Construction and application of feed amino acid engineering strain based on multi-constraint network model

Abstract

This invention belongs to the field of bioengineering technology and application, specifically relating to the prediction and application of the transcription factor MalR, which is related to the production of feed amino acids, based on a multi-constraint network model. This invention is the first to simulate and predict the transcription factor MalR, which is related to lysine production, using the multi-constraint metabolic network model system mcCGL1. Further experimental verification confirmed the role of the transcription factor MalR in the production of feed amino acids, particularly lysine. Experiments showed downregulation of the transcription factor gene in Corynebacterium glutamicum. malR The expressed strains produced lysine at a yield approximately 9.8%–55.6% higher than the control strains. This strategy can be applied to the modification of production strains for other aspartic acid family feed amino acids such as L-methionine, L-threonine, and L-isoleucine, demonstrating good versatility and promising industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and application, specifically relating to MalR, a transcription factor related to the production of feed amino acids, and its application based on prediction using a multi-constraint network model. Background Technology

[0002] Lysine (L-lysine) is one of the eight essential amino acids that humans and animals cannot synthesize on their own. It is widely used in the feed industry as the first limiting amino acid additive, playing a vital role in promoting growth, improving meat quality, and increasing protein utilization. Currently, global annual lysine production exceeds 2 million tons, and the market continues to expand. Microbial fermentation has become the mainstream process for the industrial production of lysine due to its environmental friendliness, mild conditions, and renewable raw materials.

[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum L-lysine (L-lysine) is a Gram-positive, non-pathogenic soil bacterium that has been widely used in the industrial fermentation of L-lysine and L-glutamic acid since its discovery. Its metabolic regulation mechanism is clear and its genetic manipulation tools are mature, making it the most important chassis strain for lysine production.

[0004] Significant progress has been made in metabolic engineering of the lysine biosynthesis pathway (aspartate pathway) in Corynebacterium glutamicum. Researchers have gradually increased lysine accumulation levels through strategies such as classical mutagenesis, gene knockout, deactivation of key enzyme feedback inhibition (e.g., the T311I mutation of aspartate kinase LysC), enhanced supply of precursors (oxaloacetate, NADPH), weakening of byproduct pathways (lactic acid, acetic acid, alanine, etc.), and overexpression of the lysine export protein LysE. In recent years, synthetic biology tools such as CRISPR interference (CRISPRi), riboswitch and biosensor regulation, plug-in repressor library technology, and multi-gene co-repression have also been widely applied to the rational modification of lysine-producing strains, significantly improving breeding efficiency. However, the above strategies mainly focus on the direct modification of pathway enzymes, and research on the systematic prediction and regulation of global transcription factors remains relatively limited.

[0005] Transcription factors can simultaneously regulate the expression of multiple target genes at the transcriptional level, possessing the potential for global remodeling of metabolic networks and serving as important targets for overcoming strain yield bottlenecks. Traditional transcription factor screening often relies on literature reviews or low-throughput experiments, making it difficult to systematically uncover hidden regulatory elements. In recent years, computer-aided prediction strategies based on metabolic network models have provided new ideas for efficiently identifying transcription factors related to product synthesis. In 2017, Kim et al. used the *E. coli* metabolic network model iJO1366 as a foundation, integrated 3672 transcriptional regulatory relationships obtained from the RegulonDB database, constructed an *E. coli* transcriptional regulation metabolic network model, and used the BeReTa algorithm to predict transcription factors that can improve the synthesis of various products in *E. coli*, such as arginine synthesis transcription factors ArgR, TdcA, ArgP, and Rob. In 2019, Shen et al. used the Yeast 7.6 metabolic network model as the chassis model and combined the transcriptional regulatory network and metabolic network model using the IDREAM method. They used the OptRAM algorithm to predict genes and transcription factors related to high succinic acid production. Among them, the upregulation of PHO4 and PDR1 transcription factors significantly increased succinic acid production.

[0006] Based on the above case studies and the current state of lysine production research, using network models to predict and screen transcription factors associated with high lysine production can accurately elucidate metabolic regulatory mechanisms and guide the modification of targeted strains, effectively improving the biosynthesis efficiency of feed amino acids. This provides key targets and theoretical support for large-scale industrial production and metabolic engineering optimization. However, there are currently no reports on MalR, a transcription factor related to feed amino acid production, including lysine, methionine, threonine, and isoleucine, based on network model predictions. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention uses a network model to predict the transcription factor MalR, which is related to the production of feed amino acids including lysine, methionine, threonine, and isoleucine, and further verifies the role of transcription factor MalR in the production of feed amino acids, especially lysine, through experiments.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] One of the technical solutions provided by this invention is the application of transcription factor MalR in regulating the production of feed amino acids, and further, the production of feed amino acids is increased by downregulating or inhibiting the expression of transcription factor MalR in the production strain. Furthermore, the feed amino acids include, but are not limited to: lysine, methionine, threonine, and isoleucine; Furthermore, the downregulation or inhibition of the expression of transcription factor MalR means that the expression level of MalR is significantly lower than the original level, for example, significantly reduced by at least 50%, 60%, 70%, 80%, 90%, or 100%. The downregulation of the protein expression level by gene editing on the genome of the production strain can be achieved by conventional means in the art, including but not limited to CRISPRi technology, gene knockout, gene knock-in, point mutation, deletion mutation, and combinations thereof. Furthermore, the host used for the production strain includes, but is not limited to: Corynebacterium glutamicum, Escherichia coli, Bacillus spp., and yeast spp.; Corynebacterium glutamicum is preferred. Furthermore, the amino acid sequence of the transcription factor MalR is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0010] The second technical solution provided by this invention is a genetically engineered bacterium that increases lysine production by regulating the expression level of transcription factor MalR. The engineered bacterium uses a lysine-producing strain as a host and is obtained by downregulating the expression level of transcription factor MalR. Furthermore, the method for downregulating the expression level of transcription factor MalR is to construct a CRISPRi recombinant plasmid that downregulates MalR expression using CRISPRi technology, and then introduce the CRISPRi recombinant plasmid into a lysine-producing strain. Furthermore, the CRISPRi recombinant plasmid that downregulates MalR expression uses pdCas9gRNA as a vector backbone and is inserted into Corynebacterium glutamicum. malR The sgRNA expression cassette of the gene can stably replicate in Corynebacterium glutamicum and specifically inhibit its expression. malR Gene expression; Furthermore, the CRISPRi recombinant plasmid that downregulates MalR expression is the pCRISPRi-high-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.3; Furthermore, the CRISPRi recombinant plasmid that downregulates MalR expression is the pCRISPRi-mid-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.4; Furthermore, the CRISPRi recombinant plasmid that downregulates MalR expression is the pCRISPRi-low-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.5; Furthermore, the lysine-producing strain is Corynebacterium glutamicum SAT9-M6, and the genotype of the SAT9-M6 strain is: C. glutamicum ATCC13032 ldhA pta pqo catzwf A243T gnd S361F pyc P458S mqo T224 lysC T311I hom V59A .

[0011] The third technical solution provided by this invention is the application of the genetically engineered bacteria described in the second technical solution in lysine production; Furthermore, the application includes a process of fermenting and culturing the genetically engineered bacteria and obtaining lysine from the culture.

[0012] Beneficial effects: This invention is the first to predict the transcription factor MalR, which is associated with lysine production, based on the multi-constraint metabolic network model system mcCGL1. Experiments demonstrate that this invention downregulates transcription factor genes in Corynebacterium glutamicum. malR The lysine production of the expressed strain SAT9-M6-high / mid / low-malR during shake-flask fermentation was 1.95±0.09 g / L, 1.71±0.1 g / L, and 1.68±0.13 g / L, respectively, which were approximately 27.5%, 11.8%, and 9.8% higher than those of the control strain SAT9-M6-pXi. The transcription factor gene of wild-type Corynebacterium glutamicum ATCC 13032 was downregulated. malR The expressed strain ATCC-high-malR produced 0.28 ± 0.18 g / L of lysine during shake-flask fermentation, an increase of approximately 55.6% compared to the control strain ATCC-pXi. Given the potential regulatory scope of MalR as a global transcriptional regulator, the transcription factor MalR and the corresponding CRISPRi attenuation strategy provided in this invention can also be reasonably expected to be applied to the modification of production strains for other aspartic acid family feed amino acids such as L-methionine, L-threonine, and L-isoleucine, demonstrating good versatility and industrial application prospects. Attached Figure Description

[0013] Figure 1 This is the pCRISPRi-high-malR plasmid map.

[0014] Figure 2 This is the pCRISPRi-mid-malR plasmid map.

[0015] Figure 3 The pCRISPRi-low-malR plasmid map.

[0016] Figure 4 pD-zwf A243T Plasmid map.

[0017] Figure 5 pD-gnd S361F Plasmid map.

[0018] Figure 6 pD-pyc P458S Plasmid map.

[0019] Figure 7 pD-mqo T224 Plasmid map.

[0020] Figure 8 pD-lysC T311I Plasmid map.

[0021] Figure 9 pD-hom V59A Plasmid map. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. The following embodiments are provided to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0023] 1. Sources of experimental materials such as strains or plasmids involved in this invention and its embodiments The original strain Corynebacterium glutamicum used in this invention ( Corynebacterium glutamicum ATCC13032 originated from ATCC (The Global Bioresource Center) and was purchased in October 2012. The starting strain used in this invention is the recombinant strain SAT9 of Corynebacterium glutamicum. SAT9 and its construction method have been disclosed in the published doctoral dissertation: Zhu Nianqing. Metabolic engineering modification of Corynebacterium glutamicum to produce high succinic acid [D]. Tianjin University, 2013. The genotype of the recombinant Corynebacterium glutamicum strain SAT9 is: C. glutamicum ATCC13032 ldhA pta pqo cat ;in, pta Representative knockout pta - ackA Manipulator.

[0024] The integrated carrier pD-zwf involved in this invention A243T pD-gnd S361F pD-pyc P458S pD-mqo T224 ( This indicates that the codon at this site has mutated to a stop codon), pD-lysC T311I pD-hom V59A The construction method is detailed in Example 3. The above mutations are referenced in the following literature: Mitsuhashi S, Hayashi M, Ohnishi J, Ikeda M. Disruption of malate:quinone oxidoreductase increases L-lysine production by Corynebacteriumglutamicum. Bioscience, Biotechnology, and Biochemistry, 2006, 70(11): 2803-2806. Becker J, Klopprogge C, Herold A, Zelder O, Bolten CJ, Wittmann C. Metabolic flux engineering of L-lysine production in Corynebacteriumglutamicum——over expression and modification of G6P dehydrogenase. Journal of Biotechnology, 2007, 132(2): 99-109. Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, IkedaM. A novel methodology employing Corynebacterium glutamicum genomeinformation to generate a new L-lysine-producing mutant. Applied Microbiologyand Biotechnology, 2002, 58(2): 217-223. Ohnishi J, Katahira R, Mitsuhashi S, Kakita S, Ikeda M. A novel gndmutation leading to increased L-lysine production in Corynebacteriumglutamicum. FEMS Microbiology Letters, 2005, 242(2): 265-274. The recombinant strain of Corynebacterium glutamicum, SAT9-M6, has the following genotype: C. glutamicum ATCC13032 ldhA pta pqo catzwf A243T gnd S361F pyc P458S mqo T224 lysC T311I hom V59A .

[0025] The specific operation methods for the traceless manipulation technique of Corynebacterium glutamicum and the construction of the tool vector pD-sacB involved in this invention have been disclosed in the authorized patent "High-yield chiral D-(-)-acetoin Corynebacterium glutamicum strain, construction and application" (application number: CN201710215459.7).

[0026] For the preparation of pdCas9gRNA plasmid, please refer to the following reference: Liu J, Liu M, Shi T, Sun G, Gao N, Zhao X, Guo X, Ni X, Yuan Q, Feng J, et al: CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction. Nat Commun 2022, 13:891. 2. Sources of some reagents involved in this invention and its embodiments L-lysine standard was purchased from Sigma.

[0027] The restriction endonucleases, DNA ligases and other molecular biology reagents used were purchased from Thermo Fisher Scientific, and other biochemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0028] 3. Composition of some culture media involved in this invention and its embodiments LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 2% agar powder added to LB solid medium.

[0029] BHI liquid medium: Brain and heart infusion broth powder 38.5 g / L. BHI solid medium with 2% agar powder added.

[0030] CGIII medium: yeast extract 10 g / L, tryptone 10 g / L, MOPS 21 g / L, NaCl 2.5 g / L, pH adjusted to 7 with 5 M NaOH aqueous solution.

[0031] CGXII medium: ammonium sulfate 20 g / L, urea 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate trihydrate 1.3 g / L, 3-(N-morpholino)propanesulfonic acid (MOPS) 42 g / L, calcium chloride 0.01 g / L, magnesium sulfate heptahydrate 0.25 g / L, ferrous sulfate heptahydrate 0.002 g / L, manganese sulfate monohydrate 0.002 g / L, zinc sulfate heptahydrate 0.0002 g / L, copper sulfate 0.0004 g / L, nickel chloride hexahydrate 0.00004 g / L, protocatechuic acid 0.006 g / L, biotin 0.0004 g / L, vitamin B1 0.00002 g / L, adjusted to pH 7 with 5 mol / L NaOH aqueous solution.

[0032] The antibiotic concentrations were: kanamycin 50 μg / mL and chloramphenicol 10 μg / mL.

[0033] 4. Some of the test methods involved in this invention and its embodiments E. coli DH5α competent cells were prepared using the conventional CaCl2 method; Method for detecting lysine: Sample pretreatment (extraction, purification, pH adjustment) ensures lysine is in a reactive state before derivatization: Take 100 μL of the sample solution to be tested, add 100 μL of 0.5 mol / L sodium bicarbonate solution (4.2 g NaHCO3 dissolved in 100 mL water), and 100 μL of acetonitrile solution of 2,4-dinitrofluorobenzene (weigh 1 g 2,4-dinitrofluorobenzene, diluted to 100 mL with acetonitrile). Shake well, incubate in a 60℃ water bath in the dark for 60 min, remove and allow to stand at room temperature. Add 700 μL of 0.05 mol·L⁻¹ phosphate buffer (pH 7.0), centrifuge at 12000 r·min⁻¹ for 10 min, filter the supernatant through a membrane, and perform liquid chromatography analysis under the following conditions: Chromatographic column: C18 column; Mobile phase: Phase A: Sodium acetate-acetic acid (pH 6.2), Phase B: 100% acetonitrile; Column temperature: 30℃; Detection wavelength: 210 nm; Injection volume: 10 μL; Flow rate: 1.0 mL / min gradient progress The gradient elution program is as follows: within 0-30 min, the proportion of phase B increases linearly from 5% to 40%; within 30-37 min, 40% isocratic elution is maintained; within 37-40 min, the proportion of phase B increases linearly from 40% to 70%; within 40-47 min, 70% isocratic elution is maintained; within 47-50 min, the proportion of phase B decreases linearly from 70% to 5%; within 50-52 min, the column is equilibrated with 5% phase B.

[0034] 5. Some primer sequences involved in the present invention and its embodiments are shown in Table 1.

[0035] Table 1 Primer sequences used for strain construction

[0036] The present invention will be further explained and illustrated below through specific embodiments.

[0037] Example 1: Lysine production-related transcription factor MalR predicted based on a multi-constraint network model Based on the *Corynebacterium glutamicum* multi-constraint network model mcCGL1 constructed by our research group, the BeReTa algorithm was used to predict lysine synthesis-related transcription factor targets on the multi-constraint network model in ETGEMs2.0 format. Utilizing integrated *Corynebacterium glutamicum* transcriptome data and transcriptional regulatory relationships, the model was designed to maximize the flux of biomass synthesis and lysine exchange reactions, respectively, to simulate and predict lysine synthesis-related transcription factors. The *Corynebacterium glutamicum* regulatory relationship file was read in DataFrame format and input as parameters along with the multi-constraint network model into the `BeReTa_use` function. The BeReTa algorithm was then used to obtain the result matrix. Transcription factors with P < 0.05, effective genes / reaction count ≥ 2, and effective gene percentage > 0.1 were selected as lysine synthesis-related transcription factors. Ultimately, the transcription factor MalR was found to be highly correlated with lysine production, with a correlation coefficient of -2.343.

[0038] Subsequent examples will further verify the relationship between the expression level of transcription factor MalR and lysine production in engineered bacteria.

[0039] Example 2 Construction of gene expression vector To downregulate the expression level of transcription factor MalR in glutamate rods at different intensities using the CRISPRi technique, this example systematically investigates the effect of MalR expression on lysine synthesis. malR Three CRISPRi interference plasmids with different inhibition efficiencies were designed and constructed: pCRISPRi-high-malR, pCRISPRi-mid-malR, and pCRISPRi-low-malR.

[0040] Studies have shown that in the CRISPRi system, when sgRNA targets and binds near the start codon (5' end), the binding of the dCas9-sgRNA complex effectively blocks transcription initiation or elongation of RNA polymerase, exhibiting the highest inhibitory efficiency. Targeting the middle of the coding region reduces the inhibitory efficiency, while targeting the 3' end results in the weakest inhibition of transcriptional elongation and the lowest inhibitory efficiency. Based on this principle, this embodiment targets... malRThree sgRNAs were designed from the 5' high, mid, and 3' low regions of the gene to achieve varying degrees of expression inhibition. The sgRNA sequences were designed using the Benchling online platform, with a target sequence length of 20 bp, ensuring it was located on the sense or antisense strand immediately adjacent to the PAM site (5'-NGG-3'). To avoid off-target effects, all candidate sequences underwent genome-wide alignment screening, retaining only highly specific target sequences. After design, the three sgRNA sequences were synthesized using primers and introduced into expression vectors to construct three plasmids: pCRISPRi-high-malR, pCRISPRi-mid-malR, and pCRISPRi-low-malR. After sequencing verification, these plasmids were transformed into engineered strains for functional testing.

[0041] All the above plasmids use pdCas9gRNA as the basic backbone. This backbone vector carries a catalytically inactivated Cas9 mutant gene (D10A and H840A double-point mutation), encoding a dCas9 protein that has lost its DNA cleavage activity but retains its DNA binding ability; it also contains an sgRNA expression element (composed of the constitutive promoter P...). 11 The plasmids, including the driver gene, the Corynebacterium glutamicum replicon ori pBL1, the Escherichia coli replicon ori pUC, and the chloramphenicol resistance gene (cat), can stably replicate and be screened in Corynebacterium glutamicum and Escherichia coli. After construction, each plasmid is introduced into the host strain SAT9-M6, where it binds specifically to the dCas9-sgRNA complex. malR Different regions of the gene block the transcriptional elongation of RNA polymerase with varying efficiencies, thereby achieving... malR Gene expression levels are downregulated in a gradient. The specific construction steps are as follows: Using pdCas9gRNA plasmid as a template, PCR amplification was performed with high-malR-F1 and cas-R1, mid-malR-F1 and cas-R1, and low-malR-F1 and cas-R1 as upstream and downstream primer combinations, respectively, to obtain linearized vector fragments 1-high, 1-mid, and 1-low. Each fragment contains the dCas9 coding sequence and the sgRNA scaffold region. The difference lies in the 20 bp target sequence introduced at the 5' end of the sgRNA, which corresponds to the high, medium, and low target sites of the malR gene, respectively. Using pdCas9gRNA plasmid as a template, and ori-F and CRISPRi-R2 as upstream and downstream primers, PCR amplification was performed to obtain linearized vector fragment 2, which contains plasmid replication origin ori pBL1, ori pUC and chloramphenicol resistance gene cat.

[0042] PCR amplification system: 1 μL template, 1 μL each of forward and reverse primers, 25 μL phanta buffer, 21 μL sterile double-distilled water, 1 μL DNA polymerase, total volume 50 μL. PCR reaction conditions: 95 ℃ pre-denaturation for 3 min, 30 cycles (95 ℃ denaturation for 20 s, 58 ℃ annealing for 20 s, 72 ℃ extension for 1 min), 72 ℃ final extension for 10 min, and storage at 4 ℃. PCR products were recovered and purified using a gel extraction kit.

[0043] Using 2× MultiF Seamless Assembly Mix reagent, the linearized vector fragments 1-high, 1-mid, and 1-low were seamlessly ligated with vector fragment 2, respectively. These fragments were then transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing chloramphenicol (final concentration 10 μg / mL) and incubated at 37°C for 12–16 h. Single colonies were picked, and colony PCR was performed using primers test-cri-F / test-cri-R for verification. Positive transformants were sequenced to confirm the correct sgRNA sequences. Plasmids were then extracted to obtain pCRISPRi-high-malR (…). Figure 1 ), pCRISPRi-mid-malR ( Figure 2 ) and pCRISPRi-low-malR ( Figure 3 ) plasmid.

[0044] Example 3 Construction of SAT9-M6 strain To systematically enhance the synthesis capacity of target metabolites in lysine-producing chassis strains, and to provide a host strain with a clear genetic background and good stability for subsequent transcription factor regulation research, this invention uses the recombinant strain SAT9 of Corynebacterium glutamicum as the starting strain. Through traceless site-directed mutagenesis, beneficial mutations in six key genes related to high lysine production are integrated into the chromosome to replace wild-type copies, thereby constructing a genetically stable engineered strain SAT9-M6.

[0045] 1. Construction of the integrated carrier (1) pD-zwf A243T Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, and with zwf-up-F and zwf-up-R (containing zwf A243T The point mutation (G→A, position 727) was used as a primer to amplify the upstream homologous arm fragment zwf-U of the zwf gene; using the genome of Corynebacterium glutamicum ATCC13032 as a template, zwf-down-F (containing zwf A243TPoint mutation (G→A, position 727) and zwf-down-R were used as primers to amplify the downstream homologous arm fragment zwf-D of the zwf gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using zwf-U and zwf-D fragments as templates, and zwf-up-F and zwf-down-R as primers, up-zwf was obtained by fusion PCR amplification. A243T -down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into *E. coli* DH5α. Colony PCR was performed on the obtained transformants using primer pairs test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-zwf was obtained. A243T ( Figure 4 ).

[0046] (2) pD-gnd S361F Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, gnd-up-F and gnd-up-R (containing gnd S361F The upstream homologous arm fragment gnd-U of the gnd gene was amplified using a point mutation (C→T, position 1082) as a primer; the genome of Corynebacterium glutamicum ATCC13032 was used as a template, and gnd-down-F (containing gnd) was used as a primer. S361F Point mutation (C→T, position 1082) and gnd-down-R were used as primers to amplify the downstream homologous arm fragment gnd-D of the gnd gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using gnd-U and gnd-D fragments as templates, and gnd-up-F and gnd-down-R as primers, up-gnd was obtained by fusion PCR amplification. S361F -down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into E. coli DH5α. The obtained transformants were verified by colony PCR using primer pair test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-gnd was obtained. S361F ( Figure 5 ).

[0047] (3) pD-pyc P458S Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, and pyc-up-F and pyc-up-R (containing pyc P458SThe point mutation (C→T, position 1372) was used as a primer to amplify the upstream homologous arm fragment pyc-U of the pyc gene; using the Corynebacterium glutamicum ATCC13032 genome as a template, pyc-down-F (containing pyc P458S Point mutation (C→T, position 1372) and pyc-down-R were used as primers to amplify the downstream homologous arm fragment pyc-D of the pyc gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using pyc-U and pyc-D fragments as templates, and pyc-up-F and pyc-down-R as primers, up-pyc was obtained by fusion PCR amplification. P458S -down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into *E. coli* DH5α. Colony PCR was performed on the obtained transformants using primer pairs test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-pyc was obtained. P458S ( Figure 6 ).

[0048] (4) pD-mqo T224 Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, mqo-up-F and mqo-up-R (containing mqo T224 A point mutation (G→A, position 672) was used as a primer to amplify the upstream homologous arm fragment mqo-U of the mqo gene; using the genome of Corynebacterium glutamicum ATCC13032 as a template, mqo-down-F (containing...) was used as a primer. mqo T224 Point mutation (G→A, position 672) and mqo-down-R were used as primers to amplify the downstream homologous arm fragment mqo-D of the mqo gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using mqo-U and mqo-D fragments as templates, and mqo-up-F and mqo-down-R as primers, up-mqo was obtained by fusion PCR amplification. T224 -down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into *E. coli* DH5α. Colony PCR was performed on the obtained transformants using primer pairs test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-mqo was obtained. T224 ( Figure 7 ).

[0049] (5) pD-lysC T311I Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, and lysC-up-F and lysC-up-R (containing lysC T311I The point mutation (C→T, position 932) was used as a primer to amplify the upstream homologous arm fragment lysC-U of the lysC gene; using the Corynebacterium glutamicum ATCC 13032 genome as a template, lysC-down-F (containing lysC T311I Point mutation (C→T, position 932) and lysC-down-R were used as primers to amplify the downstream homologous arm fragment lysC-D of the lysC gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using lysC-U and lysC-D fragments as templates, and lysC-up-F and lysC-down-R as primers, up-lysC was obtained by fusion PCR amplification. T311I -down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into *E. coli* DH5α. Colony PCR was performed on the obtained transformants using primer pairs test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-lysC was obtained. T311I ( Figure 8 ).

[0050] (6) pD-hom V59A Construction of integrated carrier Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, hom-up-F and hom-up-R (containing hom V59A The point mutation (T→C, position 176) was used as a primer to amplify the upstream homologous arm fragment hom-U of the hom gene; using the genome of Corynebacterium glutamicum ATCC13032 as a template, hom-down-F (containing hom V59A Point mutation (T→C, position 176) and hom-down-R were used as primers to amplify the downstream homologous arm fragment hom-D of the hom gene; pD-sacB was used as a template, and pD-F and pD-R were used as primers to amplify the pD vector backbone fragment. Using hom-U and hom-D fragments as templates, and hom-up-F and hom-down-R as primers, up-hom was obtained by fusion PCR amplification. V59A-down fragment. The above fusion fragment was seamlessly cloned with the pD vector backbone fragment, ligated, and transformed into E. coli DH5α. The obtained transformants were verified by colony PCR using primer pair test-pD-F / test-pD-R. After sequencing verification, the integration vector pD-hom was obtained. V59A ( Figure 9 ).

[0051] 2. Construction of strain SAT9-M6 Integrating the pD-zwf carrier A243T pD-gnd S361F pD-pyc P458S pD-mqo T224 pD-lysC T311I pD-hom V59A The bacteria were sequentially introduced into the recombinant strain SAT9 of Corynebacterium glutamicum via electroporation, and then integrated sequentially into the chromosome of the recombinant strain SAT9 of Corynebacterium glutamicum using the traceless operation method described in the authorized patent CN201710215459.7. zwf A243T ,gnd S361F pyc P458S mqo T224 lysC T311I hom V59A Mutations were performed, and the resulting positive clones were used as recipient bacteria for the next round of electroporation. Each integration step was verified by sequencing to ensure that the target site mutation was correct, ultimately yielding strain SAT9-M6.

[0052] Example 4: Construction and application of recombinant bacteria containing the pCRISPRi-high / mid / low-malR plasmid. (1) Construction of recombinant bacteria containing the pCRISPRi-high / mid / low-malR plasmid The plasmids pCRISPRi-high-malR, pCRISPRi-mid-malR, and pCRISPRi-low-malR, which had correct sequencing results from Example 2, were electroporated and integrated into *Corynebacterium glutamicum* SAT9-M6 obtained in Example 3. The plasmids were then evenly plated on chloramphenicol-resistant (final concentration 10 μg / mL) BHI solid plates. Single colonies were picked and verified by PCR using the following primers: test-cri-F / test-cri-R. Colonies with correct sequencing were considered successfully inserted, yielding plasmids capable of downregulating transcription factor genes. malRThe expression strains are SAT9-M6-high-malR, SAT9-M6-mid-malR, and SAT9-M6-low-malR.

[0053] The pdCas9gRNA plasmid was introduced into the recombinant strain SAT9-M6 of Corynebacterium glutamicum via electroporation to obtain strain SAT9-M6-pXi, which served as the control strain.

[0054] (2) Shake-flask fermentation of the production strain Shake-flask fermentation was performed on strains SAT9-M6-high-malR, SAT9-M6-mid-malR, SAT9-M6-low-malR, and SAT9-M6-pXi.

[0055] First, strains SAT9-M6-high-malR, SAT9-M6-mid-malR, SAT9-M6-low-malR, and SAT9-M6-pXi were streaked onto chloramphenicol-resistant BHI solid medium and incubated at 30 °C for approximately 18 h. Single colonies from the plates were then inoculated into 5 mL of BHI liquid medium (with a final concentration of 10 μg / mL chloramphenicol) and incubated at 30 °C and 220 rpm for approximately 12 h. One mL of this liquid medium was then transferred to CGIII medium and incubated for another 12 h. The initial OD was used as the starting point for the culture. 600 The culture medium was approximately 0.5, transferred to CGXII medium with a final glucose concentration of 50 g / L, and cultured in a constant temperature shaker at 30 ℃ and 220 rpm for 72 h.

[0056] Lysine production was measured during the culture period (see Table 2). The results showed that after 72 hours of culture, transcription factor genes in Corynebacterium glutamicum were downregulated. malR The expression strains SAT9-M6-high-malR, SAT9-M6-mid-malR, and SAT9-M6-low-malR produced lysine at 1.95±0.09 g / L, 1.71±0.1 g / L, and 1.68±0.13 g / L respectively during shake-flask fermentation. The production of lysine by the control strain SAT9-M6-pXi was 1.53±0.12 g / L. The lysine production of SAT9-M6-high / mid / low-malR was higher than that of the control strain SAT9-M6-pXi, indicating that inhibiting the expression of the transcription factor MalR in the producing strains to different degrees is beneficial to increasing lysine production.

[0057] Table 2 Lysine production of malR strains with different intensities under shake-flask conditions.

[0058] Example 5: Construction and application of an engineered strain with high-intensity inhibition of the malR gene in strain ATCC 13032 (1) Construction of wild-type strain containing pCRISPRi-high-malR plasmid The plasmid pCRISPRi-high-malR, whose sequencing results were correct, was electroporated and integrated into Corynebacterium glutamicum ATCC 13032. The plasmid was then evenly plated on chloramphenicol-resistant (final concentration 10 μg / mL) BHI agar plates. Single colonies were picked and verified by PCR using the primers test-cri-F / test-cri-R. Colonies with correct sequencing were considered successfully inserted, yielding cells capable of downregulating transcription factor genes. malR The expression strain is ATCC-high-malR.

[0059] The pdCas9gRNA plasmid was introduced into Corynebacterium glutamicum ATCC13032 via electroporation to obtain strain ATCC-pXi, which served as the control strain.

[0060] (2) Shake-flask fermentation of the production strain Shake-flask fermentation was performed on strains ATCC-high-malR and ATCC-pXi.

[0061] First, streaks of ATCC-high-malR and ATCC-pXi strains were performed on chloramphenicol-resistant BHI solid medium and incubated at 30 °C for approximately 18 h. Single colonies from the plates were then inoculated into 5 mL of BHI liquid medium (with a final concentration of 10 μg / mL chloramphenicol) and incubated at 30 °C and 220 rpm for approximately 12 h. One mL of this inoculum was then transferred to CGIII medium and incubated for another 12 h. The initial OD was used as the starting point for the culture. 600 The culture medium was approximately 0.5, transferred to CGXII medium with a final glucose concentration of 50 g / L, and cultured in a constant temperature shaker at 30 ℃ and 220 rpm for 72 h.

[0062] Lysine production was measured during the culture period (see Table 3). The results showed that after 72 hours of culture, transcription factor genes in Corynebacterium glutamicum were downregulated. malR The lysine yield of the expression strain ATCC-high-malR during shake-flask fermentation was 0.28±0.18 g / L, while that of the control strain ATCC-pXi was 0.18±0.12 g / L. ATCC-high-malR achieved a yield 1.56 times that of the control strain ATCC-pXi.

[0063] Table 3 Lysine production of wild-type strain with high-intensity weakened expression of malR under shake-flask conditions.

[0064] The results in Tables 2 and 3 show that inhibiting the expression of transcription factor MalR to varying degrees in different Corynebacterium glutamicum hosts (strains ATCC13032 and SAT9-M6) can achieve the technical effect of increasing lysine production.

[0065] Meanwhile, the above examples, using lysine as an example amino acid, verified the effectiveness of weakening the transcription factor MalR in increasing the yield of aspartic acid family amino acids. L-lysine, L-methionine, L-threonine, and L-isoleucine belong to the aspartic acid family of amino acids. They share an upstream synthetic pathway from oxaloacetate through aspartic acid, aspartic-4-phosphate, aspartic semialdehyde to key branch intermediates (such as tetrahydropyridine dicarboxylic acid and homoserine). Only in the downstream branch steps are they catalyzed by different enzymes to enter their respective specific synthetic branches. As a global transcriptional regulator, MalR's regulatory targets may cover one or more key enzymes (such as aspartate kinase and aspartic semialdehyde dehydrogenase) in the upstream shared pathway of the aspartic acid family. Therefore, when MalR expression is weakened by CRISPRi, the overall flux of the upstream shared pathway is increased, and the carbon flux entering each branch product may increase accordingly. Based on this metabolic network structure principle, those skilled in the art can reasonably expect that introducing the pCRISPRi-high / mid / low-malR plasmid of the present invention or a similar MalR attenuation / inhibition expression regulatory element into the corresponding aspartic acid production chassis strains such as L-methionine, L-threonine, or L-isoleucine, and culturing them under the conventional fermentation conditions for each amino acid, will also achieve the same yield enhancement effect. In specific applications, only the parameters such as carbon source, precursor addition, and culture conditions need to be adjusted according to the fermentation process of the target product; no substantial changes to the MalR regulation strategy itself are required.

[0066] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. The application of transcription factor MalR in regulating the production of feed amino acids, characterized in that, The production of feed amino acids is increased by downregulating or inhibiting the expression of transcription factor MalR in the producing strain. The feed amino acids include: lysine, methionine, threonine, and isoleucine; The amino acid sequence of the transcription factor MalR is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The hosts used for the production strains include: Corynebacterium glutamicum, Escherichia coli, Bacillus, and yeast.

3. A genetically engineered bacterium that increases lysine production by regulating the expression level of transcription factor MalR, characterized in that... The engineered bacteria were obtained by downregulating the expression of the transcription factor MalR using a lysine-producing strain as the host.

4. The genetically engineered bacteria as described in claim 3, characterized in that, The method for downregulating the expression of transcription factor MalR is to construct a CRISPRi recombinant plasmid that downregulates MalR expression using the CRISPRi technology, and then introduce the CRISPRi recombinant plasmid into a lysine-producing strain.

5. The genetically engineered bacteria as described in claim 4, characterized in that, The CRISPRi recombinant plasmid that downregulates MalR expression uses pdCas9gRNA as a vector backbone and is inserted into Corynebacterium glutamicum. malR The sgRNA expression cassette of the gene was obtained.

6. The genetically engineered bacteria as described in claim 5, characterized in that, The CRISPRi recombinant plasmid that downregulates MalR expression is the pCRISPRi-high-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.3; or, The CRISPRi recombinant plasmid that downregulates MalR expression is the pCRISPRi-mid-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.4; or, The CRISPRi recombinant plasmid that downregulates MalR expression is pCRISPRi-low-malR plasmid, and its nucleotide sequence is shown in SEQ ID NO.

5.

7. The genetically engineered bacteria as described in claim 3, characterized in that, The lysine-producing strain is Corynebacterium glutamicum.

8. The genetically engineered bacteria as described in claim 7, characterized in that, The lysine-producing strains include *Corynebacterium glutamicum* ATCC13032 or *Corynebacterium glutamicum* SAT9-M6; the genotype of *Corynebacterium glutamicum* SAT9-M6 is: C. glutamicum ATCC13032 ldhA pta pqo catzwf A243T gnd S361F pyc P458S mqo T224 lysC T311I hom V59A .

9. The application of the genetically engineered bacteria according to claim 3 in the production of lysine.

10. The application as described in claim 9, characterized in that, This includes the process of fermenting and culturing the genetically engineered bacteria and obtaining lysine from the culture.

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