Application of soybean SAM-dependent methyltransferase GmMET gene in regulation and control of protein content of soybean seeds

Abstract

The invention provides an application of a soybean SAM-dependent methyltransferase GmMET gene in regulating and controlling the protein content of soybean seeds, the nucleotide sequence of the GmMET gene is as shown in SEQ ID NO.1, and the sequence of the coded protein is as shown in SEQ ID NO.2. The invention further provides an application of the soybean SAM-dependent methyltransferase GmMET gene in regulating and controlling the protein content of soybean seeds. The gene and the protein coded by the gene positively regulate the protein content in seeds; the protein content in the soybean seeds is increased through overexpression, and the protein content in the soybean seeds is reduced due to early termination of the gene; and overexpression and early termination do not obviously influence the normal growth of the soybeans. Therefore, a feasible scheme can be provided for cultivating soybeans with high protein content based on the gene.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to soybean SAM-dependent methyltransferases. GmMET Application of genes in regulating protein content in soybean seeds. Background Technology

[0002] As an important oilseed crop globally and a key source of plant protein for humans, increasing the oil and protein content of soybeans is a crucial task for agricultural research and development.

[0003] Due to the extremely high nutritional value and functional properties of soybean protein (such as gelling and emulsifying properties), its application in meat products, snack foods, plant-based protein beverages, and health foods is constantly expanding. The protein and oil content of existing soybean varieties cannot yet meet my country's current needs, and there is a gap compared to international varieties, requiring large-scale imports to satisfy domestic requirements. Therefore, increasing the protein and oil content of soybeans is a crucial goal in my country's current soybean breeding efforts. Soybean protein and oil content are quantitative traits, influenced by multiple genes and the current environment; studies have also shown a negative correlation between soybean protein and fat content, which is a major factor limiting the simultaneous increase of both. Furthermore, the molecular mechanisms of soybean protein and oil synthesis are not yet clear, and the genes regulating the balance between the two are still unknown.

[0004] Therefore, there are certain challenges in cultivating soybean varieties that are high in oil and high in protein. Summary of the Invention

[0005] In view of the above challenges, this invention provides a new breeding direction by providing a soybean SAM-dependent methyltransferase. GmMET Application of genes in regulating soybean seed protein content GmMET Genes play a positive role in regulating the protein content in seeds.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a soybean SAM-dependent methyltransferase. GmMET The application of genes in regulating soybean seed protein content, the aforementioned GmMET The gene nucleotide sequence is shown in SEQ ID NO.1; The specific details of SEQ ID NO.1 are as follows:

[0007] The second objective of this invention is to provide the above-mentioned GmMET The application of a gene-encoded protein in regulating the protein content of soybean seeds, the amino acid sequence of which is shown in SEQ ID NO.2; SEQ ID NO.2 is as follows: MCKEAAPPLPQIHCDFSSTRPHVPAKNRALRVQSTRLWAAKVLSFSLLFRDHLLLRAAAFNHSCVLCVSAGAGQEVAALRLLGIDDVTGVEILESPPLVRRADPHNLPFFDGVFDLAFTARFDEALFAAEMERVIRPGGACVVLVAESGEDEVREVVGLFRNSRLVRWSNVSDWNENGQCSFEN.

[0008] The third objective of this invention is to provide a method for regulating the protein content of soybean seeds, wherein: Increase the protein content of soybean seeds through overexpression GmMET Genetic realization; Reducing the protein content of soybean seeds by knocking out GmMET Genetic implementation.

[0009] Furthermore, through overexpression GmMET The specific steps for obtaining high-protein soybean seeds are as follows: Construct an overexpression vector and transfect soybeans with Agrobacterium to obtain overexpressing plants, thus obtaining high-protein soybean seeds. The primers used to construct the overexpression vector are as follows: GmMET -F:ATGTGTAAAGAAGCCGCACCTC; GmMET -R: CTAGTTCTCAAAAGAACACTGGCCA.

[0010] Furthermore, by knocking out GmMET The specific steps for obtaining low-protein soybean seeds are as follows: Construct a CRISPR / Cas9 multi-target knockout vector and transfect soybeans with Agrobacterium tumefaciens to obtain mutant plants, thus obtaining low-protein soybean seeds. The specific target fragments for constructing the CRISPR / Cas9 multi-target knockout vector are as follows: GmMET Gene target 1: GCAGGGCGCGGTTCTTGGCG; GmMET Gene target 2: AGTGGTTGAAGGCGGCGGCG.

[0011] In some specific embodiments, the primers used to construct the CRISPR / Cas9 multi-target knockout vector are preferably as follows: GmMET sgRNA1-F: GGATTGCAGGGCGCGGTTCTTGGCG; GmMET sgRNA1-R: AAACCGCCAAGAACCGCGCCCTGCA; GmMET sgRNA2-F: GGATTGAGTGGTTGAAGGCGGCGGCG; GmMET sgRNA-2-R: AAACCGCCGCCGCCTTCAACCACTCA.

[0012] The fourth objective of this invention is to provide a gene expression cassette, wherein the gene expression cassette comprises the above-mentioned... GmMET Gene.

[0013] The fifth objective of this invention is to provide a recombinant expression vector, which includes the gene expression cassette described above.

[0014] In some specific embodiments, preferably, the above-mentioned recombinant expression vector is a Gateway system vector and a binary Agrobacterium vector, such as Fu28, pBWA(V)BS, EHA105, etc. During construction, any kind of enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as the constitutive promoter 35S of cauliflower mosaic virus, etc., which can be used alone or in combination with other plant promoters.

[0015] Furthermore, genes encoding enzymes or luminescent compounds that can be expressed in plants, as well as antibiotic resistance markers, are added to the aforementioned recombinant expression vectors.

[0016] The sixth objective of this invention is to provide an engineered bacterium, which includes the recombinant expression vector described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention screened and obtained soybean SAM-dependent methyltransferases. GmMET This gene, along with its encoded protein, positively regulates the protein content in soybean seeds. Overexpression of this gene increases the protein content in soybean seeds, while premature termination of the gene leads to a decrease in protein content. Neither overexpression nor premature termination significantly affects the normal growth of soybeans. Therefore, this gene could provide a feasible approach for cultivating soybeans with high protein content. Attached Figure Description

[0018] Figure 1 The results of the knockout strain test in Example 2 of this invention are shown.

[0019] Figure 2 The results are the detection results of the overexpressing plants in Example 2 of this invention.

[0020] Figure 3 The results show the total nitrogen content of soybean grains from T3 generation knockout and T2 generation overexpression plants in Example 2 of this invention.

[0021] Figure 4 The results show the total nitrogen content of soybean grains from T4 generation knockout and T3 generation overexpression plants in Example 2 of this invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0023] Example 1 This embodiment provides GmMET The acquisition of genes and the construction of expression vectors are detailed below: 1.1 GmMET Acquisition of genes This study used cultivated soybean Suinong 14 and wild soybean ZYD00006 (both materials were obtained from the Soybean Genetic Improvement Laboratory of the College of Agriculture, Northeast Agricultural University) as parents to construct chromosome segment substitution lines (CSSLs) as experimental materials. Based on QTL analysis of soybean seed protein content in CSSLs, and the Meta-QTL results from Qi et al. (see article "Meta-analysis and transcriptome profiling reveal hub genes for soybean seed storage composition during seed development"), combined with CSSLs whole-genome resequencing data, self-crossing was performed on single plants (R7) with the target segment introduced heterozygous. Secondary segregating populations were constructed, and a gene related to soybean seed protein content, Glyma.06G301100, was screened using expression level analysis and haplotype analysis. Its gene annotation is S-adenosyl-L-methionine-dependent methyltransferase, a core gene that transfers methyl groups to substrate methylation sites during methylation. GmMET Gene.

[0024] Furthermore, primers were designed to clone the target gene from DN50 (the Dongnong 50 material came from DN50 seeds harvested that year at the Xiangyang Base by the Soybean Genetic Improvement Laboratory of the College of Agriculture, Northeast Agricultural University). This gene was then knocked out and overexpressed in DN50 for functional verification. The results showed that the candidate gene Glyma.06G301100 positively regulates the protein content of soybean seeds. Soybean plants overexpressing Glyma.06G301100 had significantly higher seed protein content than the control, while soybean plants with knockout Glyma.06G301100 had significantly lower seed protein content than the control.

[0025] 1.2 Construction of CRISPR / Cas9 multi-target knockout vector Total RNA was extracted from DN50 seedlings, and the RNA was reverse transcribed using reverse transcriptase to synthesize cDNA. Based on the soybean genome sequence from Phytozome and the full-length cDNA sequence of Glyma.06G301100 from the DN50 genome sequence we determined, primers were designed, and the primer sequences are as follows: GmMET -F:ATGTGTAAAGAAGCCGCACCTC; GmMET -R:CTAGTTCTCAAAAGAACACTGGCCA.

[0026] Using DN50 cDNA as a template, PCR amplification was performed using GmMET-F and GmMET-R primers to obtain a 555bp PCR product.

[0027] The PCR reaction system is shown in the table below:

[0028] PCR reaction program: 95℃ for 30s, 95℃ for 10s, 58℃ for 5s, 69℃ for 10s, for a total of 35 cycles, 78℃ for 5min, 4℃ for 10min.

[0029] The target sites for GmMET-CDS sequences were designed using the CRISPR-GE website. The two target sequences are as follows: GmMET Gene target 1: GCAGGGCGCGGTTCTTGGCG; GmMET Gene target 2: AGTGGTTGAAGGCGGCGGCG.

[0030] The two primer pairs are as follows: GmMET sgRNA1-F: ggattGCAGGGCGCGGTTCTTGGCG; GmMET sgRNA1-R: AAACCGCCAAGAACCGCGCCCTGCA; GmMET sgRNA2-F: ggattgAGTGGTTGAAGGCGGCGGCG; GmMET sgRNA-2-R: AAACCGCCGCCGCCTTCAACCACTCA.

[0031] After annealing the target primers, they were ligated into pGES201 and then transformed into Agrobacterium tumefaciens. The specific steps are as follows: CRISPR / Cas9 vector construction uses WMC021 The vector, provided by Weimi Biotechnology (Jiangsu) Co., Ltd., was used to anneal the oligonucleotides into double strands using the reaction system shown in the table below.

[0032]

[0033] Repeat the mixing of the prepared annealing buffer, centrifuge at 3000 rpm for 30 seconds, and then place on a standard PCR instrument. Run the following PCR program: 95℃ for 4 min, RAMP 0.1℃ / s (95℃ to 16℃), and store at 16℃. The vector was linearized in advance by Weimi Biotechnology Co., Ltd., and the T4 ligation system was configured as shown in the table below. The ligation reaction was carried out at room temperature or in a metal bath at 16°C for 2 hours.

[0034]

[0035] The ligation product was transformed into E. coli DH5α competent cells, and positive clones were detected by colony PCR and purified and sequenced.

[0036] Take normal, mature soybean seeds, add them to a 150mL Erlenmeyer flask containing 75% ethanol, and let stand for 5 minutes. Then add 15% H2O2 solution and immerse in a shaker at 28°C and 100rpm for 15 minutes for sterilization. Finally, wash with sterile ddH2O. Add 50-100mL of ddH2O and incubate at 4°C for 1 day. Spread Agrobacterium carrying the target gene plasmid onto the appropriate resistant medium for initial activation. After culturing for 48 hours, collect the cells for reactivation. After culturing for 24 hours, collect the cells and add them to the infiltration solution. Vortex to mix well and adjust the OD value to 0.6 using a spectrophotometer for later use.

[0037] 1.3 Construction of overexpression vectors Obtain using the same steps described above GmMET The CDS sequences were ligated into the entry vector Fu28, and then into the expression vector pBWA(V)BS via an LR reaction system. The main steps are as follows: (1) Extraction of Fu28 plasmid: The Fu28 empty vector was removed from -80°C and activated with bacterial culture. Plasmid extraction was performed according to the instructions of Novizan Biotech's 8minFastPure Plasmid Mini Kit, and the plasmid was stored at -20°C.

[0038] (2) Construction of Fu28-GmMET introductory carrier The purified PCR product and the extracted Fu28 plasmid were subjected to double enzyme digestion at 37°C for 5 hours using a metal bath apparatus. The reaction system shown in the table below was prepared on ice:

[0039] After the enzyme digestion reaction is complete, the Fu28 vector is combined with... GmMET The CDS fragments were ligated for a reaction at 16°C for 16 h. The reaction systems shown in the table below were prepared on ice.

[0040] (3) Specific steps for the transformation of DH5α Escherichia coli: Step 1.2 (4) LR reaction The constructed introductory platform Fu28-GmMET The reaction system, constructed into the expression vector pBWA(V)BS using Gateway recombination technology, was prepared on ice:

[0041] After performing the LR reaction for 5 hours in a 25℃ metal bath instrument, the product was transformed into Escherichia coli according to method (3), and then the plasmid of the positive clone was extracted according to method (1).

[0042] (5) Transformation of Agrobacterium tumefaciens EHA105 pBWA(V)BS-GmMET with correct sequencing results was transformed into Agrobacterium tumefaciens EHA105 for subsequent infection of plants.

[0043] 1) Clean the electric rotary cup multiple times with anhydrous ethanol, dry it on a clean bench sterilized with ultraviolet light, and pre-cool it at a low temperature; 2) Add 1 μL of plasmid to the melted EHA105 Agrobacterium competent bacterial culture, mix thoroughly, and let stand at low temperature for 5 min; 3) Add the mixture to the bottom gap of the pre-prepared electroporation cup in the electroporator and perform conversion at 1800V; 4) After transformation, add 200 μL of antibiotic-free LB liquid medium to the electroporation cuvette, mix well by pipetting, and then aspirate it into a sterile 1.5 mL centrifuge tube. Repeat this step twice to ensure that the EHA105 competent cells are completely aspirated. 5) Place the above bacterial culture in a shaker and incubate at 28°C for 2.5 hours; 6) After centrifugation, remove part of the culture medium, resuspend the culture, and spread it evenly on LB solid medium containing double resistance. After there are no obvious liquid traces on the surface of the solid medium, seal the medium, invert it and place it in a 37℃ incubator for 2-3 days. 7) Select single colonies from the solid culture medium for PCR identification.

[0044] Example 2 This embodiment provides the acquisition of mutant plants and the detection of mutant plant phenotypes and soybean seed protein content, as detailed below: 2.1 Obtaining mutant plants The two Agrobacterium species obtained in Example 1 were subjected to Agrobacterium-mediated genetic transformation of soybean cotyledonary nodes. The specific steps are as follows: (1) Infection: After scalpels are cut into the germinated soybeans, they are poured into the prepared bacterial solution for infection. Then the explants are placed on a co-culture medium lined with filter paper and co-cultured in the dark at 23°C for 5 days.

[0045] (2) Recovery culture: Select the end of the hypocotyl from the healthy explants that have grown well after co-culture, insert it into the recovery solid culture medium, and recover culture for 7 days.

[0046] (3) Screening culture: Explants with clustered buds after recovery were inoculated onto screening culture medium and cultured for 21 days with 16 hours of light and 8 hours of darkness.

[0047] (4) Elongation culture: The selected shoots were transferred to a new elongation culture medium and cultured for 21 days with 16 hours of light and 8 hours of darkness.

[0048] (5) Rooting culture: When the buds grow to about 5cm, they are transferred to the rooting culture medium for the next screening. They are cultured for 21 days with 16 hours of light and 8 hours of darkness.

[0049] (6) Bar test strip detection of positive seedlings: When the first trifoliate compound leaf unfolds, the leaves of the transgenic plant are directly identified using bar test strips.

[0050] (7) Hardening off seedlings: Add sterile water to the culture medium of seedlings that test positive with Bar test strips and culture them with the lid off. After 3 days of adaptation, remove the culture medium, gently wash the culture medium off the roots, and transplant them into flower pots filled with nutrient soil. Use plastic wrap to build a simple greenhouse. Culture for 16 hours in light and 8 hours in darkness for 5-10 days.

[0051] (8) Remove the culture chamber and place it in the outdoor environment for regular potted plant management.

[0052] 2.2 Detection of knockout lines DNA was extracted from the mutant T2 generation, and specific primers were designed upstream and downstream of the gene editing site for PCR amplification. The edited recipient DN50 soybean variety was used as a negative control, and the amplified unpurified product was sent to Sanger sequencing. GmMET Two mutation types were detected in the gene sequence, named KO-1 and KO-2 according to the number of bases deleted, respectively. The results are shown below. Figure 1 .

[0053] Among them, the KO-1 sequence (SEQ ID NO.3): MCKEAAPPLPOIHCDFSSTRPHVPAKNRALRVOSTRLWAAKVLSFSLLFRDHLLLRARLOPLLRPLRLRWRRIGGRRAPDPRHRRRHRRRDDRVPAARIPRRSAOPPVLRRRVRPRLHRAVRRGAVCRRDGARDPWWRRVRCSRGGVWGG; KO-2 sequence (SEQ ID NO. 4): MCKEAAPPLPOIHCDFSSTRPHVDATAICAFSPRASGIPRSSPSDSSSATTSSSADDPSTTDASSASPLAPARRSPRSASSASTTSPASRSSSPRRSSAAPIRTTSRSSTACSTSPSPRGSTRRCLPPRWSA.

[0054] The specific steps for detecting knockout strains are as follows: Primers were designed within a 150 bp region upstream and downstream of the editing site to perform PCR detection of the mutant. The specific primers used are as follows: MET -F: ACCACGCGCCGAGGAAG; MET -R:TGACGTCGTCGATGCCGAG.

[0055] Total DNA was extracted from the transgenic material using the CTAB method and amplified using the primers described above. The PCR reaction system is shown in the table below:

[0056] PCR reaction program: 95℃ for 30s, 98℃ for 10s, 58℃ for 5s, 68℃ for 10s, for a total of 35 cycles, 68℃ for 5min, 4℃ for 10min.

[0057] 2.3 Detection of overexpressing plants Overexpression seeds harvested from T0 were sown in the phenotyping area of ​​Northeast Agricultural University. After the trifoliate compound leaves emerged, samples were taken for DNA and RNA extraction. GmMET Specific primers were designed based on the full-length CDS sequence of the target gene and GFP. PCR was performed using Dongnong 50 as a control to verify whether the gene and expression vector were completely fused. Positive plants amplified the following: GmMET protein bands ， Wild-type plants, lacking the GFP fragment, showed no bands. The expression levels of overexpressing plants were detected at the RNA level using qRT-PCR. Finally, nine GmMET overexpressing plants were tested in the T1 generation, and the results are shown below. Figure 2 .

[0058] The verification process is as follows: RNA was extracted from different soybean tissues using the Trizol method. The extracted RNA was reverse transcribed into cDNA using HiScript® III Reverse Transcriptase. The reaction system shown in the table below was prepared in an RNase-free PCR tube.

[0059] Add 2 μL of 5×qRT SuperMix II to each tube of the above reaction solution and follow the reaction program: 37℃ for 15 min, 85℃ for 5 s, 4℃ for 10 min to synthesize cDNA.

[0060] Total DNA was extracted from transgenic materials using the CTAB method, and the design was carried out. GmMET Specific quantitative primers were selected. GmActin2 ( Glyma.02g091900 ) was used as an internal reference gene, employing 2 -ΔCT The qRT-PCR data were analyzed using computational methods to obtain the relative expression levels of genes. The specific primers are as follows: MET CDS-F: TGAGGAGGTTGTTGGATTATT; GFP-R:GTCCTTGAAGAAGATGGTGC.

[0061] The qRT-PCR reaction system is shown in the table below:

[0062] qRT-PCR reaction program: 95℃ for 30s, 95℃ for 3-10s, 60℃ for 10-30s, for a total of 40 cycles, followed by 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s.

[0063] 2.2 Detection of mutant plant phenotype and soybean seed protein content 2.2.1 Phenotypic Identification The phenotypes of the control, two knockout lines, and three overexpression lines were examined under normal conditions. Under normal conditions, the control and... GmMET There were no significant differences in phenotypes, such as plant height, between the knockout and overexpression lines.

[0064] 2.2.2 Detection of Soybean Seed Protein Content Furthermore, the DN50 of the control wild-type and GmMET Protein content of knockout and overexpression lines was measured. The method is as follows: FOSS near-infrared grain quality analyzer determined the protein and oil content of soybean grains: The protein and oil content of genetically modified soybean grains were measured using a FOSS near-infrared grain quality analyzer (INFRATEC1241TM), ensuring that the grain moisture content remained within safe limits during measurement. Three technical replicates were performed, and the average of the three replicates was used as the final data.

[0065] Dumas nitrogen determination: Soybean seeds grown in uniform environments were sun-dried and then dried in a 65℃ oven for 24 hours. After removing the samples, the seeds were ground and sieved using a mortar and pestle that had been dried in the oven. 30-50 mg of each sample was weighed and recorded, with each sample being tested three times in total. The protein content of the samples was then determined using the Dumas nitrogen determination method, and the results were reported.

[0066] result Figure 3 , 4 This indicates that the protein content of the two knockout lines was significantly lower than that of the DN50 wild type, while the protein content of the three overexpression lines was significantly higher than that of the DN50 wild type.

[0067] The above research indicates that the soybean SAM-dependent methyltransferase GmMET positively regulates the protein content in soybean seeds. Overexpression of the encoding gene GmMET increases the protein content in soybean seeds, while premature termination of this gene leads to a decrease in the protein content. Neither overexpression nor premature termination significantly affects normal soybean growth. Therefore, this gene can be used as a target gene to increase the protein content in soybean seeds, providing a feasible approach for cultivating high-protein soybeans.

[0068] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0069] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Soybean SAM-dependent methyltransferase GmMET The application of genes in regulating the protein content of soybean seeds is characterized by, The GmMET The gene nucleotide sequence is shown in SEQ ID NO.

1.

2. The claim 1 GmMET Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A method for regulating the protein content of soybean seeds, characterized in that, In the method: Increase the protein content of soybean seeds through overexpression GmMET Genetic realization; Reducing the protein content of soybean seeds by knocking out GmMET Genetic implementation.

4. The method according to claim 3, characterized in that, Through overexpression GmMET The specific steps for obtaining high-protein soybean seeds are as follows: Construct an overexpression vector and transfect soybeans with Agrobacterium to obtain overexpressing plants, thus obtaining high-protein soybean seeds. The primers used to construct the overexpression vector are as follows: GmMET -F:ATGTGTAAAGAAGCCGCACCTC; GmMET -R:CTAGTTCTCAAAAGAACACTGGCCA。 5. The method according to claim 3, characterized in that, By knocking GmMET The specific steps for obtaining low-protein soybean seeds are as follows: Construct a CRISPR / Cas9 multi-target knockout vector and transfect soybeans with Agrobacterium tumefaciens to obtain mutant plants, thus obtaining low-protein soybean seeds. The specific target fragments for constructing the CRISPR / Cas9 multi-target knockout vector are as follows: GmMET Gene target 1: GCAGGGCGCGGTTCTTGGCG; GmMET Gene target 2: AGTGGTTGAAGGCGGCGGCG.

6. The method according to claim 5, characterized in that, The primers used to construct the CRISPR / Cas9 multi-target knockout vector are as follows: GmMET sgRNA1-F:GGATTGCAGGGCGCGGTTCTTGGCG; GmMET sgRNA1-R:AAACCGCCAAGAACCGGCCCCTGCA; GmMET sgRNA2-F: GGATTGAGTGGTTGAAGGCGGCGGCG; GmMET sgRNA-2-R:AAACCGCCGCCGCCTTCAACCACTCA。 7. A gene expression cassette, characterized in that, The gene expression cassette includes the one described in claim 1. GmMET Gene.

8. A recombinant expression vector, characterized in that, The recombinant expression vector includes the gene expression cassette as described in claim 7.

9. An engineered bacterium, characterized in that, The engineered bacteria include the recombinant expression vector described in claim 8.

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