A rothia mucilaginosa engineering strain for synthesizing aminomethyl phosphonic acid by utilizing co2 and a construction method thereof

Abstract

This invention discloses an engineered strain of *Rochelys rochleinii* that synthesizes aminomethylphosphonic acid using CO2 and its construction method, belonging to the field of metabolic engineering technology. This invention utilizes the knockout of key components in the synthesis of polyhydroxybutyrate (PHB). phaCAB The gene cluster of *D. rochleinii*, a true eutroph, was the starting strain, which synthesized aminomethylphosphonic acid. alp Gene clusters were disassembled and constructed into pBAD and pSEVA551 plasmids, and introduced into the host in a dual-plasmid co-expression manner to obtain a recombinant engineered bacterium capable of synthesizing aminomethylphosphonic acid. Based on this, a multi-module synergistic strategy involving metabolic pathway enhancement, cofactor optimization, and global regulatory factor modification was employed to improve CO2 conversion efficiency, integrating various optimization modules to obtain a highly efficient engineered bacterium. This engineered bacterium can synthesize aminomethylphosphonic acid using CO2 as the sole carbon source, achieving a yield of 200 mg / L under optimized fermentation conditions.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering technology, specifically relating to an engineered strain of Rochecholesterolus that synthesizes aminomethylphosphonic acid using CO2 and its construction method. Background Technology

[0002] Glyphosate is the most widely used broad-spectrum herbicide globally and holds the largest market share among pesticides. Traditional chemical synthesis suffers from environmental pollution and high energy consumption, making green biomanufacturing a current research hotspot. Aminomethylphosphonate (AMP) is a key precursor to glyphosate, differing from it only by a carboxylmethyl group. Currently, heterologous biosynthesis of AMP in *Streptomyces lividans* has been reported, achieving a yield of 52 mg / L. However, using *Streptomyces* as a production host has several limitations: ① *Streptomyces* is a heterotrophic microorganism, requiring organic carbon sources such as glucose as substrates, resulting in high raw material costs and competition with food production; ② *Streptomyces* has a long growth cycle, typically requiring 3-5 days from spore germination to product synthesis, requiring improvements in fermentation efficiency; ③ *Streptomyces* has a complex genetic background and a large genome (approximately 8 Mb), making genetic manipulation relatively difficult and limiting the depth and breadth of metabolic engineering. Therefore, exploring microbial hosts more suitable for industrial production and realizing green bio-manufacturing of glyphosate using CO2, a third-generation biorefining raw material, as a carbon source is key to promoting the practical application of this technology.

[0003] *Cupriavidus necator* H16, a true aerotrophic bacterium, possesses strong carbon metabolism plasticity and a promising foundation for industrial applications, making it an ideal chassis cell for achieving microbial CO2 fixation and the synthesis of high-value chemicals. *C. necator* H16 efficiently fixes CO2 and synthesizes organic matter through the Calvin-Benson-Bassham (CBB) cycle. Under autotrophic culture conditions, the specific growth rate of *C. necator* H16 can reach 0.2–0.3 h⁻¹. -1 C. necator H16 is one of the fastest-growing chemoautotrophic bacteria known. Its strong central carbon metabolism capacity provides favorable conditions for the construction of heterologous metabolic pathways. Currently, C. necator cell factories that convert CO2 into various high-value-added products have been developed, including alcohols, fatty acids, isoprene-like compounds, food additives, and drug precursors. Based on these advantages, a Roche chemoautotrophic engineered strain was constructed using synthetic biology strategies to efficiently synthesize the glyphosate precursor aminomethylphosphonic acid using CO2 as the sole carbon source, laying a solid foundation for the green and sustainable biomanufacturing of aminomethylphosphonic acid. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide an engineered strain of Rochelys rochelys that synthesizes aminomethylphosphonic acid using CO2, as well as its construction method and application, providing a new route for the carbon-neutral biomanufacturing of aminomethylphosphonic acid and laying a theoretical foundation for the green transformation of the glyphosate industry.

[0005] The technical solution provided by this invention is as follows:

[0006] A method for constructing an engineered strain of *Rochelys oryzae* that synthesizes aminomethylphosphonic acid using CO2, comprising the following steps:

[0007] (1) Using Rochefoucauld as the chassis host bacteria, the phaCAB gene cluster was knocked out by homologous recombination technology to obtain the recombinant host bacteria;

[0008] (2) The first recombinant plasmid carrying the alpKL gene cluster and the second recombinant plasmid carrying the alpGHIJ gene cluster or the alpGoptHIJ gene cluster were introduced into the recombinant host bacteria to obtain the engineered strain of Roche's true trophic bacteria that synthesizes aminomethylphosphonic acid using CO2.

[0009] The alpKL gene cluster includes the alpK gene and the alpL gene in sequence along the 5'→3' direction; the alpGHIJ gene cluster includes the alpG gene, the alpH gene, the alpI gene, and the alpJ gene in sequence along the 5'→3' direction; and the alpGoptHIJ gene cluster includes the alpGopt gene, the alpH gene, the alpI gene, and the alpJ gene in sequence along the 5'→3' direction.

[0010] The nucleotide sequences of the alpG, alpH, alpI, alpJ, alpK, alpL, and alpGopt genes are shown in SEQ ID NO.1 to SEQ ID NO.7, respectively.

[0011] Furthermore, the first recombinant plasmid mentioned above uses pBAD plasmid as its backbone, and the second recombinant plasmid mentioned above uses pSEVA551 plasmid as its backbone.

[0012] The nucleotide sequences of pBAD plasmid and pSEVA551 plasmid are shown in SEQ ID NO.8~SEQ ID NO.9, respectively.

[0013] Furthermore, the first recombinant plasmid also carries at least one of the following genes: mdh, gdh, pgam, and eno.

[0014] The nucleotide sequences of the mdh, gdh, pgam, and eno genes are shown in SEQ ID NO.10 to SEQ ID NO.13, respectively.

[0015] Furthermore, the first recombinant plasmid also carries at least one of the following genes: rpoN1, rpoN2, rpoS, uspA, and fnr.

[0016] The nucleotide sequences of the rpoN1, rpoN2, rpoS, uspA, and fnr genes are shown in SEQ ID NO.14 to SEQ ID NO.18, respectively.

[0017] Furthermore, the first recombinant plasmid mentioned above carries a long polycistronic fragment composed of the alpKL gene cluster, pgam gene, eno gene, mdh gene, and rpoN1 gene; the element arrangement order of the long polycistronic fragment along the 5'→3' direction is: alpKL gene cluster, RBS1, pgam gene, RBS2+linker, eno gene, RBS2+linker, mdh gene, RBS2+linker, and rpoN1 gene;

[0018] The nucleotide sequence of RBS1 is 5'-tttaagaaggagatatacat-3', and the nucleotide sequence of RBS2+ linker is 5'-AAAGGAGGACAACC-3'.

[0019] Furthermore, step (1) above also includes the step of knocking out the glnA gene in the recombinant host bacteria;

[0020] The nucleotide sequence of the glnA gene is shown in SEQ ID NO.19.

[0021] An engineered strain of Rochechoi pseudotrophic bacteria obtained by the above construction method.

[0022] The above-mentioned engineered strain of Rochelys oryzae is used in the fermentation production of aminomethylphosphonic acid.

[0023] Furthermore, the above application specifically involves: inoculating the engineered strain of *Rochelys rochelys* into MSM medium containing 10 g / L fructose for activation culture, then transferring it to a sealed culture flask containing fresh MSM medium, introducing syngas to completely replace the air in the flask, purging at a relative pressure of 11 kPa, and fermenting at 30°C and 160 rpm.

[0024] Furthermore, the syngas mentioned above is a mixture of CO2, H2, and O2 in a volume ratio of 1:7:2; the fresh MSM culture medium mentioned above contains L-glutamate and / or NAD. + .

[0025] The significant advantages of this invention are:

[0026] This invention successfully constructed a highly efficient engineered *Rochelys rochelys* strain that synthesizes aminomethylphosphonic acid (AMPA) using CO2 as the sole carbon source through modular assembly and systematic optimization of the heterologous synthesis pathway. Under optimized fermentation conditions, the engineered strain achieved an AMPA yield of 200 mg / L, realizing direct biotransformation from inorganic carbon to organic phosphonic acid. This invention provides a feasible technical route for producing AMPA, a key precursor of glyphosate, using microbial CO2 fixation, and is of great significance for the development of a green circular economy. Attached Figure Description

[0027] Figure 1 The engineered Roche propagules Reh01 to Reh06 utilize CO2 as the sole carbon source to ferment and synthesize aminomethylphosphonic acid. 31 P NMR spectrum.

[0028] Figure 2 Schematic diagram of the construction of engineered true trophic bacteria Reh07 to Reh16.

[0029] Figure 3 The engineered fungus Reh16, a true trophic bacterium, utilizes CO2 as the sole carbon source to ferment and synthesize aminomethylphosphonic acid. 31 P NMR spectrum.

[0030] Figure 4 Structural diagram of recombinant plasmid pb-alpKL-pgam-eno-mdh-rpoN1.

[0031] Figure 5 Structural diagram of recombinant plasmid p5-alpGoptHIJ. Detailed Implementation

[0032] To facilitate understanding of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0033] The genes and gene clusters of this invention can be obtained by PCR amplification using the primers described in the text, or they can be directly synthesized artificially using nucleotide sequences listed in the sequence listing.

[0034] The formulation of the LB liquid culture medium of the present invention is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH adjusted to 7.0.

[0035] The formulation of the LB solid culture medium of the present invention is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, and pH adjusted to 7.0.

[0036] The formulation of the MSM culture medium of this invention is as follows: KH2PO4 2.3 g / L, Na2HPO4·12H2O 7.3 g / L, NH4Cl 1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.1 g / L, Fe(NH4)3(C6H5O7)2 0.05 g / L, trace element solution 1 mL / L, adjusted pH to 7.0. The trace element solution formulation is as follows: ZnSO4·7H2O 0.10 g / L, MnCl2·4H2O 0.03 g / L, CoCl2·6H2O 0.20 g / L, CuCl2·2H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, NaMoO4·2H2O 0.03 g / L, H3BO3 0.30 g / L.

[0037] The *Cupriavidus necator* DSM428 of this invention was purchased from the German National Culture Collection Center (DSMZ).

[0038] In the following embodiments, after fermentation, the fermentation broth was collected by centrifugation, and the supernatant was collected. The obtained supernatant was transferred to the evaporation flask of a rotary evaporator, the circulating water vacuum pump was turned on, the vacuum degree was controlled at -0.08~-0.095MPa, the water bath temperature was set at 30℃, the rotation speed was 80~120rpm, and the concentration time was 1~3h until all water was evaporated. The concentrated liquid was collected for later use. 31 P nuclear magnetic resonance spectroscopy (P nuclear magnetic resonance spectroscopy) 31 P NMR was used to quantitatively detect aminomethylphosphonic acid in the concentrate.

[0039] Example 1:

[0040] (1) The alpGHIJ, alpGHIJKL, and alpKL gene clusters originated from *Streptomyces monomycini* NRRL B-24309. In the genome of this strain, the six genes alpG, alpH, alpI, alpJ, alpK, and alpL are arranged in a continuous cis configuration. The alpGHIJ gene cluster contains the first four genes alpG, alpH, alpI, and alpJ in the 5'→3' direction. The alpGHIJKL gene cluster covers a complete continuous segment and contains all six genes alpG, alpH, alpI, alpJ, alpK, and alpL in the 5'→3' direction. The alpKL gene cluster only contains the last two continuous genes alpK and alpL. The alpGopt gene was obtained by optimizing the alpG gene for codon bias in *Streptomyces monomycini*. The alpKLGHIJ gene cluster is obtained by rearranging the gene sequence based on the alpGHIJKL gene cluster, and its 5'→3' direction contains six genes: alpK, alpL, alpG, alpH, alpI, and alpJ. The alpGoptHIJ gene cluster is obtained by replacing the alpG gene with the alpGopt gene based on the alpGHIJ gene, and its 5'→3' direction contains four genes: alpGopt, alpH, alpI, and alpJ. The nucleotide sequences of the alpG, alpH, alpI, alpJ, alpK, and alpL genes are shown in SEQ ID NO.1~SEQ ID NO.6, and the nucleotide sequence of the alpGopt gene is shown in SEQ ID NO.7.

[0041] The alpGHIJKL, alpKLGHIJ, and alpKL gene clusters were cloned into the pBAD plasmid to construct recombinant plasmids pb-alpGHIJKL, pb-alpKLGHIJ, and pb-alpKL (hereinafter, pb-alpKL is abbreviated as pb-KL). These gene clusters are located downstream of the araBAD promoter and are linked to the araBAD promoter via the RBS1 sequence inherent in the plasmid, with expression driven by the araBAD promoter. The alpGHIJKL, alpGHIJ, and alpGoptHIJ gene clusters were cloned into the pSEVA551 plasmid to construct recombinant plasmids p5-alpGHIJKL, p5-alpGHIJ, and p5-alpGoptHIJ (hereinafter, p5-alpGoptHIJ is abbreviated as p5-GoptHIJ). These gene clusters are located downstream of the PHAC1 promoter and are linked to the PHAC1 promoter via the RBS sequence inherent in the plasmid, with expression driven by the PHAC1 promoter. Note: The prefix pb in plasmid naming represents vector pBAD, and p5 represents vector pSEVA551. The nucleotide sequence of pBAD plasmid is shown in SEQ ID NO.8, and the nucleotide sequence of pSEVA551 plasmid is shown in SEQ ID NO.9.

[0042] (2) Using the true trophic bacterium Cupriavidus necator DSM428 as the chassis host bacterium, the phaCAB gene cluster (GenBank accession number MH558939.1) was knocked out using homologous recombination technology to obtain the engineered bacterium Reh△CAB. The recombinant plasmid constructed in step (1) was transformed into the engineered bacterium Reh△CAB through a parental conjugation method. The specific steps are as follows: using E. coli S17-1 λpir carrying the recombinant plasmid as the donor bacterium and Reh△CAB as the recipient bacterium, the OD... 600 Donor and recipient bacterial cultures with a pH of 2-3 were mixed at a 1:1 volume ratio, centrifuged at 4000 rpm for 10 min to collect the cells, and the supernatant was discarded. The cells were washed twice with LB liquid medium and resuspended, then added dropwise to LB solid medium plates lined with sterile filter paper and co-cultured at 30°C for 24 h. All bacterial growth on the plates was collected and resuspended in sterile ultrapure water, then serially diluted to 10⁻⁶. -2 The plasmids were then evenly spread onto LB agar plates containing 450 μg / mL kanamycin and 40 μg / mL gentamicin, and incubated upside down at 30°C for approximately 48 h. After single colonies grew, plasmids were extracted from the strains. The pBAD series recombinant plasmids were identified using primer pair YP2M-F / YP2M-R. Positive strains amplified a specific fragment of approximately 5000 bp, while negative strains showed no amplification band. The pSEVA551 series recombinant plasmids were identified using primer pair YP5-F / YP5-R. Positive strains amplified a specific fragment of approximately 5000 bp, while negative strains showed no amplification band.

[0043] The following engineered bacteria were finally obtained: Reh△CAB(pb-alpGHIJKL) carrying the recombinant plasmid pb-alpGHIJKL, named Reh02; Reh△CAB(p5-alpGHIJKL) carrying the recombinant plasmid p5-alpGHIJKL, named Reh03; Reh△CAB(p5-alpKLGHIJ) carrying the recombinant plasmid p5-alpKLGHIJ, named Reh04; Reh△CAB(pb-KL+p5-alpGHIJ) carrying both recombinant plasmids pb-KL and p5-alpGHIJ, named Reh05; and Reh△CAB(pb-KL+p5-GoptHIJ) transformed with both recombinant plasmids pb-KL and p5-GoptHIJ, named Reh06. The engineered strain Reh△CAB(pb+p5), which co-carries the empty vector pBAD and the empty vector pSEVA551, was used as a blank control strain and named Reh01.

[0044] (3) To verify the ability of the above-mentioned engineered bacteria to synthesize aminomethyl phosphate using CO2 as the sole carbon source, each engineered bacterium was first inoculated into MSM medium containing 10 g / L fructose and activated at 30°C and 160 rpm with shaking until the OD value was reached. 600 The value was 3.0; 4 vol% inoculum was transferred to a 500 mL sealed culture flask containing 50 mL of fresh MSM medium. Syngas with a ratio of V(CO2):V(H2):V(O2) = 1:7:2 was introduced to completely replace the air in the flask, maintaining a relative pressure of 11 kPa. Fermentation was carried out at 30°C and 160 rpm for 120 h; the aeration and pressurization process was repeated every 12 h during fermentation. After fermentation, the supernatant was collected and processed using… 31 P nuclear magnetic resonance spectroscopy (P nuclear magnetic resonance spectroscopy) 31 Quantitative determination of aminomethyl phosphate yield using P-NMR. Results showed that the target product aminomethyl phosphate was not detected in engineered bacteria Reh01, Reh02, Reh03, and Reh04; however, it was detected in engineered bacteria Reh05 and Reh06, with yields of 25.82 mg / L and 37.07 mg / L, respectively. The aminomethyl phosphate synthesis capacity of Reh06 was significantly superior to that of Reh05. This demonstrates that the present invention can successfully construct a complete pathway for the synthesis of aminomethyl phosphate using CO2 in *Rochetomyces*.

[0045] Example 2:

[0046] (1) Cofactor optimization: The engineered strain Reh06 was inoculated into MSM medium containing 10 g / L fructose and activated at 30℃ and 160 rpm to OD. 600The value was 3.0; at an inoculum rate of 4 vol%, the cells were transferred to fresh MSM medium supplemented with different exogenous substances. The experimental groups were supplemented with 1 mM NAD. + The fermentation vessel, gas mixture ratio, pressure, temperature, rotation speed, fermentation time, gas exchange cycle, and product detection method were all consistent with step (3) of Example 1, using 2 mM pyridoxal phosphate (PLP), 5 mM L-glutamic acid, and 5 mM L-aspartic acid. 31 P-NMR quantitative analysis showed that NAD + The aminomethyl phosphate yields for the group, PLP group, L-glutamate group, and L-aspartate group were 40.05 mg / L, 23.99 mg / L, 49.45 mg / L, and 33.36 mg / L, respectively. Exogenous NAD+ supplementation... + L-glutamic acid can significantly promote the synthesis of aminomethyl phosphate by the Reh06 strain.

[0047] (2) Metabolic pathway enhancement: The mdh, gdh, pgam, and eno genes are derived from Cupriavidusnecator H16, and their nucleotide sequences are shown in SEQ ID NO.10~SEQ ID NO.13.

[0048] The mdh and gdh genes were spliced ​​with the alpKL gene cluster to obtain polycistronic fragments alpKL-mdh and alpKL-gdh. alpKL-mdh contains the alpKL gene cluster, the RBS1 sequence (5'-tttaagaaggagatatacat-3'), and the mdh gene along the 5'→3' direction; alpKL-gdh contains the alpKL gene cluster, the RBS1 sequence (5'-tttaagaaggagatatacat-3'), and the gdh gene along the 5'→3' direction. The two polycistronic fragments were cloned into the pBAD plasmid to obtain recombinant plasmids pb-KL-mdh and pb-KL-gdh. The polycistronic fragments are located downstream of the araBAD promoter and are linked to the araBAD promoter via the RBS1 sequence inherent in the plasmid, and their expression is driven by the araBAD promoter.

[0049] The pgam and eno genes were spliced ​​with the alpKL gene cluster to construct the polycistronic fragment alpKL-pgam-eno. alpKL-pgam-eno contains, along the 5'→3' direction, the alpKL gene cluster, the RBS1 sequence (5'-tttaagaaggagatatacat-3'), the pgam gene, the RBS2+ linker sequence (5'-AAAGGAGGACAACC-3'), and the eno gene. This polycistronic fragment was cloned into the pBAD plasmid to construct the recombinant plasmid pb-KL-pgam-eno. The polycistronic fragment is located downstream of the araBAD promoter and is linked to the araBAD promoter via the plasmid's own RBS1 sequence, with expression driven by the araBAD promoter.

[0050] (3) Global regulatory factor modification: The rpoN1, rpoN2, rpoS, uspA, and fnr genes are derived from the true trophozoite Cupriavidus necator H16, and their nucleotide sequences are shown in SEQ ID NO.14~SEQ ID NO.18. Each gene was spliced ​​with the alpKL gene cluster to obtain polycistronic fragments alpKL-rpoN1, alpKL-rpoN2, alpKL-rpoS, alpKL-uspA, and alpKL-fnr. Taking alpKL-rpoN1 as an example, it contains the alpKL gene cluster, the RBS1 sequence (5'-tttaagaaggagatatacat-3'), and the rpoN1 gene sequentially from 5' to 3'. The arrangement of the other polycistronic fragment elements is completely consistent.

[0051] The polycistronic fragments were cloned into the pBAD plasmid to construct recombinant plasmids pb-KL-rpoN1, pb-KL-rpoN2, pb-KL-rpoS, pb-KL-uspA, and pb-KL-fnr. The polycistronic fragments are located downstream of the araBAD promoter and are linked to the araBAD promoter by the RBS1 sequence inherent in the plasmid, and are expressed by the araBAD promoter.

[0052] (4) Referring to the parental conjugation transformation method described in Example 1, the pBAD series recombinant plasmids constructed in steps (2) and (3) of this example were introduced into the basic engineered bacterium Reh06 to obtain a series of dual-plasmid co-expression engineered bacteria: Reh06 (pb-KL-mdh+p5-GoptHIJ) carrying both pb-KL-mdh and p5-GoptHIJ, named Reh07; Reh06 (pb-KL-gdh+p5-GoptHIJ) carrying both pb-KL-gdh and p5-GoptHIJ, named Reh08; Reh06 (pb-KL-pgam-eno+p5-GoptHIJ) carrying both pb-KL-pgam-eno and p5-GoptHIJ, named Reh09; and Reh06 (pb-KL-rpoN1 and p5-GoptHIJ) carrying both pb-KL-rpoN1 and p5-GoptHIJ. The strain carrying pb-KL-rpoN1+p5-GoptHIJ was named Reh10; the engineered strain carrying pb-KL-rpoN2 and p5-GoptHIJ was named Reh06(pb-KL-rpoN2+p5-GoptHIJ), and the engineered strain carrying pb-KL-rpoS and p5-GoptHIJ was named Reh11; the engineered strain carrying pb-KL-rpoS and p5-GoptHIJ was named Reh06(pb-KL-rpoS+p5-GoptHIJ), and the engineered strain carrying pb-KL-uspA and p5-GoptHIJ was named Reh12; the engineered strain carrying pb-KL-uspA and p5-GoptHIJ was named Reh06(pb-KL-uspA+p5-GoptHIJ), and the engineered strain carrying pb-KL-fnr and p5-GoptHIJ was named Reh13; the engineered strain carrying pb-KL-fnr and p5-GoptHIJ was named Reh06(pb-KL-fnr+p5-GoptHIJ), and the engineered strain carrying pb-KL-fnr and p5-GoptHIJ was named Reh14.

[0053] Based on Reh△CAB, the glnA gene (SEQ ID NO.19) was knocked out using homologous recombination technology to obtain the engineered strain Reh△CAB△glnA. Following the parental conjugation transformation method described in Example 1, the recombinant plasmids pb-KL-gdh and p5-GoptHIJ were co-transformed into Reh△CAB△glnA, and the resulting engineered strain was named Reh15.

[0054] (5) Activate and transfer all engineered bacteria from Reh07 to Reh15 for fermentation, including the following parameters: culture medium, inoculum size, mixed gas ratio, pressure, temperature, rotation speed, fermentation cycle, and aeration operation. 31The P-NMR detection methods were consistent with step (3) of Example 1. The results showed that the aminomethylphosphonic acid yield of Reh07 was 61.96 mg / L; the aminomethylphosphonic acid yields of Reh08 and Reh15 were 33.96 mg / L and 35.27 mg / L, respectively; the aminomethylphosphonic acid yields of Reh09, Reh10, Reh12, and Reh14 were 62.67 mg / L, 50.05 mg / L, 37.28 mg / L, and 36.68 mg / L, respectively; and aminomethylphosphonic acid was not detected in Reh11 and Reh13.

[0055] Example 3:

[0056] The pgam, eno, mdh, and rpoN1 genes were spliced ​​with the alpKL gene cluster to construct the long polycistronic fragment alpKL-pgam-eno-mdh-rpoN1; the element arrangement order along the 5'→3' direction is as follows: alpKL gene cluster, RBS1 sequence (5'-tttaagaaggagatatacat-3'), pgam gene, RBS2+ linker sequence (5'-AAAGGAGGACAACC-3'), eno gene, RBS2+ linker sequence (5'-AAAGGAGGACAACC-3'), mdh gene, RBS2+ linker sequence (5'-AAAGGAGGACAACC-3'), and rpoN1 gene. The complete polycistronic fragment was cloned into the pBAD plasmid to construct the recombinant plasmid pb-KL-pgam-eno-mdh-rpoN1. The fragment is located downstream of the araBAD promoter and is linked to the araBAD promoter by the RBS1 sequence inherent in the plasmid, and its expression is driven by the araBAD promoter.

[0057] Referring to the biparental conjugation transformation method described in Example 1, the recombinant plasmid pb-KL-pgam-eno-mdh-rpoN1 was transformed into the engineered strain Reh09, and the resulting engineered strain was named Reh16.

[0058] Reh16 was inoculated into MSM medium containing 10 g / L fructose and activated at 30°C and 160 rpm to OD. 600 The value was 3.0; 4 vol% inoculum was transferred to a 500 mL sealed culture flask containing 50 mL of fresh MSM medium supplemented with 5 mM L-glutamic acid. Syngas with a ratio of V(CO2):V(H2):V(O2) = 1:7:2 was introduced to completely replace the air in the flask, and the relative pressure was 11 kPa. Fermentation was carried out at 30℃ and 160 rpm for 120 h; the aeration and pressurization process was repeated every 12 h during fermentation. After fermentation, the supernatant was collected and processed using… 31P-NMR quantitatively determined the yield of aminomethyl phosphate. The results showed that Reh16 can efficiently synthesize the target product using CO2 as the sole carbon source, with an aminomethyl phosphate yield of up to 200 mg / L after 120 h of fermentation.

[0059] The primers used for the construction and identification of the recombinant plasmid are as follows:

[0060] Primers pb-GHIJKL-F / pb-GHIJKL-F were used to amplify alpGHIJKL in order to construct the recombinant plasmid pb-alpGHIJKL;

[0061] Primers phaC1-GHIJ-F / phaC1-GHIJ-R were used to amplify alpGHIJKL and alpGHIJ to construct recombinant plasmids p5-alpGHIJKL and p5-alpGHIJ;

[0062] Primers pb-KLGHIJ-F / KL-GHIJ-R were used to amplify alpKL, and primers KL-GHIJ-F / pb-KLGHIJ-R were used to amplify alpGHIJ, in order to construct the recombinant plasmid pb-alpKLGHIJ;

[0063] Primers pb-KLGHIJ-F / pb-GHIJKL-R were used to amplify alpKL in order to construct the recombinant plasmid pb-KL;

[0064] Primers pb-GoptHIJ-F / pb-GHIJ-R were used to amplify alpGoptHIJ to construct the recombinant plasmid p5-GoptHIJ;

[0065] Primers YP2M-F / YP2M-R were used for positive verification of pBAD series recombinant bacteria, and YP5-F / YP5-R were used for positive verification of pSEVA551 series recombinant bacteria.

[0066] Primers pb-KLGHIJ-F / KL-mdh-R were used to amplify alpKL, and primers KL-mdh-F / pb-mdh-R were used to amplify mdh, in order to construct the recombinant plasmid pb-KL-mdh;

[0067] Primers pb-KLGHIJ-F / KL-gdh-R were used to amplify alpKL, and primers KL-gdh-F / pb-gdh-R were used to amplify gdh, in order to construct the recombinant plasmid pb-KL-gdh;

[0068] Primers pb-KLGHIJ-F / KL-pgam-R were used to amplify alpKL, primers KL-pgam-F / pgam-eno-R were used to amplify pgam, and primers pgam-eno-F / pb-pgam-eno-R were used to amplify eno, in order to construct the recombinant plasmid pb-KL-pgam-eno;

[0069] Primers pb-KLGHIJ-F / KL-rpoN1-R were used to amplify alpKL, and primers KL-rpoN1-F / pb-rpoN1-R were used to amplify rpoN1, in order to construct the recombinant plasmid pb-KL-rpoN1;

[0070] Primers pb-KLGHIJ-F / KL-rpoN2-R were used to amplify alpKL, and primers KL-rpoN2-F / pb-rpoN2-R were used to amplify rpoN2 to construct the recombinant plasmid pb-KL-rpoN2.

[0071] Primers pb-KLGHIJ-F / KL-rpoS-R were used to amplify alpKL, and primers KL-rpoS-F / pb-rpoS-R were used to amplify rpoS, in order to construct the recombinant plasmid pb-KL-rpoS.

[0072] Primers pb-KLGHIJ-F / KL-uspA-R were used to amplify alpKL, and primers KL-uspA-F / pb-uspA-R were used to amplify uspA, in order to construct the recombinant plasmid pb-KL-uspA;

[0073] Primers pb-KLGHIJ-F / KL-FNR-R were used to amplify alpKL, and primers KL-FNR-F / pb-FNR-R were used to amplify fnr, in order to construct the recombinant plasmid pb-KL-fnr;

[0074] Primers pb-KLGHIJ-F / eno-mdh-R were used to amplify alpKL-pgam-eno, primers eno-mdh-F / mdh-rpoN1-R were used to amplify mdh, and primers mdh-rpoN1-F / pb-rpoN1-R were used to amplify rpoN1, in order to construct the recombinant plasmid pb-KL-pgam-eno-mdh-rpoN1.

[0075] pb-GHIJKL-R:5'-ctgagcctttcgttttatttgatgcAAGCTTtcaggggcgggtgagggc-3'

[0076] phaC1-GHIJ-F:5'-cggcagagagacaatcaaatctttaagaaggagatatacat-3'

[0077] phaC1-GHIJ-R:5'-gcaagcttgcatgcctgcagGTCGACacagataaaacgaaaggccc-3'

[0078] pb-KLGHIJ-F:5'-tttaagaaggagatatacatAAGCTTATGAACGCACAGCGTGTCCT-3'

[0079] KL-GHIJ-R:5'-gggtccggtacggacatggttgtcctccttttcaggggcgggtgagggccgtac-3'

[0080] KL-GHIJ-F:5'-acggccctcacccgcccctgaaaaggaggacaaccatgtccgtaccggaccctgcct-3'

[0081] pb-KLGHIJ-R:5'-cctttcgttttatttgatgcAAGCTTtcatcgcgcggcgccttccgtca-3'

[0082] pb-GoptHIJ-F:5'-tttaagaaggagatatacatAAGCTTATGAGCGTGCCGGACCCGGC-3'

[0083] pb-GHIJ-R:5'-gagcctttcgttttatttgatgcAAGCTTtcatcgcgcggcgcctt-3'

[0084] YP2M-F:5'-cccgccattcagagaagaaaccaat-3'

[0085] YP2M-R:5'-cgattcaggttcatcatgccgtttg-3'

[0086] YP5-F:5'-gataacaatttcacacaggaggc-3'

[0087] YP5-R:5'-aatcgtaattattggggacccctg-3'

[0088] KL-mdh-F:5'-cggccctcacccgcccctgaaaaggaggacaaccatggctaaagccccaatgcg-3'

[0089] KL-mdh-R:5'-cgcattggggctttagccatggttgtccctctttcggcgggtgagggcg-3'

[0090] pb-mdh-R:5'-cctttcgttttatttgatgcAAGCTTtcagcccagcaggtgctgca-3'

[0091] KL-gdh-F:5'-acggccctcacccgcccctgaaaaggaggacaaccgtgaacccatccagaaatc-3'

[0092] KL-gdh-R:5'-gatttctggatgggtttcacggttgtccctcctttcaggggcgggtgagggccgt-3'

[0093] pb-gdh-R:5'-cctttcgttttatttgatgcAAGCTTtcaggccgctgccttgccgt-3'

[0094] KL-pgam-F:5'-GTACGGCCCTCACCCGCCCCTGAtttaagaaggagatatacat-3'

[0095] KL-pgam-R:5'-atgtatatctccttcttaaaTCAGGGGCGGGTGAGGGCCGTAC-3'

[0096] pgam-eno-F:5'-gccagggcaaggcacgctgaaaaggacaaccatgagtgcaatcgtagatat-3'

[0097] pgam-eno-R:5'-atgatatctacgattgcactcatGGTTGTCCTCCTTTtcagcgtgccttgccctg-3'

[0098] pb-pgam-eno-R:5'-ccttcgttttatttgatgcAAGCTTtcagcgcaggttgtagaacg-3'

[0099] KL-rpoN1-F:5’-cggccctcacccgcccctgaaaaggagaacaaccatgaacttcaagatcagtgg-3’

[0100] KL-rpoN1-R:5’-ccactgatcttgaagttcatggttgtcctccttttcaggggcgggtgagggccg-3’

[0101] pb-rpoN1-R:5’-cctttcgttttatttgatgcAAGCTTtcattgctgcatttgcgctg-3’

[0102] KL-rpoN2-F:5’-cggccctcacccgcccctgaaaaggaggacaaccatgaaaccgtcgctacagct-3’

[0103] KL-rpoN2-R:5’-agctgtagcgacggtttcatggttgtcctccttttcaggggcgggtgagggccg-3’

[0104] pb-rpoN2-R:5’-cctttcgttttatttgatgcAAGCTTctacaaagacttgcggagat-3’

[0105] KL-rpoS-F:5’-cggccctcacccgcccctgaaaaggaggacaaccatgccacgccagaaaactgt-3’

[0106] KL-rpoS-R:5’-acagttttctggcgtggcatggttgtcctccttttcaggggcgggtgagggccg-3’

[0107] pb-rpoS-R:5’-cctttcgttttatttgatgcAAGCTTtcatagaacagcgtccttcc-3’

[0108] KL-uspA-F:5’-cggccctcacccgcccctgaaaaggaggacaaccatgttccaacatatcctgct-3’

[0109] KL-uspA-R:5'-agcaggatatgttggaacatggttgtccctcctttcaggggcgggtgagggcg-3'

[0110] pb-uspA-R:5'-cctttcgttttatttgatgcAAGCTTtcagcggtagacgatcaccg-3'

[0111] KL-FNR-F:5'-cggccctcacccgccctgaaaaggaggacaaccat gccggacagcggcgccc-3'

[0112] KL-FNR-R:5'-ggggcgccgctctcggcatggttgtccctcctttcggcgggtgagggcg-3'

[0113] pb-FNR-R:5'-cctttcgttttatttgatgcAAGCTTctaggccagccggggttcgc-3'

[0114] eno-mdh-F:5'-cgttctacaacctgcgctgaAAAGGAGGACAACCatggctaaagccccaatgcg-3'

[0115] eno-mdh-R:5'-cgcattggggctttagccatGGTTGTCCTCCTTTtcagcgcaggttgtagaacg-3'

[0116] mdh-rpoN1-F:5'-tgcagcacctgctgggctgaaaaaggacaaccatgaacttcaagatcagtgg-3'

[0117] mdh-rpoN1-R:5'-ccactgatcttgaagttcatggttgtccctcttccagcccagcaggtgctgca-3'

Claims

1. A method for constructing an engineered strain of *Rochelys oryzae* that synthesizes aminomethylphosphonic acid using CO2, characterized in that: Includes the following steps: (1) Using Rochefoucauld as the chassis host bacteria, the phaCAB gene cluster was knocked out by homologous recombination technology to obtain the recombinant host bacteria; (2) The first recombinant plasmid carrying the alpKL gene cluster and the second recombinant plasmid carrying the alpGHIJ gene cluster or the alpGoptHIJ gene cluster were introduced into the recombinant host bacteria to obtain the engineered strain of Roche's true trophic bacteria that synthesizes aminomethylphosphonic acid using CO2. The alpKL gene cluster includes the alpK gene and the alpL gene in sequence along the 5'→3' direction; the alpGHIJ gene cluster includes the alpG gene, the alpH gene, the alpI gene, and the alpJ gene in sequence along the 5'→3' direction; and the alpGoptHIJ gene cluster includes the alpGopt gene, the alpH gene, the alpI gene, and the alpJ gene in sequence along the 5'→3' direction. The nucleotide sequences of the alpG, alpH, alpI, alpJ, alpK, alpL, and alpGopt genes are shown in SEQ ID NO.1 to SEQ ID NO.7, respectively.

2. The construction method according to claim 1, characterized in that: The first recombinant plasmid uses pBAD plasmid as its backbone, and the second recombinant plasmid uses pSEVA551 plasmid as its backbone. The nucleotide sequences of pBAD plasmid and pSEVA551 plasmid are shown in SEQ ID NO.8~SEQ ID NO.9, respectively.

3. The construction method according to claim 1, characterized in that: The first recombinant plasmid also carries at least one of the following genes: mdh gene, gdh gene, pgam gene, and eno gene. The nucleotide sequences of the mdh, gdh, pgam, and eno genes are shown in SEQ ID NO.10 to SEQ ID NO.13, respectively.

4. The construction method according to claim 3, characterized in that: The first recombinant plasmid also carries at least one of the following genes: rpoN1, rpoN2, rpoS, uspA, and fnr. The nucleotide sequences of the rpoN1, rpoN2, rpoS, uspA, and fnr genes are shown in SEQ ID NO.14 to SEQ ID NO.18, respectively.

5. The construction method according to claim 4, characterized in that: The first recombinant plasmid carries a long polycistronic fragment composed of the alpKL gene cluster, pgam gene, eno gene, mdh gene and rpoN1 gene; the element arrangement order of the long polycistronic fragment along the 5'→3' direction is: alpKL gene cluster, RBS1, pgam gene, RBS2+linker, eno gene, RBS2+linker, mdh gene, RBS2+linker, rpoN1 gene; The nucleotide sequence of RBS1 is 5'-tttaagaaggagatatacat-3', and the nucleotide sequence of RBS2+ linker is 5'-AAAGGAGGACAACC-3'.

6. The construction method according to claim 1, characterized in that: Step (1) also includes the step of knocking out the glnA gene in the recombinant host bacteria; The nucleotide sequence of the glnA gene is shown in SEQ ID NO.

19.

7. An engineered strain of *Rochelys* obtained by the construction method according to any one of claims 1 to 6.

8. The application of the engineered strain of *Rochelys rochelys* as described in claim 7 in the fermentation production of aminomethylphosphonic acid.

9. The application according to claim 8, characterized in that: The engineered strain of *Rochelys rochelys* was inoculated into MSM medium containing 10 g / L fructose for activation and culture. Then it was transferred to a sealed culture flask containing fresh MSM medium, and syngas was introduced to completely replace the air in the flask. The relative pressure was 11 kPa, and fermentation was carried out at 30°C and 160 rpm.

10. The application according to claim 9, characterized in that: The synthesis gas is a mixture of CO2, H2, and O2 in a volume ratio of 1:7:2; the fresh MSM culture medium contains L-glutamate and / or NAD. + .

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