

[0079] 1) Screening and optimization of efficient enzyme catalysts: The present invention first screened efficient L-carnosine synthases (such as SmpepD from Serratia marcescens). These enzymes have high activity and good substrate specificity in the process of catalyzing the synthesis of L-carnosine. The enzymes are modified through genetic engineering technology to further improve their catalytic efficiency and tolerance to adapt to industrial reaction conditions such as high substrate concentration and high temperature.

[0080] 2) Enhanced histidine synthesis pathway: In order to increase the intracellular supply of L-histidine, the present invention has modified the biosynthetic pathway of histidine, and increased the accumulation of L-histidine in cells by enhancing the expression level of histidine synthesis-related enzymes. The design of this enhanced pathway can not only provide sufficient substrate for the synthesis of L-carnosine, but also improve the conversion rate and alleviate the synthesis bottleneck caused by insufficient substrate.

[0081] 3) Optimization of substrate transport system: The present invention optimizes the intracellular accumulation of substrates such as L-histidine and β-alanine through transporter engineering. Gene editing technology (such as CRISPR / Cas9 system) is used to overexpress or knock out the transporter on the cell membrane to improve the transport efficiency of the substrate and reduce the substrate inhibition effect, thereby increasing the utilization rate of the substrate by the cell and further increasing the synthesis yield of L-carnosine.

[0082] 4) Construction of β-alanine (β-Ala) biosynthetic pathway: In order to solve the problem of exogenous addition of β-alanine, the present invention constructs a β-alanine biosynthetic pathway, enabling the host bacteria to independently synthesize β-alanine, thereby providing a stable substrate supply. This construction effectively reduces production costs and avoids concentration fluctuations caused by exogenous addition of β-alanine, thereby improving the synthesis efficiency of L-carnosine.

[0083] 5) Optimization of reaction conditions: In order to further increase the yield of L-carnosine, the present invention also optimizes the reaction conditions in detail. The key optimization parameters include reaction temperature, cell density (OD 600 ), metal ion concentration, substrate ratio, etc. The experimental results show that under the optimal reaction conditions (such as temperature 30℃, Mn 2+ The concentration was 1 mM, β-Ala was added), the synthesis efficiency of L-carnosine was significantly improved, achieving a higher yield.

[0084] 6) Scale-up verification of large-scale production: The present invention further conducted scale-up experimental verification in a 5 L fermenter. By adjusting the stirring speed, medium formula and cell culture conditions, the efficient synthesis of L-carnosine was successfully achieved. Finally, the fermentation reaction system using this technical solution successfully produced 133.2 mM L-carnosine at a 5 L scale, with a conversion rate of 49.3%, reaching the highest value in the existing literature, showing the industrial production potential of this technology.

[0085] Specifically, the plan includes:

[0086] Screening of efficient L-carnosine synthetase: Through screening and genetic engineering, efficient L-carnosine synthetase (such as SmpepD, PmCAR, etc.) is obtained to significantly improve the synthesis efficiency of L-carnosine.

[0087] Enhancement of the histidine biosynthesis pathway: By overexpressing histidine synthesis-related genes (such as hisG), constructing mutants (such as hisG S143F, hisG E217K, hisGΔ209-281) and expressing multiple copies of the his operator, the intracellular supply of histidine is increased, thereby increasing the production of L-carnosine.

[0088] Construction and optimization of the β-alanine (β-Ala) biosynthetic pathway: By screening and overexpressing a variety of β-alanine synthase genes (such as BA01-BA10), the host bacteria's internal supply of β-alanine is increased, thereby increasing the synthesis yield of L-carnosine.

[0089] Optimization of substrate transport system: CRISPR / Cas9 technology is used to integrate substrate transporter genes (such as yeaS and cycA) at specific sites of the strain to optimize substrate transport efficiency, reduce substrate inhibition effects, and thus increase L-carnosine production. The combined overexpression of yeaS and cycA genes showed significant synergistic effects, and was outstanding in improving substrate utilization.

[0090] Optimization of fed-batch fermentation process: By optimizing the fed-batch fermentation strategy, cell growth and L-carnosine production were improved, achieving high-density cell culture and efficient L-carnosine production.

[0091] Green and sustainable biocatalytic methods: Using green catalysts (enzymes) and biocatalytic processes to avoid harmful chemical reagents and high-energy consumption reactions in traditional chemical synthesis is in line with the principles of green chemistry and sustainable development.

[0092] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0093] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0094] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0095] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0096] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0097] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0098] The present invention will be further described below in conjunction with the embodiments.

[0099] Example 1: Screening of L-carnosine synthetase and its effect on L-carnosine production

[0100] Implementation steps: In this embodiment, by screening L-carnosine synthetase from different sources, a highly active enzyme is selected to improve the biosynthetic efficiency of L-carnosine. The specific operation steps are as follows:

[0101] Enzyme source screening: The present invention selected aminopeptidases or carnosine synthetases from four different sources, including SmpepD (NZ_CP058353.1), CpCAR (WP_164816820.1), SmCAR (WP_016928980.1) and PmCAR (WP_034652116.1), as candidate enzyme sources for L-carnosine synthetase. Through genetic engineering technology, these enzyme genes (pepD) were cloned into the expression vector pET28a(+) and transformed into E. coli BL21 (DE3) to construct the corresponding recombinant strains: E. coli BL21 / pET28a-SmpepD (EC01), E. coli BL21 / pET28a-CpCAR (EC02), E. coliBL21 / pET28a-SmCAR (EC03) and E. coli BL21 / pET28a-PmCAR (EC04).

[0102] Expression of recombinant strains: The constructed recombinant strains were inoculated into 10 mL of Luria-Bertani (LB) medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 8-12 hours as seed solution. The seed solution was inoculated into 50 mL of LB medium containing kanamycin at a 1% inoculum.

[0103] Shake flask fermentation conditions: The recombinant strain after induced expression was inoculated into a 250 mL Erlenmeyer flask containing 10 g / L β-alanine (β-Ala) and 100 mL M9 medium. The medium components included: 13.3 g / L KH2PO4, 4 g / L (NH4)2HPO4, 1.2 g / L MgSO4·7H2O, 20 g / L glucose and 5 g / L nicotinamide (NAM). Shake flask fermentation experiments were carried out at 37°C and 200 rpm.

[0104] Shake flask fermentation induction and sample collection: 6 hours after the start of fermentation culture, 1 mM IPTG (final concentration) was added to induce expression. 10 mL of samples were taken at 24 hours and 48 hours for subsequent L-carnosine detection and analysis. The samples were separated by centrifugation (8000 rpm, 4°C, 10 minutes) to separate the supernatant and filtered with a 0.22 μm PTFE filter to ensure the purity of the fermentation broth.

[0105] Detection method of L-carnosine concentration: The concentration of L-carnosine was quantitatively analyzed by high performance liquid chromatography (HPLC, Younglin Inc. Korea). An Eclipse plus C18 column (4.6 × 250 mm, 5 μm) was used with 10% acetonitrile as the mobile phase, isocratic elution at a flow rate of 1 mL / min, and the detection wavelength was 210 nm. The standard curve was drawn based on L-carnosine standards of known concentrations.

[0106] The experimental results are shown in Table 1.

[0107] Table 1: Screening of L-carnosine synthase and its effect on L-carnosine production (g / L)

[0108]

[0109] Among the four enzymes screened, PmCAR from Priestia megaterium showed the highest L-carnosine synthesis activity. Under the same reaction conditions, the yield of L-carnosine synthesized by PmCAR was significantly higher than that of other enzyme sources. In addition, by comparing the conversion efficiency of different enzymes, it was found that PmCAR showed the best conversion rate in the L-carnosine synthesis reaction, indicating that it has significant advantages in the biosynthesis of L-carnosine. Therefore, PmCAR was selected as the key enzyme source for L-carnosine synthesis in the present invention, laying the foundation for subsequent efficient L-carnosine production.

[0110] Example 2: Effect of Enhanced Histidine Synthesis Pathway on L-Carnosine Production

[0111] In this example, the internal supply of L-histidine in the host bacteria was improved by enhancing the histidine synthesis pathway to further increase the synthesis yield of L-carnosine. To this end, various strategies were used to enhance the biosynthesis of histidine in the E. coli BL21 / pET28a-PmCAR (EC04) recombinant strain, including overexpression of the hisG gene, construction of hisG mutants (hisGS143F, hisG E217K, hisGΔ209-281), and multi-copy expression of the his operator, and their effects on the shake flask fermentation yield of L-carnosine were evaluated.

[0112] Gene editing operation: First, use the CRISPR / Cas9 system to edit the genes of the E. coli BL21 / pET28a-PmCAR strain. The specific steps are as follows:

[0113] hisG overexpression: The hisG gene was integrated into the yeeL site of the EC04 strain to obtain the strain E. coli BL21yeeL:: hisG / pET28a-PmCAR (EC05).

[0114] Construction of hisG mutants: In order to obtain mutants with potential improvement in histidine synthesis, three different mutations were made to hisG in this example, namely S143F, E217K and Δ209-281. These mutants were integrated into the genome of the host bacteria by genome editing methods.

[0115] E. coli BL21 hisG::hisGS143F / pET28a-PmCAR (EC06); and

[0116] E. coli BL21 hisG::hisGE217K / pET28a-PmCAR (EC07); and

[0117] E. coli BL21 hisG::hisGΔ209-281 / pET28a-PmCAR (EC08).

[0118] Multi-copy expression of his operon: The his operon was integrated into the yeeL site of the EC04 strain to obtain the strain E. coli BL21 yeeL:: hisGDCBHAFI / pET28a-PmCAR (EC09).

[0119] Cultivation of recombinant strains and determination of L-carnosine synthesis:

[0120] Culture conditions: The constructed recombinant strain was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 8-12 hours as seed solution. Then the seed solution was inoculated into 50 mL of LB medium containing kanamycin at a 1% inoculum and cultured until OD 600 Reach 0.8~1.0.

[0121] Shake flask fermentation: Induce the expression of the strain by adding 1 mM IPTG and culture it at 37°C and 200 rpm for 12 hours to ensure efficient expression of the enzyme. The induced strain was inoculated into a 250 mL Erlenmeyer flask, and 10 g / L β-alanine and an appropriate amount of inorganic salts were added to the culture medium to ensure the supply of substrate. The shake flask fermentation conditions were 37°C and 200 rpm.

[0122] Sample collection and detection: 10 mL of samples were taken after 24 hours and 48 hours of fermentation, and the supernatant was separated by centrifugation (8000 rpm, 4°C, 10 minutes). The concentration of L-carnosine was quantitatively analyzed using high performance liquid chromatography (HPLC).

[0123] Experimental results:

[0124] By comparing the L-carnosine production of different strains under shake flask fermentation conditions (Table 2), the following results were observed.

[0125] Table 2: Effect of enhancement of histidine biosynthesis pathway on L-carnosine production (g / L)

[0126]

[0127] Compared with the control group, hisG overexpression significantly increased the intracellular accumulation of L-histidine, and the final L-carnosine production increased by 29.4%. S143F mutant: The production of L-carnosine increased by 35.3%, indicating that this mutation helps to enhance the synthesis of histidine. E217K mutant: The increase in L-carnosine production was about 17.6%, which was slightly lower than that of the S143F mutant. The EC08 mutant showed the best effect, with an increase of 41.1% in L-carnosine production. his operator multi-copy strain: This strain showed significant histidine accumulation ability, and the production of L-carnosine increased by about 35%.

[0128] Overall, the Δ209-281 mutant showed the best effect in enhancing the supply of L-histidine, and its L-carnosine synthesis output was significantly higher than that of other strains, reaching 2.4 g / L.

[0129] Example 3: Effect of β-alanine (β-Ala) biosynthesis pathway construction on L-carnosine production

[0130] Implementation steps: In this example, the biosynthetic pathway of β-alanine was constructed to increase the internal supply of β-alanine in the host bacteria, thereby increasing the synthesis yield of L-carnosine. Seven β-alanine synthase genes were screened, and the effect of their overexpression on the yield of L-carnosine was studied.

[0131] Plasmid construction and construction of recombinant strains:

[0132] β-Alanine synthetase gene cloning: Seven β-alanine synthetase genes BA01 (A8F5K4), BA02 (B5Y862), BA03 (Q60358), BA04 (Q5JJ82), BA05 (A0B9M9), BA06 (O27188), and BA07 (Q171S0) were cloned from different sources and inserted into the pGEX-5x plasmid through the BamHI and XhoI sites.

[0133] Plasmid verification: Sequencing is used to verify whether the constructed plasmid is successfully inserted into the target gene to ensure that there are no mismatches or mutations.

[0134] Construction of recombinant strains: The successfully constructed plasmids pGEX-5x-BA01 to pGEX-5x-BA07 were introduced into the EC08 strain, respectively, to obtain recombinant strains EC10 to EC16.

[0135] Cultivation of recombinant strains and determination of L-carnosine synthesis:

[0136] Culture conditions: The recombinant strain was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 8-12 hours as seed solution. Then the seed solution was inoculated into 50 mL of LB medium containing kanamycin at a 1% inoculum and cultured until OD 600 Reach 0.8~1.0.

[0137] Induced expression: The strain was induced by adding 1 mM IPTG and cultured at 16°C and 200 rpm for 12 hours to ensure efficient expression of the enzyme.

[0138] Shake flask fermentation: The induced strain was inoculated into a 250 mL Erlenmeyer flask, and 10 g / L of L-histidine and an appropriate amount of inorganic salts were added to the culture medium to ensure the supply of substrate. The shake flask fermentation conditions were 37°C and 200 rpm. Each strain was fermented three times for testing.

[0139] Sample collection and detection: 10 mL of samples were taken after 24 hours and 48 hours of fermentation, and the supernatant was separated by centrifugation (8000 rpm, 4°C, 10 minutes). The concentration of L-carnosine was quantitatively analyzed using high performance liquid chromatography (HPLC).

[0140] The experimental results are as follows Figure 1 As shown. By comparing the L-carnosine yields of strains EC10~EC16 under shake flask fermentation conditions, the following results were obtained: by constructing the biosynthetic pathway of β-alanine and screening efficient β-alanine synthase, the present invention significantly increased the yield of L-carnosine. Among them, the EC13 strain (overexpressing BA04) performed best, indicating that the BA04 enzyme has a significant promoting effect on the biosynthesis of β-alanine, thereby further improving the synthesis efficiency of L-carnosine. This provides a new approach and option for subsequent large-scale production.

[0141] Example 4: Effect of Optimizing the Substrate Transport System on L-Carnosine Production

[0142] Implementation steps: In this example, the substrate transport system was optimized to increase the yield of L-carnosine. Using CRISPR / Cas9 technology, the yeaS and cycA genes were integrated into the flgG site of the EC13 strain, and recombinant strains that overexpressed yeaS alone (EC17), overexpressed cycA alone (EC18), and overexpressed yeaS and cycA together (EC19) were constructed, and the effects of these strains on the yield of L-carnosine in shake flask fermentation were evaluated.

[0143] Gene integration and construction of recombinant strains:

[0144] Gene integration: Using the CRISPR / Cas9 system, the yeaS and cycA genes were integrated into the flgG site of the EC13 strain. First, the yeaS (AJE56230.1) and cycA (ACT45869.1) genes were obtained by PCR amplification and integrated into the flgG site by homologous recombination to obtain the recombinant strains EC17 (single integration of yeaS), EC18 (single integration of cycA), and EC19 (combined integration of yeaS and cycA).

[0145] Sequencing verification: Sequencing is used to verify whether the integrated gene has been successfully inserted to ensure the accuracy of gene integration.

[0146] Cultivation of recombinant strains and determination of L-carnosine synthesis:

[0147] Culture conditions: The recombinant strain was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 8-12 hours as seed solution. Then the seed solution was inoculated into 50 mL of LB medium containing kanamycin at a 1% inoculum and cultured until OD 600 Reach 0.8~1.0.

[0148] Shake flask fermentation: The strain was induced to express by adding 1 mM IPTG and cultured at 16°C and 200 rpm for 12 hours. 10 g / L β-alanine and appropriate amount of inorganic salts were added to the culture medium to ensure the supply of substrate. The shake flask fermentation conditions were 37°C and 200 rpm. Each strain was fermented three times in parallel for testing.

[0149] Sample collection and detection: 10 mL of samples were taken after 24 hours and 48 hours of fermentation, and the supernatant was separated by centrifugation (8000 rpm, 4°C, 10 minutes). The concentration of L-carnosine was quantitatively analyzed using high performance liquid chromatography (HPLC).

[0150] The experimental results are as follows Figure 2By optimizing the substrate transport system, especially by integrating yeaS and cycA genes, this embodiment significantly increased the production of L-carnosine. Among them, the EC19 strain performed best, indicating that the synergistic effect of yeaS and cycA has a significant effect in improving substrate transport efficiency and increasing the utilization rate of substrates by cells, thereby further improving the synthesis efficiency of L-carnosine ( Figure 2 ).

[0151] Example 5: Optimization of fed-batch fermentation strategy and improvement of L-carnosine production

[0152] Implementation steps: This example optimizes the fed-batch fermentation process to improve the L-carnosine production efficiency of the E. coli recombinant strain. The specific operation steps are as follows:

[0153] 1. Selection of starting strain:

[0154] In this example, the metabolically engineered high-efficiency L-carnosine producing strain EC18 was selected for fermentation experiments.

[0155] 2. Fermentation conditions and medium preparation:

[0156] Fermentation vessel: 2.5 L fermenter;

[0157] Initial working volume: 1.5 L.

[0158] Basic culture medium (M9 medium), the components are as follows:

[0159] KH2PO4: 13.3 g / L;

[0160] (NH4)2HPO4: 4 g / L;

[0161] MgSO4·7H2O: 1.2 g / L;

[0162] Glucose: 20 g / L;

[0163] Nicotinamide (NAM): 5 g / L;

[0164] Inducer lactose: added at a concentration of 1% during the fermentation process.

[0165] The initial pH was adjusted to 6.8, the culture temperature was set to 37 °C, the stirring speed was 300 rpm, and the aeration rate was set to 1.5 vvm.

[0166] 3. Fermentation process:

[0167] Seed culture: EC18 strain was inoculated into 10 mL LB medium and cultured at 37°C and 200 rpm for 12 h as seed solution.

[0168] 5% (volume ratio) of the seed solution was inoculated into a 2.5 L fermenter, and the initial culture medium volume was 1.5 L.

[0169] Initial fermentation stage: The fermenter was cultured at 37°C, 300 rpm, and 1.5 vvm. The glucose concentration was 20 g / L and the β-Ala concentration was 10 g / L.

[0170] When the glucose in the fermenter was exhausted (approximately 6 hours), the fed-batch phase was initiated.

[0171] Fed-batch phase: Feed solution composition: 600 g / L glucose, 60 g / L yeast extract, 60 g / L tryptone.

[0172] Feeding strategy: Phase 1 (6-12 hours): The feeding rate is 10 mL / h to ensure rapid cell proliferation while maintaining the dissolved oxygen level above 40%.

[0173] Phase 2 (12-24 h): The feed rate was adjusted to 20 mL / h to increase the synthesis rate of carnosine while controlling acetate accumulation.

[0174] Stage 3 (24 hours later): When OD 600 When the pH value reached above 150, the feeding rate was maintained at 20 mL / h, and the expression of genes related to L-carnosine synthesis was induced by adding 10 g / L lactose.

[0175] Sample collection: 10 mL of sample was taken every 5 h after the start of fermentation.

[0176] The samples were centrifuged (8000 rpm, 4°C, 10 min) to separate the supernatant, which was then filtered through a 0.22 μm PTFE filter membrane for L-carnosine concentration detection.

[0177] 4. L-Carnosine concentration detection method:

[0178] High performance liquid chromatography (HPLC, Younglin Inc. Korea) was used to quantify the concentration of carnosine. An Eclipse plus C18 column (4.6 × 250 mm, 5 μm) was used with a mobile phase of 10% acetonitrile at a flow rate of 1 mL / min and a detection wavelength of 260 nm. The concentration of L-carnosine was quantitatively analyzed based on the standard curve.

[0179] The experimental results are as follows Figure 3 By analyzing the production data of EC16 strain under two fermentation strategies, the results showed that the production of fed-batch fermentation strategy could reach 21.3 g / L within 50 hours.

[0180] Conclusion: This example shows that by optimizing the fed-batch fermentation process, the L-carnosine production efficiency of the E. coli recombinant strain can be significantly improved. The fed-batch fermentation strategy of the present invention has a wide range of application prospects in the industrial-scale L-carnosine production, which can significantly reduce the production cost and increase the L-carnosine yield.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the enzyme with NCBI accession number WP_034652116 in synthesizing L-carnosine or improving the synthesis rate of L-carnosine.

2. A genetically engineered bacterium, characterized in that: include: (A1), adding the gene of the enzyme with NCBI accession number WP_034652116; (A2), hisG with Δ209-281 mutation; (A3), adding the gene of the enzyme with UniProt accession number Q5JJ82; (A4), adding yeaS gene and cycA gene.

3. The genetically engineered bacterium according to claim 2, characterized in that: Include: (B1), an expression vector having a gene for an enzyme with NCBI accession number WP_034652116; and / or (B2) An expression vector having a gene for an enzyme with UniProt accession number Q5JJ82.

4. The genetically engineered bacterium according to claim 2 or 3, characterized in that: The chassis is E. coli.

5. The genetically engineered bacterium according to claim 4, characterized in that The adding of the yeaS gene and the cycA gene comprises: integrating the yeaS gene and the cycA gene into the flgG site.

6. Use of the genetically engineered bacteria as claimed in any one of claims 2 to 5 in the synthesis of L-carnosine.

7. A method for synthesizing L-carnosine, characterized in that: include: The genetically engineered bacteria according to any one of claims 2 to 5 are subjected to fed-batch fermentation.

8. The synthesis method according to claim 7, characterized in that The following steps are involved: S1. Inoculate the seed liquid of the genetically engineered bacteria according to any one of claims 2 to 5 into a culture medium, and culture at a temperature of 37°C, a rotation speed of 300 rpm, and a ventilation volume of 1.5 vvm; S2. When the glucose in the culture medium is exhausted, feed at a rate of 10 mL / h and continue culturing for 6 to 12 hours; S3, change the feed rate to 20 mL / h and continue culturing for 12-24 hours; S4. When OD 600 When the concentration reaches above 150, lactose is induced and fermented to obtain L-carnosine.

9. The synthesis method according to claim 8, characterized in that include: (C1), the culture medium comprising: 13.3 g / L KH2PO4, 4 g / L (NH4)2HPO4, 1.2 g / L MgSO4·7H2O, 20 g / L glucose and 5 g / L nicotinamide (NAM); and / or (C2) The feeding solution used in the feeding comprises: 600 g / L glucose, 60 g / L yeast extract and 60 g / L tryptone.

10. A method for increasing L-carnosine production, characterized in that: include: (D1) synthesizing L-carnosine based on the enzyme with NCBI accession number NZ_CP058353 to increase the yield of L-carnosine; or (D2) overexpressing the hisG gene in the L-carnosine producing strain to increase the L-carnosine production; or (D3) introducing S143F mutation, E217K mutation or Δ209-281 mutation into the hisG gene of the L-carnosine producing strain to increase the L-carnosine production; or (D4) expressing multiple copies of the his operon in the strain producing L-carnosine to increase the yield of L-carnosine; or (D5) introducing a gene of an enzyme with UniProt accession number A8F5K4, B5Y862, Q60358, Q5JJ82, A0B9M9, O27188 or Q171S0 into a strain producing L-carnosine to increase the yield of L-carnosine; or (D6) Adding the yeaS gene and the cycA gene to the L-carnosine producing strain can increase the L-carnosine production.

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