An engineered bacterium for producing s-adenosylmethionine-rich single-cell protein and a construction method and application thereof

Abstract

This disclosure provides an engineered bacterial strain for producing S-adenosylmethionine (SAM)-rich single-cell protein, its construction method, and its applications, relating to the field of microbial metabolic engineering technology. Using *Tricholoma mater* as a host, the invention seamlessly integrates the PgroE1-metK-rrnB expression cassette into the neutral gene region [5,315,702-5,317,309] bp of the *Tricholoma mater* chromosome. Marker-free, stable genetically engineered bacteria are obtained through double-crossover homologous recombination and two rounds of screening. Heat-induced, staged expression is employed to achieve synergistic effects between bacterial growth and efficient SAM accumulation. This disclosure is applicable to the feed industry and can be used to produce SAM-rich, nutritionally fortified single-cell protein raw materials, showing promising application prospects in livestock, poultry, and aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering technology, and specifically relates to an engineered bacterium that produces single-cell protein rich in S-adenosylmethionine, its construction method, and its application. Background Technology

[0002] Single-cell protein (SCP), also known as microbial cell protein, is a microbial protein product made from inexpensive raw materials such as industrial and agricultural waste, sugars, and one-carbon compounds through large-scale microbial fermentation of bacteria, yeasts, molds, and microalgae. The resulting cell bodies are harvested, dried, and processed. In addition to protein, the cell bodies are rich in amino acids, vitamins, nucleic acids, and minerals. Currently, the core microbial strains used in SCP production are primarily yeasts, including *Saccharomyces cerevisiae* and *Candida utilis*; bacteria include *Clostridium ethanolans* and methyltrophic bacteria (such as *Methylobacterium tumefaciens*). Methylorubrum extorquens SCP contains Bacillus subtilis, as well as some molds and microalgae such as Spirulina and Chlorella. SCP is primarily used in the feed industry, widely replacing soybean meal and fishmeal as a protein raw material for livestock, poultry, and aquaculture. A small amount, after nucleic acid removal and refining, can be used as a food protein additive and nutritional fortifier. It also has some applications in biofertilizers and microbial preparations.

[0003] S-Adenosylmethionine (SAM) is a key methyl donor and sulfur metabolism intermediate in organisms. It participates in the methylation modification of proteins and nucleic acids during animal metabolism, and plays a crucial role in immune function regulation, gut health maintenance, and growth performance enhancement, making it a highly valuable nutritional fortifier in animal feed. Currently, industrial production utilizes high-density fermentation with Saccharomyces cerevisiae to prepare pharmaceutical-grade SAM salts by adding L-methionine precursors, metabolic regulation, and downstream separation and purification. This technology is mature and has concentrated production capacity. Research-stage processes include in vitro enzymatic catalysis, whole-cell catalysis, and synthetic biology-modified fermentation, aiming to improve conversion rates and reduce production costs.

[0004] Among the chassis strains synthesized by SAM, *Saccharomyces cerevisiae* is the only industrially produced strain, while *Pichia pastoris*, *Yersinia lipolytica*, *Corynebacterium glutamicum*, and *Escherichia coli* are the main strains studied for chassis modification. The methyltrophic bacterium *Typhonometabolum* (…) Methylorubrum extorquens While it has a theoretical advantage in utilizing a single carbon source, it is limited by multiple shortcomings, including insufficient methionine and ATP precursors, vigorous endogenous degradation of SAM, formaldehyde metabolic stress, poor intracellular stability, bacterial endotoxin defects, high difficulty in modification, and high overall cost. In addition, since Saccharomyces cerevisiae has established a mature industrial production system, Twisting Methylbacillus can only be used as a niche chassis for basic metabolic research and does not have the application potential for industrial production of SAM.

[0005] Furthermore, in methyltrophic bacteria, there is significant metabolic antagonism between cell growth and SCP synthesis, as well as intracellular SAM enrichment. Carbon flux allocation, precursor energy supply, and endogenous metabolic priority all compete with each other. The fermentation regulation conditions required by both are completely opposite and cannot be adapted synergistically, thus making it difficult to achieve simultaneous maximum production. Summary of the Invention

[0006] This invention provides an engineered bacterium for producing S-adenosylmethionine (SAM)-rich single-cell protein, its construction method, and its applications. Using *Tricholoma mater* as a chassis, this invention constructs a staged expression regulation system based on the heat-responsive promoter PgroE1. Combined with optimized chromosomal integration sites and a traceless site-specific integration strategy, it enhances the accumulation level of SAM within the bacteria while ensuring the host's ability to utilize methanol for growth and the bacterial cell's growth capacity. This results in a SAM-rich functionalized single-cell protein product to meet the demand for high-value-added, nutritionally fortified feed ingredients in animal husbandry.

[0007] On the one hand, the present invention provides an engineered bacterium for producing single-cell proteins rich in S-adenosylmethionine, wherein the engineered bacterium is obtained by inserting the metK gene into the genome of Bacillus thuringiensis as the starting strain;

[0008] The sequence of the metK gene is shown in SEQ ID NO:1.

[0009] Furthermore, the engineered bacteria were constructed by site-specific integration of the metK gene expression cassette into the chromosome of *Tricholoma matrophilia* using homologous recombination.

[0010] Furthermore, the site of targeted integration is the neutral intergenic region of the *Thunb. methyltrimonium* chromosome [5,315,702-5,317,309] bp.

[0011] Furthermore, the nucleotide sequence of the neutral intergenic region of the *Thunb. methyltrimonium* chromosome [5,315,702-5,317,309] bp is shown in SEQ ID NO:12.

[0012] Furthermore, the metK gene expression cassette includes a heat-inducible promoter PgroE1 and a terminator rrnB.

[0013] Furthermore, the PgroE1 originates from the upstream regulatory region of the groEL1 / groESL1 operon, and its nucleotide sequence is shown in SEQ ID NO:2; the rrnB terminator sequence is shown in SEQ ID NO:3.

[0014] On the other hand, the present invention provides a method for constructing engineered bacteria rich in S-adenosylmethionine single-cell protein, comprising the following steps: (1) The PgroE1 promoter, metK coding sequence, and rrnB terminator are sequentially connected to form an integrated expression box: PgroE1–metK–rrnB; (2) An upstream homologous arm and a downstream homologous arm are set on both sides of the expression box to form an integrated fragment: upstream homologous arm – PgroE1 – metK – rrnB – downstream homologous arm; (3) Using the broad-host seamless integration vector pCM433 as the vector backbone, the obtained integration fragments were assembled into a vector to construct a recombination integration vector for chromosome site-directed seamless integration. (4) The recombinant integration vector was introduced into the host strain of *Thunb.*; (5) By combining double crossover homologous recombination with two rounds of screening, the metK gene expression cassette was site-directedly integrated into the neutral intergenic region [5,315,702-5,317,309] bp of the chromosome of *Thizoctonia solani*, and a traceless integration positive engineered bacteria was obtained by screening. Furthermore, the upstream homologous arm sequence is shown in SEQ ID NO:10, and the downstream homologous arm sequence is shown in SEQ ID NO:11.

[0015] In another aspect, the present invention provides the application of the above-mentioned engineered bacteria in the production of single-cell protein rich in S-adenosylmethionine.

[0016] Furthermore, the above-mentioned method involves culturing the engineered bacteria at a temperature of 30°C during the cell growth stage; once the cells have grown to the OD... 600 When the value is greater than 3.0, the culture conditions are switched to a heat induction temperature of 37°C to obtain a functionalized single-cell protein product rich in SAM.

[0017] Compared with the prior art, this application has the following advantages: This invention uses *Thunbergia nigra* as a host and constructs a PgroE1-driven target expression cassette. A stable engineered strain is obtained by integrating the cassette at a specific site. Further, staged heat-induced culture is employed to enhance target gene expression, resulting in a high-yield single-cell protein product rich in S-adenosylmethionine (SAM). Compared to the wild-type strain, the SAM content is increased by 17.02 times, and the yield of the single-cell product is increased by 23.87%. The SAM-rich single-cell protein product obtained by this invention can be used as a protein raw material or as a functional nutritional fortification ingredient in animal feed systems, and is suitable for further development of functional feed ingredients for aquatic animals, poultry, or livestock. Attached Figure Description

[0018] Figure 1The image shows the spectrum of the recombinant plasmid pBBR1-DW5.1. Figure 2 The standard curve for quantitative detection of SAM (Logistic nonlinear regression fitting); Figure 3 The image shows the spectrum of the recombinant plasmid pCM172-DW5.1. Figure 4 This is a map of the recombinant plasmid pCM433-DW5.5. Detailed Implementation

[0019] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. The components of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0020] The industrial production of S-adenosylmethionine (SAM) by *Thizobium truncatum* suffers from the following technical defects: 1. Insufficient precursor supply. As a C1 methyltrophic bacterium, this bacterium faces limited supply of L-methionine and ATP precursors, with carbon metabolism preferentially allocated to the cell proliferation pathway, failing to meet the substrate requirements for SAM synthesis; 2. Vigorous intracellular degradation of SAM. The high activity and short half-life of endogenous SAM hydrolases lead to rapid consumption of the synthesized product and low accumulation; 3. Formaldehyde metabolic stress. Formaldehyde intermediates produced during methanol metabolism trigger oxidative stress, inhibiting the expression of the key enzyme in SAM synthesis (MetK); 4. Poor intracellular stability of the product. SAM is prone to non-enzymatic degradation, and bacterial endotoxin control is difficult, resulting in high product separation and purification costs and insufficient process reproducibility. These technical defects lead to low SAM synthesis yields by this strain, making it difficult to meet the requirements of industrial production. The high difficulty in modification and high overall cost, among other shortcomings, have resulted in the lack of research reports on the synthesis of SAM using *Thizobium truncatum*.

[0021] Furthermore, *Methylobacterium tumefaciens* faces metabolic antagonism when used as a single-cell protein carrier for SAM co-production: methanol carbon source competes for allocation between bacterial protein synthesis and SAM synthesis pathways, with L-methionine and ATP precursors preferentially meeting proliferation needs, leading to insufficient supply of SAM synthesis substrates; the high specific growth rate and nitrogen-enriched culture conditions required for high-yielding cells conflict with the nitrogen-limiting stress and precursor enhancement required for SAM enrichment, manifesting as nitrogen-enriched culture promoting growth but low SAM accumulation, while adding precursors inhibits growth, and high dissolved oxygen and high pH enhancing biomass inhibits SAM. SAM proliferation is halted due to nitrogen-limited induction of synthetic enzyme activity; in addition, formaldehyde metabolism causes oxidative stress, and endogenous SAM degradation enzymes accelerate product consumption, making it impossible to achieve synergistic yield increase for both objectives through conventional fermentation regulation; existing strain modification is constrained by the complexity of the methyl metabolism network and the instability of exogenous gene expression, resulting in high costs and poor process reproducibility. Furthermore, the short intracellular half-life of SAM, the difficulty in controlling endotoxins, and the complexity of separation and purification limit the large-scale application of feed protein additives; there is an urgent need to reconstruct the metabolic network through precise genome modification to coordinate the metabolic flux distribution between single-cell protein synthesis and SAM enrichment.

[0022] In this regard, this application provides an engineered bacterium that produces single-cell protein rich in S-adenosylmethionine. The engineered bacterium is obtained by inserting the metK gene into the genome of Bacillus thuringiensis as the starting strain. The sequence of the metK gene is shown in SEQ ID NO:1.

[0023] The *Thiazariae* strain described in this invention, strain number DSM-1338, was purchased from the German Microbiological and Cell Culture Collection (DSMZ).

[0024] Specifically, to obtain engineered bacteria capable of producing single-cell proteins rich in S-adenosylmethionine, *Tricholoma mater* can be selected as the host strain. *Tricholoma mater* was chosen for its unique ability to utilize one-carbon compounds; it can efficiently grow using inexpensive carbon sources such as methanol, thus providing an economic basis for single-cell protein production. In practice, wild-type strains of *Tricholoma mater* or strains that have undergone preliminary domestication or adaptive evolution can be selected as the starting strain.

[0025] Subsequently, to enrich SAM in *Typhonium tumefaciens*, the metK gene needs to be introduced into its genome. The metK gene encodes SAM synthase, a key rate-limiting enzyme in the SAM synthesis pathway. Inserting this gene into the genome enhances the strain's endogenous SAM synthesis capacity. Various strategies can be employed for gene insertion. For example, transposon-mediated random insertion can be used to randomly integrate the metK gene into the *Typhonium tumefaciens* genome. Alternatively, a linear DNA fragment containing the metK gene can be constructed, and the gene can be integrated into a specific region of the genome using the host strain's own homologous recombination mechanism. These insertion methods aim to stably introduce the metK gene into the genetic material of *Typhonium tumefaciens*, enabling it to be inherited during strain replication.

[0026] The sequence of the metK gene is shown in SEQ ID NO:1. This sequence was determined based on the screening and optimization of highly efficient SAM synthase genes. In the actual construction process, the metK gene can be obtained through gene synthesis technology based on the nucleotide sequence shown in SEQ ID NO:1, or obtained from known organisms containing this gene through molecular biology methods such as PCR amplification. After obtaining the gene, it is ligated with appropriate expression elements (such as promoters and terminators) to form a complete expression cassette, which is then inserted into the genome of *Tricholoma matsutake*.

[0027] Furthermore, this invention discovered that recombinant *Thiazora truncatella* strains corresponding to different candidate sites exhibited differences in specific growth rate, final biomass, and intracellular SAM levels under the same fermentation and induction conditions. After comparing and screening multiple candidate sites, the applicant selected the neutral intergenic region [5,315,702-5,317,309] bp of *Thiazora truncatella* chromosome as the integration site. This site can achieve good target expression cassette carrying, target gene expression, and target product accumulation while taking into account both host growth and methanol utilization, and therefore was identified as the integration site of this invention.

[0028] The following examples will provide a more detailed explanation of the above technical solutions: Example 1: Pre-validation scheme for expression boxes This embodiment uses a preliminary free expression validation protocol to rapidly verify the initiation and expression effects of the PgroE1-metK expression cassette, as well as its SAM accumulation and enhancement capabilities. This provides an expression basis for subsequent chromosome integration modification and is not intended as the final industrial production strain construction process. The pBBR1MCS-2 vector used in this embodiment is a broad-host-range free plasmid, which possesses broad-host replication capabilities and a kanamycin resistance selection marker.

[0029] (1) Using the genomic DNA of *Thizoctonia solani* as a template, specific primers PgroE1-pBBR-F and metK-pBBR-R were designed based on the multiple cloning site of the vector. The metK gene coding fragment corresponding to SEQ ID NO:1 and the PgroE1 promoter fragment corresponding to SEQ ID NO:2 were amplified by high-fidelity DNA polymerase. The amplified products were purified and recovered after being identified by gel electrophoresis.

[0030] (2) The purified PgroE1 promoter fragment was seamlessly linked to the metK gene coding sequence by overlap PCR technology to assemble a complete PgroE1–metK fusion expression cassette.

[0031] (3) The PgroE1–metK fusion expression cassette and the pBBR1MCS-2 vector were subjected to double enzyme digestion. The digestion products were recovered and ligated to construct the recombinant expression plasmid pBBR1MCS-2-PgroE1-metK (named pBBR1-DW5.1, plasmid map as shown). Figure 1 As shown in the figure, the PgroE1 promoter is located upstream of the metK gene and achieves functional tandem.

[0032] (4) The ligation product was transformed into an Escherichia coli clone host for plasmid amplification. The sequence of recombinant plasmid elements, insertion direction and metK gene sequence were confirmed to be completely correct by colony PCR, restriction endonuclease digestion analysis and bidirectional sequencing.

[0033] (5) The recombinant plasmid that has passed the sequencing verification is introduced into the original host strain of *Thiago methylobacterium* to obtain a free expression engineered bacterium carrying the PgroE1-metK expression cassette.

[0034] (6) The control strain and the recombinant strain were inoculated into a shake flask culture system containing methanol carbon source and corresponding screening antibiotics, respectively, and cultured with shaking at the conventional culture temperature of 30℃ for 50 h until the bacterial cells grew to the OD. 600 When the OD value was greater than 3.0, a portion of the samples was taken as pre-induction samples; subsequently, the remaining culture system was transferred to 37℃ for further induction culture for 6 h, and samples were taken after induction as post-induction samples. The control strain was named CK-1, and the recombinant strain was named OE-a. The OD values ​​of each sample were measured. 600 The bacterial cells were collected and lysate was prepared. The intracellular SAM concentration was detected using the S-Adenosylmethionine (SAM) ELISA Kit.

[0035] The specific steps for SAM testing are as follows: Take 10 mL of the sample before induction and 10 mL of the sample after induction, and centrifuge at 4 °C to collect the bacterial cell pellet. Discard the supernatant and resuspend the bacterial cell pellet in 10 mL of phosphate-buffered saline (PBS) to obtain a bacterial cell resuspension. Place the bacterial cell resuspension in an ice bath for ultrasonic disruption. After disruption, centrifuge at 4 °C and collect the supernatant as the sample solution to be tested.

[0036] The content of S-adenosylmethionine in the test sample solution was determined by competitive enzyme-linked immunosorbent assay (ELISA). The test sample solutions were prepared at 1×10⁻⁶... 4 times, 3×10 4 times, 5×10 4 times, 1×10 5 times, 1.5×10 5 Doubled to 2×10 5 Perform gradient dilution, and if necessary, further adjust the dilution factor based on the test results to ensure that the response value of the test sample falls within the effective range of the standard curve.

[0037] A series of standard solutions were prepared using S-adenosylmethionine (SAM) standard, with standard curve concentrations of 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 nM. Microplates coated and blocked with SAM conjugate were used for detection. 50 μL of standard solution or test sample was added to each well, and the plate was incubated at room temperature with shaking for 10 min. Then, 50 μL of diluted anti-SAM antibody was added to each well, and the plate was incubated at room temperature with shaking for 1 h. After washing, 100 μL of diluted HRP-labeled secondary antibody was added to each well, and the plate was incubated at room temperature with shaking for 1 h. After washing again, 100 μL of chromogenic substrate was added to each well, and the plate was incubated at room temperature. Finally, 100 μL of stop solution was added to terminate the reaction, and the absorbance was measured at 450 nm. Parallel replicates were prepared for the standard solution, blank control, and test sample.

[0038] According to the standard curve of the same plate (e.g.) Figure 2 Calculate the S-adenosylmethionine concentration (nM) in each diluted sample, and multiply it by the corresponding dilution factor to obtain the concentration C in the original lysate. O (nM), that is:

[0039] Based on the relative molecular mass of S-adenosylmethionine (398.44), the mass concentration C of S-adenosylmethionine in the original lysate is... m (g / L) is:

[0040] The results showed that the intracellular SAM concentration of the recombinant strain OE-a was higher than that of the control strain CK-1 before and after induction, and further increased after induction at 37℃ for 6 h. This indicates that the constructed PgroE1–metK–rrnB free expression system can promote the accumulation of intracellular SAM in the host. The results are shown in Table 1 below.

[0041] Table 1: Effects of free expression system (pBBR1-DW5.1) on the growth of *Bacillus thuringiensis* and intracellular SAM accumulation.

[0042] Example 2: Construction of engineered bacteria using pCM172 as a carrier framework (1) Constructing the PgroE1–metK–rrnB integrated expression box Using *Thunb. genomic DNA* as a template, the PgroE1 promoter fragment and metK coding sequence fragment were amplified; the rrnB terminator fragment was amplified using a vector or reference sequence as a template.

[0043] Specific primers with overlapping complementary sequences were designed, and the PgroE1 promoter, metK coding sequence, and rrnB terminator were sequentially spliced ​​together using overlap extension PCR technology to form a complete PgroE1–metK–rrnB integrated expression cassette.

[0044] The spliced ​​products were identified by agarose gel electrophoresis, and the target fragment was purified and recovered using a gel recovery kit.

[0045] (2) Constructing a pCM172 recombination and integration vector The pCM172 vector backbone used in this embodiment pre-carries the upstream and downstream homologous arm sequences of the M. extorquens katA gene, which can mediate the site-specific insertion of exogenous fragments into the katA site on the M. extorquens chromosome via homologous recombination, thereby achieving site-specific integration into the genome.

[0046] The pCM172 vector backbone was linearized by single or double enzyme digestion, and the linearized vector fragment was purified and recovered.

[0047] The integrated fragment obtained in step (1) was seamlessly cloned and ligated with the linearized pCM172 vector, and the ligation product was transformed into an E. coli cloning host.

[0048] The culture was plated on LB agar plates containing tetracycline and incubated overnight at 35°C. Single colonies were picked, and colony PCR, enzyme digestion verification, and sequencing analysis confirmed the correct construction of the recombinant vector. The PgroE1–metK–rrnB expression cassette in the recombinant vector was correctly inserted between the upstream and downstream homologous arms of katA, yielding the recombinant integrative vector pCM172-integrated fragment (named pCM172-DW5.1, plasmid map as shown). Figure 3 (As shown).

[0049] (3) Introduce the recombinant integration vector into Bacillus thuringiensis The recombinant integrative vector, which was verified by sequencing, was introduced into competent cells of *Thunb. methyl* using electroporation.

[0050] Specifically, the original strain of *Methylobacterium tumefaciens* was first inoculated into a defined mineral salt medium for *Methylobacterium tumefaciens* without antibiotics, and activated using succinate as a carbon source. After the cells reached the logarithmic growth phase, they were collected and washed with sterile water and glycerol solution at low temperature to prepare electroporation competent cells. The pCM172-DW5.1 recombinant integrative vector was mixed with the competent cells and electroporated. Immediately after electroporation, antibiotic-free succinate resuscitation medium was added, and the cells were resuscitated at 30°C.

[0051] After resuscitation, the bacterial culture was plated on tetracycline-containing succinate-defined mineral salt solid medium and incubated at 30°C until single colonies formed. Candidate single colonies capable of growing on tetracycline-selective plates were selected and further purified by streaking on tetracycline-containing succinate solid medium to obtain candidate transformants with consistent genetic backgrounds. Subsequently, using the candidate transformants as templates, PCR identification was performed using primers for identifying the expression cassette interior, the integration site boundary, and the wild-type site control, confirming that the PgroE1-metK-terminator expression cassette had been inserted into the expected chromosomal region, and that the insertion direction, connection boundary, and expression cassette sequence were correct. Following the above screening and identification, the pCM172-DW5.1-mediated *Methylobacterium truncatum* chromosome insertion engineered strain was obtained.

[0052] (4) Measurement of SAM expression level The control strain and recombinant strain were inoculated into shake flask culture systems containing methanol carbon source and corresponding screening antibiotics, respectively, and cultured with shaking at standard culture temperature for 50 h until the bacterial cells reached OD. 600 When the OD value was greater than 3.0, a portion of the samples was taken as pre-induction samples; subsequently, the remaining culture system was transferred to 37℃ for further induction culture for 6 h, and samples were taken after induction as post-induction samples. The control strain was named CK-1, and the recombinant strain was named OE-1. The OD values ​​of each sample were measured.600 The bacterial cells were collected and lysate was prepared. The intracellular SAM concentration was detected using the S-Adenosylmethionine (SAM) ELISA Kit.

[0053] Example 3: Shake-flask culture to verify the effect of different candidate sites on the target expression cassette carrying capacity. (1) Constructing the PgroE1–metK–rrnB integrated expression box Using *Thunb. genomic DNA* as a template, the PgroE1 promoter fragment and metK coding sequence fragment were amplified; the rrnB terminator fragment was amplified using a vector or reference sequence as a template.

[0054] Design specific primers with overlapping complementary sequences, and sequentially splice the PgroE1 promoter, metK coding sequence, and rrnB terminator using overlap extension PCR or seamless cloning technology to form a complete PgroE1–metK–rrnB integrated expression cassette.

[0055] The spliced ​​products were identified by agarose gel electrophoresis, and the target fragment was purified and recovered using a gel recovery kit.

[0056] (2) Constructing an integrated fragment containing homologous arms Using *Thunb. genomic DNA* as a template, fragments of the upstream homologous arms (SEQ ID NO: 4, 6, 8, 10) and the downstream homologous arms (SEQ ID NO: 5, 7, 9, 11) were amplified.

[0057] Through seamless cloning or multi-round PCR splicing, the upstream homologous arm, the integration expression cassette, and the downstream homologous arm are assembled sequentially to form a complete upstream homologous arm –PgroE1 –metK –rrnB – downstream homologous arm integrated fragment.

[0058] Purify and recover the full-length integrated fragment, ensuring no contamination from impurities.

[0059] (3) Constructing a pCM433 recombination and integration vector The pCM433 vector backbone was linearized by single or double enzyme digestion, and the linearized vector fragment was purified and recovered.

[0060] The integrated fragment obtained in step (2) was seamlessly cloned and ligated with the linearized pCM433 vector, and the ligation product was transformed into the E. coli cloning host.

[0061] The culture was plated on LB agar plates containing chloramphenicol and incubated overnight at 35°C. Single colonies were picked, and colony PCR, enzyme digestion verification, and sequencing analysis confirmed the correct construction of the recombinant vector, yielding the recombinant integrative vector pCM433-integration fragment, named as follows: pCM433-DW5.2, pCM433-DW5.3, pCM433-DW5.4, and pCM433-DW5.5. The plasmid map of pCM433-DW5.5 is shown below. Figure 4 As shown.

[0062] (4) Introduce the recombinant integration vector into Bacillus thuringiensis The recombinant integrative vector, which was verified by sequencing, was introduced into competent cells of *Thunb. methyl* using electroporation.

[0063] Competent cells of *Typhonium methylobacterium* were prepared, mixed with a recombinant vector, and then electroporated and revived before being plated on a selective plate.

[0064] Incubate at 30℃ until a single colony grows to obtain a single-exchange strain with the recombinant vector introduced (the vector backbone has been integrated into the chromosome).

[0065] (5) Two rounds of screening combined with double crossover homologous recombination were used to obtain positive engineered bacteria without leaving a trace. The *Methylobacterium tumefaciens* strains introduced into the recombinant integration vector underwent two rounds of screening—a first round of positive selection and a second round of sacB negative selection—combined with double crossover homologous recombination to achieve seamless integration. The first round was a preliminary positive screening of integrons, using a positive selection marker carried by the pCM433 vector for resistance selection, identifying candidate integrons that underwent the first round of single crossover homologous recombination. These strains had the vector backbone and selection marker inserted entirely into their genomic locus. PCR molecular identification of the integration junction region verified that the vector and expression cassette had integrated near the target chromosomal locus. The single crossover candidate strains that passed the first round of identification were passaged without resistance to induce the second round of double crossover homologous recombination. A second round of negative selection was then performed using the sacB sucrose reverse selection marker, killing strains that retained the vector backbone and surviving only those that underwent double crossover recombination. After double crossover, the strains were divided into revertant strains that restored the original genome and target seamless integration strains that retained the metK expression cassette but lost the vector backbone. Finally, the integration site region was amplified by PCR and sequenced for verification. The PgroE1-metK expression cassette was successfully integrated into the target site on the chromosome, the vector backbone and selection markers were completely removed, and the sequence structure was intact. The construction results are shown in Table 2.

[0066] Table 2: Construction of Recombination and Integration Vectors at Different Chromosomal Loci

[0067] (6) Measurement of SAM expression level The control strain and recombinant strain were inoculated into shake flask culture systems containing methanol carbon source and corresponding screening antibiotics, respectively, and cultured with shaking at a standard culture temperature of 30℃ for 50 h until the bacterial cells reached OD. 600 When the OD value was greater than 3.0, a portion of the samples were taken as pre-induction samples; subsequently, the remaining culture system was transferred to 37℃ for further induction culture for 6 h, and samples were taken after induction as post-induction samples. The control strain was named CK-1, and the recombinant strains were named OE-2~OE-5. The OD values ​​of each sample were measured. 600 The bacterial cells were collected and lysate was prepared. The intracellular SAM concentration was detected using the S-Adenosylmethionine (SAM) ELISA Kit. The results are shown in Table 3.

[0068] Table 3: Growth and intracellular SAM concentration of recombinant bacteria at different integration sites during shake-flask culture

[0069] Example 4: Fermentation in a fermenter to verify the growth capacity and SAM yield of strains corresponding to different candidate sites. To compare the effects of different candidate integration sites on the growth capacity and S-adenosylmethionine (SAM) accumulation capacity of engineered bacteria, recombinant strains that completed site-directed integration of the PgroE1-metK expression cassette at different candidate sites were selected and fed-batch cultured in a 5 L fermenter to evaluate their growth characteristics and differences in product levels.

[0070] The strains used in this embodiment include: OE-1: Recombinant strain (pCM172-DW5.1 recombinant strain); OE-5: Recombinant strain (pCM433-DW5.5 recombinant strain).

[0071] (1) Fermentation culture conditions Each recombinant strain was inoculated into seed culture medium and cultured to the logarithmic growth phase. Then, it was transferred to a 5 L fermenter at the same inoculation rate for further cultivation. The fermentation medium was an inorganic salt medium with methanol as the main carbon source. A fed-batch method was used to maintain methanol supply during cultivation to support cell growth and the synthesis of the target product. All strains were cultured in parallel under the same process conditions to ensure comparability between strains corresponding to different candidate sites.

[0072] (2) Monitoring of growth process and calculation of specific growth rate During fermentation, samples were taken periodically to measure cell growth. Each strain entered the logarithmic growth phase after the lag phase, preferably when the cell growth reached the OD value.600 After approximately 1, continuous sampling begins, and the specific growth rate of each strain is calculated based on the growth data during the logarithmic growth phase. Simultaneously, the OD of each strain at the final placement in the container is recorded. 600 This study aims to evaluate the impact of different candidate sites on the overall growth capacity and biomass accumulation capacity of the strain.

[0073] (3) Thermal induction and sampling After all strains were cultured uniformly for 36 hours, the fermentation temperature was raised to 37°C for heat induction culture. The heat induction lasted for 6 hours before being transferred to the fermentation tank.

[0074] Samples were taken before heating and induction, serving as pre-induction samples; samples were taken again when the samples were removed from the container after induction, serving as post-induction samples. The OD of each sample was measured. 600 The bacterial cells were collected for determination of intracellular SAM content.

[0075] (4) SAM content detection Bacterial samples were collected before and after induction, and cell lysates were prepared. Intracellular SAM levels were detected using an S-Adenosylmethionine (SAM) ELISA Kit. The results were expressed as SAM concentration in the cell lysates. Changes in SAM levels before and after induction were compared among strains corresponding to different candidate sites to evaluate the impact of different integration sites on the accumulation capacity of the target product.

[0076] (5) Results and Analysis Different candidate sites corresponding to engineered strains exhibited varying growth characteristics and product accumulation levels under 5L fermenter conditions. The specific growth rate and OD results at the bottom of the fermenter showed differences in cell growth capacity among strains corresponding to each site. Furthermore, a comparison of SAM levels before and after induction further indicated differences in the carrying capacity of the PgroE1-metK expression cassette and the accumulation capacity of the target product at different integration sites. The strains corresponding to the preferred sites performed better in balancing growth and product accumulation, demonstrating that the selection of chromosome integration sites has a significant impact on the implementation effect of the technical solution of this invention. The detection results are shown in Table 4 below: Table 4: Growth and SAM yield of recombinant strains with different integration sites in the fermenter

[0077] The results of this embodiment show that recombinant strains corresponding to different candidate sites exhibit differences in specific growth rate, final biomass, and intracellular SAM levels under the same fermentation and induction conditions. Among them, strains corresponding to the preferred site achieve higher SAM levels and better induction responses without significantly affecting host growth, indicating that the selection of the preferred integration site plays a crucial role in effectively carrying the PgroE1-driven expression cassette, maintaining strain growth capacity, and promoting the accumulation of the target product. These results further validate the effectiveness of the technical route of "candidate site screening—preferred site determination—seamless site-specific integration—thermal induction to enhance expression" in this invention.

[0078] Experimental results showed that when the neutral intergenic region of the *Methylobacterium tumefaciens* chromosome [5,315,702-5,317,309] bp was selected as the integration site, the maximum biomass and specific growth rate of the resulting metK-integrated engineered bacteria during fermentation were 10² and 0.178, respectively, and the SAM yield after heat induction reached 393.1 mg / L. This confirms that the selected chromosomal integration site is a safe and neutral site, and that the site-directed integration and induced expression of the metK gene will not adversely affect the host's normal growth and methanol utilization metabolism, meeting the basic requirements for bacterial growth in the preparation of functional single-cell proteins. Simultaneously, it significantly increases the amount of SAM synthesized, making it suitable for producing SAM-rich single-cell proteins.

[0079] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An engineered bacterium that produces single-cell protein rich in S-adenosylmethionine, characterized in that, The engineered bacteria were obtained by inserting the metK gene into the genome of *Methylobacterium truncatum* as the starting strain. The nucleotide sequence of the metK gene is shown in SEQ ID NO:1; The integration site of the metK gene is the neutral intergenic region of the *Thunb. methyltrimonium* chromosome [5,315,702-5,317,309] bp. The nucleotide sequence of the neutral intergenic region of the [5,315,702-5,317,309]bp chromosome of the *Thunb.* chromosome is shown in SEQ ID NO:

12.

2. The engineered bacteria according to claim 1, characterized in that, The engineered bacteria were constructed by site-specific integration of the metK gene expression cassette into the chromosome of *Thunb. methyl* using homologous recombination.

3. The engineered bacteria according to claim 2, characterized in that, The metK gene expression cassette contains the promoter PgroE1, which is a heat-inducible promoter capable of inducing upregulation of metK gene expression at 35-37℃. The PgroE1 is derived from the upstream regulatory region of the groEL1 / groESL1 operon, and its nucleotide sequence is shown in SEQ ID NO:

2.

4. The engineered bacteria according to claim 2, characterized in that, The metK gene expression cassette contains the rrnB terminator, which is a bidirectional transcription terminator.

5. The engineered bacteria according to claim 2, characterized in that, The homologous recombination uses pCM433 as the vector backbone.

6. A method for constructing engineered bacteria rich in S-adenosylmethionine single-cell protein, characterized in that, Includes the following steps: (1) Connect the PgroE1 promoter, metK coding sequence, and rrnB terminator in sequence to form an integrated expression box: PgroE1–metK–rrnB; (2) An upstream homologous arm and a downstream homologous arm are set on both sides of the expression box to form an integrated fragment: upstream homologous arm – PgroE1 – metK – rrnB – downstream homologous arm; (3) Using the broad-host seamless integration vector pCM433 as the vector backbone, the obtained integration fragments were assembled into a vector to construct a recombination integration vector for chromosome site-directed seamless integration. (4) The recombinant integration vector was introduced into the host strain of *Thunb.*; (5) By combining double crossover homologous recombination with two rounds of screening, the metK gene expression cassette was site-directedly integrated into the neutral intergenic region [5,315,702-5,317,309] bp of the chromosome of *Thizoctonia solani*, and a traceless integration positive engineered bacteria was obtained by screening. The upstream homologous arm sequence is shown in SEQ ID NO:10, and the downstream homologous arm sequence is shown in SEQ ID NO:

11.

7. The use of the engineered bacteria of claim 1 or 2 in the production of single-cell protein rich in S-adenosylmethionine.

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