L-tryptophan genetically engineered bacterium, construction method and application thereof

Abstract

The application provides an L-tryptophan genetically engineered bacterium and a construction method and application thereof. The L-tryptophan genetically engineered bacterium is obtained by using metabolic engineering means to edit genes of an Escherichia coli as a starting strain: the genes of tnaA trpR tyrR trpL ackA poxB ldhA adhE aroG trpE tktA talB yddG aroE aroD trpBA trpC The strain has a clear genetic background, can stably and efficiently produce L-tryptophan by biological fermentation, has very low by-products, and especially has very low organic acids in the related metabolic pathways, and greatly improves the carbon source utilization rate.​​​​​​​​​​​​​​​​

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an L-tryptophan genetically engineered bacterium, its construction method, and its application. Background Technology

[0002] L-Tryptophan (L-trp) is a versatile aromatic amino acid that not only participates in protein synthesis but also plays a crucial role in human immune regulation. It is commonly used as a feed additive and a precursor in pharmaceutical synthesis. In recent years, with a deeper understanding of the bioactivity of L-Tryptophan and the expansion of its applications, global demand for L-Tryptophan has increased dramatically, leading to a supply shortage.

[0003] The primary method for producing L-tryptophan is microbial fermentation, which uses inexpensive raw materials such as glucose as carbon sources. In recent years, significant progress has been made in constructing tryptophan-producing strains in *E. coli* using gene editing technology. *E. coli* possesses advantages such as a clear genetic background, mature gene editing technology, rapid growth, and low nutrient requirements, making it an excellent starting strain for the microbial fermentation industry. However, the tryptophan synthesis pathway in *E. coli* is lengthy, involving the shikimic acid pathway, branched acid pathways, and complex key enzyme feedback mechanisms. This leads to the accumulation of byproducts, especially organic acids from related metabolic pathways, such as 3-dehydroshikimic acid, shikimic acid, and anthranilic acid, during L-tryptophan production. Furthermore, the difficulty in controlling industrial fermentation processes easily generates byproducts such as acetic acid, lactic acid, and ethanol.

[0004] Therefore, how to increase L-tryptophan production while reducing the accumulation of byproducts, thereby improving production efficiency, is an urgent problem to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an L-tryptophan genetically engineered bacterium.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-tryptophan genetically engineered bacteria.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-tryptophan genetically engineered bacteria.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A type of L-tryptophan genetically engineered bacterium, based on Escherichia coli Escherichia coli W3110 was obtained through gene editing using metabolic engineering techniques, starting with the gene knockout of [a specific gene]. tnaA Gene, trpR Gene,tyrR Gene, trpL Gene, ackA Gene, poxB Gene, ldhA Gene, adhE Genes; overexpression to understand feedback aroG Genes, de-feedback trpE Gene, tktA Gene, talB Gene, yddG Genes, mutations aroE Genes, mutations aroD In genes, in the tryptophan operon trpBA Genes, Aspergillus niger ( Aspergillus niger (Source) trpC Gene, wherein the mutated aroE Genes and mutations aroD Genes are overexpressed in the genome in the form of miniature artificial operons. aroED .

[0009] The above-mentioned L-tryptophan genetically engineered bacteria will trpE Genes undergo site-directed mutagenesis to resolve feedback. trpE The serine residue at position 40 of the gene is mutated to phenylalanine; aroG Genes undergo site-directed mutagenesis to resolve feedback. aroG The 146th amino acid residue of the gene is mutated from aspartic acid to asparagine; aroE Genes undergo site-directed mutations. aroE The gene mutates threonine at position 61 to tryptophan and leucine at position 241 to isoleucine; aroD Genes undergo site-directed mutations. aroD The serine residue at position 141 of the gene is mutated to proline.

[0010] Preferably, the L-tryptophan genetically engineered bacteria mentioned above are Escherichia coli strains. Escherichia coli W3110 is Escherichia coli W3110 ATCC 27325 (commercially available strain).

[0011] Preferably, the above-mentioned L-tryptophan genetically engineered bacteria, the mutated aroE Genes and mutations aroD The gene is overexpressed in the genome via a TRC promoter and is tandemly integrated with... aroE Genes and aroD A gene, with an RBS sequence inserted in the middle of the gene sequence, forms a miniature artificial operon. aroED The artificial manipulator aroED The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing, and it can be expressed in tandem.aroE Gene-encoded 3-phosphoglycerate dehydrogenase, aroD The nucleotide sequence of the trc promoter is shown in SEQ ID NO.1; the nucleotide sequence of the RBS is shown in SEQ ID NO.20.

[0012] The artificial manipulator aroED ,include aroE , aroD Genes, transcribed by the same trc control operon and terminated by the same rrnB T1 terminator, were obtained by optimizing gene linkage and gene placement within the operon. Specifically, the operon gene sequence is Ptrc, aroE RBS aroD ,rrnB T1 terminator, the Ptrc and aroE The genes are directly linked end to end. aroD It is directly linked to the rrnB T1 terminator at both ends; the nucleotide sequence of the rrnB T1 terminator is shown in SEQ ID NO.21 of the sequence listing.

[0013] Preferably, the above-mentioned L-tryptophan-engineered bacteria are enhanced using the trc promoter. aroG Gene, trpE Gene, tktA Gene, talB Gene, yddG Gene, trpBA Gene transcription; heterologous introduction of Aspergillus niger-derived gene trpC Genes were transcribed using the trc promoter; artificial operons were overexpressed on the genome. aroED Transcription was initiated using the trc promoter.

[0014] Preferably, the L-tryptophan-engineered bacteria described above, through site-directed mutagenesis... aroG The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; site-directed mutagenesis. trpE The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing; tktA The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing; talB The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing; yddG The nucleotide sequence of the gene is shown in SEQ ID NO. 6 of the sequence listing; site-directed mutagenesis. aroE The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing; site-directed mutagenesis. aroD The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing; trpBAThe nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing; it is derived from Aspergillus niger. trpC The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing.

[0015] Preferably, the above-mentioned L-tryptophan genetically engineered bacteria, tnaA The nucleotide sequence of the gene is shown in SEQ ID NO. 12 of the sequence listing; trpR The nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing; tyrR The nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing; trpL The nucleotide sequence of the gene is shown in SEQ ID NO.15 of the sequence listing; ackA The nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing; poxB The nucleotide sequence of the gene is shown in SEQ ID NO.17 of the sequence listing; ldhA The nucleotide sequence of the gene is shown in SEQ ID NO.18 of the sequence listing; adhE The nucleotide sequence of the gene is shown in SEQ ID NO.19 of the sequence listing.

[0016] The above-mentioned method for constructing L-tryptophan-engineered bacteria involves starting with the strain... Escherichia coli The following are the specific steps for targeted modification based on the W3110: (1) Knockout of the enzyme encoding tryptophanase tnaA Gene; (2) Knockout of the protein encoding repressor trpR Gene, and insert at that site to relieve feedback inhibition aroG Genes are started using the trc promoter; (3) Knockout of the protein encoding repressor tyrR Gene, and insert at that site tktA Genes are started using the trc promoter; (4) Knockout of the leader peptide trpL Genes and Genome Origins trpE and at that point, use the solution feedback trpE Gene( trpE fbr (Gene) Replace the original trpLE Genes, and started using the trc promoter; trpLE Genes include the original trpL and the original trpE Gene; (5) Overexpressed on the genome talB Gene; (6) Overexpressed on the genome yddG Gene; (7) Knockout ackA Gene, poxB Gene, ldhA Gene, adhE Gene; (8) Overexpression of artificial operons on the genome aroED ; (9) Overexpression of the tryptophan operon in the genome trpBA Gene; (10) Overexpression of Aspergillus niger-derived gene on the genome trpC Gene.

[0017] The application of the above-mentioned L-tryptophan genetically engineered bacteria in the production of L-tryptophan.

[0018] Preferably, the above-mentioned application involves producing L-tryptophan using a fermentation method in a shake flask or fermenter.

[0019] Preferably, in the above application, a mechanically stirred fermenter is used for fermentation, and the specific steps are as follows: (1) Slant culture: The genetically engineered strain was inoculated on LB solid medium as the first generation slant culture and cultured at 32-34℃ for 10-14h; the colonies of the first generation slant culture were inoculated on another LB slant medium as the second generation slant culture and cultured at 32-34℃ for 10-12h. (2) Seed culture: The second generation slant was washed with sterile water into the seed culture medium. The culture temperature was 36℃. The pH of the seed culture was maintained at 6.7±0.2 and the dissolved oxygen value was 35-45% by automatically adding ammonia solution through the bioreactor. When the OD600nm of the seed culture reached 18-20, the next fermentation culture was carried out. (3) Fermentation culture: The inoculum amount is 20-30%, the culture temperature is 34℃, and the pH of the seed culture is maintained at 7.0±0.2 and the dissolved oxygen value is 45%-55% by automatically adding ammonia solution through the bioreactor.

[0020] Preferably, in the above application, the seed culture medium used in step (2) is: glucose 30-40 g / L, yeast extract powder 4-6 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-6 g / L, anhydrous magnesium sulfate 1-4 g / L, V H 0.5-2 mg / L, V B1 0.5-2 mg / L, the remainder is water.

[0021] Preferably, in the above application, the fermentation medium used in step (3) is: glucose 20-30 g / L, yeast extract powder 4-8 g / L, ammonium sulfate 2-6 g / L, potassium dihydrogen phosphate 6-10 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 40-80 mg / L, manganese sulfate monohydrate 10-20 mg / L, V H 0.1-1 mg / L, V B1 1-2 mg / L, the remainder is water.

[0022] Preferably, in the above application, after the glucose in the culture medium is consumed in step (3), a glucose solution is added to maintain the glucose concentration in the fermentation culture medium at 0.1-1 g / L.

[0023] All of the above-mentioned culture media can be prepared using standard methods.

[0024] Beneficial effects: The aforementioned L-tryptophan-engineered bacteria, through the redirection of tryptophan-related metabolic fluxes in *E. coli*, used metabolic engineering techniques to gene-edit wild-type *E. coli*, deleting [a specific substance / phenomenon]. tnaA , trpR , tyrR , trpL The gene blocks the tryptophan degradation pathway, inactivates the repressor proteins trpR and tyrR, and removes the leader peptide regulating the tryptophan operon; overexpression enables feedback. aroG Genes, de-feedback trpE Gene, tktA Gene, talB Gene, yddG Genes, on trpE The gene underwent site-directed mutation to relieve tryptophan feedback inhibition; the TRC promoter was used to enhance [the gene's function]. talB , tktA Gene transcription enhanced the metabolic flux of the cladistic acid pathway and the supply of the precursor erythrose 4-phosphate, resulting in good tryptophan synthesis capacity. Subsequently, the metabolic flux of related organic acids in the tryptophan-engineered bacteria was optimized, and [the following was observed]: [deletion of certain components]. ackA Gene, poxB Gene, ldhA Gene, adhE Genes, overexpressing the mutated form aroE Genes, mutations aroD Gene, trpBA Aspergillus niger source trpCThe resulting strain exhibits a clear genetic background and can stably and efficiently produce L-tryptophan via bio-fermentation with extremely low byproduct levels, particularly organic acids involved in related metabolic pathways, such as 3-dehydroshikimic acid, shikimic acid, and anthranilic acid, significantly improving carbon source utilization. Furthermore, it does not accumulate organic acids, such as acetic acid, lactic acid, and ethanol, which are easily produced as byproducts during industrial fermentation. Ultimately, after 40 hours of fermentation in a 5L fermenter, 60.7 g / L of tryptophan was produced, achieving a sugar-acid conversion rate of 23%, demonstrating significant economic advantages for industrial production. Attached Figure Description

[0025] Figure 1 Bar chart showing tryptophan yield and conversion rate for L-tryptophan producing strains Y7, Y11, and Y12.

[0026] Figure 2 Bar chart showing tryptophan yield and conversion rate for L-tryptophan producing strains Y13 and Y14. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0028] Unless otherwise specified, the percentage sign "%" in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0029] The starting strain used in the examples was wild-type. Escherichia coli W3110 ATCC 27325 (commercially available), the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0030] Table 1 Primers used in strain construction

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] The gene editing methods used in the gene manipulations in this embodiment are referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0040] Example 1 This embodiment aims to illustrate the specific steps for constructing the strain. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0041] (1) Knockout tnaA Genes: Escherichia coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using tnaA-US / tnaA-UA and tnaA-DS / tnaA-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using tnaA-US and tnaA-DA primers. Using pGRB-tnaA-s and pGRB-tnaA-a primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain tnaA-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragments and tnaA-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain Y1.

[0042] (2) Knockout trpR Genes and integration aroG Gene: Escherichia coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by amplification using trpR-US / trpR-UA and trpR-DS / trpR-DA, respectively. Then, the mutant nucleotide sequence was designed onto aroGtb-A and aroGtb-S, with homologous sequences. PCR amplification was performed using primers aroG-1 / aroGtb-A and aroGtb-S / aroG-2 to obtain fragment 1 and fragment 2, respectively. Finally, using fragment 1 and fragment 2 as templates, overlap PCR was performed using primers aroG-1 / aroG-2 to obtain the target fragment P. trc -aroGfbr Using upstream and downstream homologous arms and the target gene fragment as templates, and trpR-US and trpR-DA as primers, overlapping fragments were amplified by overlapping PCR to obtain overlapping fragments. Using pGRB-trpR-s and pGRB-trpR-a as primers, gRNA fragments were annealed and ligated to the pGRB vector to obtain trpR-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragments and trpR-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain Y2.

[0043] (3) Knockout tyrR Genes and integration tktA Genes: The same operation method as in (2) is used, except that the primers used are tyrR-US / tyrR-UA, tyrR-DS / tyrR-DA, tktA-trc-s / tktA-trc-a, pGRB-tyrR-s, pGRB-tyrR-a, the competent cells are Y2, and strain Y3 is obtained.

[0044] (4) Knockout trpLE The gene integrates Ptrc-trpE at this site. fbr Using the E. coli W3110 genome as a template, the DNA sequence of the exogenous cleavage site was segmented and added to primers trpLE-2 and trpLE-3. Upstream and downstream homologous arms were obtained by PCR amplification using primers trpLE-1 and trpLE-2, and trpLE-3 and trpLE-4, respectively. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed using primers trpLE-1 and trpLE-4 to obtain the knockout gene trpLE along with the required complement fragment from the exogenous cleavage site. Using pGRB-LE-s and pGRB-LE-a as primers, the gRNA fragment was annealed and ligated into the pGRB vector to obtain pGRB-LE. Y3 electroporation competent cells were prepared, and the target fragment and pGRB-LE were electroporated into competent cells. Positive transformants were screened to obtain strain Y4. Next, using *E. coli* W3110 as a template, PCR amplification was performed using primers trpE-1 and trpE-2, trpE-5 and trpE-6 to obtain upstream and downstream homologous arms. The mutant nucleotide sequence was designed to be located on trpEtb-A and trpEtb-S, and homologous sequences were present. PCR amplification was performed using primers trpE-3 / trpEtb-A and trpEtb-S / trpE-4 to obtain fragment 1 and fragment 2, respectively. Then, using fragment 1 and fragment 2 as templates, overlap PCR was performed using primers trpE-3 / trpE-4 to obtain the target fragment P. trc -trpE fbrUsing pGRB-1-s and pGRB-1-a as primers, gRNA fragments were annealed and ligated with the pGRB vector to obtain pGRB-1. YTP04 electrotransformation competent cells were prepared, and the target fragment and pGRB-1 were electrotransformed into competent cells together. Positive transformants were screened to obtain strain Y5.

[0045] (5) In ilvG Pseudogene sites are controlled using the TRC promoter. talB Gene overexpression: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms and the target gene fragment were amplified by PCR using ilvG-US / ilvG-UA, ilvG-DS / ilvG-DA, and talB-s / talB-a, respectively. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were amplified by overlapping PCR using ilvG-US and ilvG-DA as primers. The gRNA fragment was annealed using pGRB-ilvG-s and pGRB-ilvG-a as primers and ligated into the pGRB vector to obtain pGRB-ilvG. YTP06 electrotransformation competent cells were prepared, and the target fragment and pGRB-ilvG were electrotransformed into competent cells together. Positive transformants were screened to obtain strain Y6.

[0046] (6) In rph Pseudogene sites are controlled using the TRC promoter. yddG Gene overexpression: The same operation method as in (5) is used, except that the primers used are rph-US, rph-UA, rph-DS, rph-DA, yddG-s, yddG-a, pGRB-rph-s, and pGRB-rph-a. The competent cells are Y6, and strain Y7 is obtained.

[0047] (7) Knockout ackA Gene: It has the same operation method as in (1), except that the primers used are ackA-US / ackA-UA, ackA-DS / ackA-DA, pGRB-ackA-s, pGRB-ackA-a, the competent cells are Y7, and the strain Y8 is obtained.

[0048] (8) Knockout poxB Gene: It has the same operation method as in (1), except that the primers used are poxB-US / poxB-UA, poxB-DS / poxB-DA, pGRB-poxB-s, pGRB-poxB-a, the competent cells are Y8, and the strain Y9 is obtained.

[0049] (10) Knockout ldhAGene: It has the same operation method as in (1), except that the primers used are ldhA-US / ldhA-UA, ldhA-DS / ldhA-DA, pGRB-ldhA-s, pGRB-ldhA-a, the competent cells are Y9, and the strain obtained is Y10.

[0050] (11) Knockout adhE Gene: It has the same operation method as in (1), except that the primers used are adhE-US / adhE-UA, adhE-DS / adhE-DA, pGRB-adhE-s, pGRB-adhE-a, the competent cells are Y10, and the strain Y11 is obtained.

[0051] (12) In dhaL Gene loci use TRC promoters to control artificial operons aroED Overexpression: Escherichia coli W3110 Using the genome as a template, upstream and downstream homologous arms were obtained by amplification using dhaL-US / dhaL-UA and dhaL-DS / dhaL-DA, respectively. Then, the mutant nucleotide sequence was designed onto aroEtb-1, aroEtb-2, aroEtb-3, and aroEtb-4, with homologous sequences. PCR amplification was performed using primers aroE-1 / aroEtb-1, aroEtb-2 / aroEtb-3, and aroEtb-4 / aroE-4 to obtain fragments 1, 2, and 3, respectively. Then, using fragments 1, 2, and 3 as templates, overlap PCR was performed using primers aroE-1 / aroE-4 to obtain the target fragment P. trc -aroE-RBS; The mutant nucleotide sequence was designed on aroDtb-A and aroDtb-S, and homologous sequences were present. PCR amplification was performed using primers aroD-3 / aroDtb-A and aroDtb-S / aroD-4 to obtain fragment 4 and fragment 5, respectively. Then, using fragment 4 and fragment 5 as templates, overlap PCR was performed using primers aroD-3 / aroD-4 to obtain the target fragment RBS-aroD; overlap PCR was performed using primers aroE-3 / aroD-4 to obtain the target fragment P. trc -aroED; Overlap PCR was performed using primers dhaL-US / dhaL-DA to obtain overlapping fragments; using pGRB-dhaL-s and pGRB-dhaL-a as primers, gRNA fragments were annealed and ligated into the pGRB vector to obtain trpR-pGRB; Y11 electrotransformed competent cells were prepared, and the overlapping fragments and dhaL-pGRB were electrotransformed into competent cells together, and positive transformants were screened to obtain strain Y12.

[0052] (13) In yjiVPseudogene sites are controlled using the TRC promoter. trpBA Gene overexpression: Escherichia coli W3110 Using the genome as a template, upstream and downstream homologous arms and the target gene fragment were amplified by PCR using yjiV-US / yjiV-UA, yjiV-DS / yjiV-DA, and trpBA-trc-s / trpBA-trc-a, respectively. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were amplified by overlapping PCR using yjiV-US and yjiV-DA as primers. Using pGRB-yjiV-s and pGRB-yjiV-a as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain pGRB-yjiV. Y12 electroporation competent cells were prepared, and the target fragment and pGRB-yjiV were electroporated together into competent cells. Positive transformants were screened to obtain strain Y13.

[0053] (14) In yjiT Pseudogene sites are controlled using the TRC promoter. trpC Gene overexpression: Escherichia coli W3110 Using yjiT-US / yjiT-UA and yjiT-DS / yjiT-DA as templates, upstream and downstream homologous arms were obtained by PCR amplification, respectively, using Aspergillus niger synthesized by Genewiz Biotechnology Co., Ltd. trpC Using the gene as a template, the target gene fragment was amplified by PCR using trpC-trc-s / trpC-trc-a. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, and yjiT-US and yjiT-DA as primers, overlapping fragments were amplified by overlapping PCR to obtain overlapping fragments. Using pGRB-yjiT-s and pGRB-yjiT-a as primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain pGRB-yjiT. Y13 electroporation competent cells were prepared, and the target fragment and pGRB-yjiT were electroporated together into competent cells. Positive transformants were screened to obtain strain Y14.

[0054] Example 2 L-tryptophan was produced by fermentation of strains Y7, Y11, and Y12 described in Example 1 in a 5L fermenter, with the specific steps as follows: (1) Slant culture: L-tryptophan producing strains were inoculated onto slant culture medium as first-generation slant culture and cultured at 34℃ for 12h; colonies from the first-generation slant culture were inoculated onto new slant culture medium as second-generation slant culture and cultured at 34℃ for 10h; the slant culture medium used was general LB solid medium. (2) Seed culture: The second-generation slant was washed with sterile water into the seed culture medium. The culture temperature was 36℃. A 25% ammonia solution was automatically added via a 5L bioreactor to maintain the pH of the seed culture at 6.7 and the dissolved oxygen value at 45%. The OD of the seed culture was... 600nm Once the concentration reaches 18, the next fermentation stage begins. The seed culture medium used is: glucose 30 g / L, yeast extract 5 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 6 g / L, anhydrous magnesium sulfate 1 g / L, and vitamin C. H 1mg / L, V B1 2 mg / L, the remainder being water; (3) Fermentation culture: The inoculum size was 20%, and the culture temperature was 34℃. A 25% ammonia solution was automatically added via a bioreactor to maintain the seed culture pH at 7.0 and dissolved oxygen at 45%. The fermentation medium used was: glucose 20g / L, yeast extract powder 6g / L, ammonium sulfate 3g / L, potassium dihydrogen phosphate 7g / L, anhydrous magnesium sulfate 2g / L, ferrous sulfate heptahydrate 40mg / L, manganese sulfate monohydrate 10mg / L, and V... H 1mg / L, V B1 1 mg / L, the remainder being water; after the glucose in the culture medium is consumed, add 80% (m / v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L; the fermentation cycle is 40 h.

[0055] like Figure 1 As shown in Table 2, strains Y7, Y11, and Y12 achieved final L-tryptophan yields of 49.5 g / L, 50.1 g / L, and 52.6 g / L, respectively, in a 5 L fermenter; the sugar-acid conversion rates were 19%, 20.1%, and 21.5%, respectively; the organic acid contents are shown in Table 2. Y11 had lower accumulations of acetic acid, lactic acid, and ethanol than Y7, while Y12 had lower accumulations of 3-dehydroshikimic acid and shikimic acid than Y11.

[0056] Table 2

[0057] Example 3 The strain Y12-1 was constructed using the same method as steps (1) to (12) in Example 1, except that in step (12)... dhaL Gene loci use TRC promoters to control artificial operons aroDE (Its nucleotide sequence is shown in SEQ ID NO. 138 of the sequence listing) Overexpression: Escherichia coli W3110Using the genome as a template, upstream and downstream homologous arms were obtained by amplification using dhaL-US / dhaL-UA and dhaL-DS / dhaL-DA, respectively. Then, the mutant nucleotide sequences were designed into aroDtb-A and aroDtb-S, and PCR amplification was performed using primers aroD-3s / aroDtb-A and aroDtb-S / aroD-4a to obtain fragments 4 and 5, respectively. Finally, using fragments 4 and 5 as templates, overlap PCR was performed using primers aroD-3s / aroD-4a to obtain the target fragment P. trc -aroD-RBS; Mutant nucleotide sequences were designed on aroEtb-1, aroEtb-2, aroEtb-3, and aroEtb-4, with homologous sequences. PCR amplification was performed using primers aroE-1 / aroEtb-1, aroEtb-2 / aroEtb-3, and aroEtb-4 / aroE-4 to obtain fragments 1, 2, and 3, respectively. Then, using fragments 1, 2, and 3 as templates, PCR was performed using primers aroE-1s / aroE-4a. Overlap PCR was performed to obtain the target fragment RBS-aroE; overlap PCR was performed using primers dhaL-US / dhaL-DA to obtain the overlap fragment; using pGRB-dhaL-s and pGRB-dhaL-a as primers, the gRNA fragment was annealed and ligated with the pGRB vector to obtain trpR-pGRB; Y11 electrotransformed competent cells were prepared, and the overlap fragment and dhaL-pGRB were electrotransformed into competent cells together, and positive transformants were screened to obtain strain Y12-1.

[0058] The 3-dehydroshikimic acid of strain Y12 and strain Y12-1 is compared in Table 3 below.

[0059] Table 3 Y12 1.97 0.05 Y12-1 2.41 0.11

[0060] As shown in the table above, the accumulation of 3-dehydroshikimic acid in Y12(aroED) is 1.97 g / L and the shikimic acid is 0.05 g / L, both lower than those in Y12-1(aroDE). Therefore, subsequent genomic operations were performed in Y12.

[0061] Example 4 L-tryptophan was produced by fermentation of strains Y13 and Y14 described in Example 1 in 5L fermenters, with the specific steps being the same as in Example 2.

[0062] like Figure 2As shown, strains Y13 and Y14 produced L-tryptophan in a 5L fermenter, with maximum yields of 55.1 g / L and 60.7 g / L, respectively. The sugar-acid conversion rates at the end of fermentation were 22% and 23%, respectively. Table 4 shows a comparison of various organic acids; Y14 exhibited almost no organic acid accumulation, demonstrating a clear advantage.

[0063] Table 4

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An L-tryptophan-engineered bacterium, characterized in that: E. coli E. coli W3110 was obtained through gene editing using metabolic engineering techniques, starting with the gene knockout of [a specific gene]. tnaA Gene, trpR Gene, tyrR Gene, trpL Gene, ackA Gene, poxB Gene, ldhA Gene, adhE Gene; overexpressed aroG Gene, trpE Gene, tktA Gene, talB Gene, yddG Genes, mutations aroE Genes, mutations aroD In genes, in the tryptophan operon trpBA Genetic, Aspergillus niger-derived trpC Gene, wherein the mutated aroE Genes and mutations aroD Genes are overexpressed in the genome in the form of miniature artificial operons. aroED .

2. The L-tryptophan-engineered bacterium according to claim 1, characterized in that: The mutation aroE The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing. The mutated... aroD The nucleotide sequence of the gene is shown in SEQ ID NO. 8 of the sequence listing, and the artificial operon aroED The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing.

3. The L-tryptophan-engineered bacterium according to claim 2, characterized in that: Enhanced using the TRC promoter aroG Gene, trpE Gene, tktA Gene, talB Gene, yddG Gene, trpBA Gene transcription; Heterogeneous introduction of Aspergillus niger source trpC Genes were transcribed using the trc promoter; artificial operons were overexpressed on the genome. aroED Transcription was initiated using the trc promoter, the nucleotide sequence of which is shown in SEQ ID NO.1 of the sequence listing.

4. The L-tryptophan-engineered bacterium according to claim 3, characterized in that: The aroG The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing; trpE The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing; tktA The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing; talB The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing; yddG The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing; trpBA The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing; it is derived from Aspergillus niger. trpC The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing.

5. The L-tryptophan-engineered bacterium according to claim 1, characterized in that: The tnaA The nucleotide sequence of the gene is shown in SEQ ID NO.12 of the sequence listing; trpR The nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing; tyrR The nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing; trpL The nucleotide sequence of the gene is shown in SEQ ID NO. 15 of the sequence listing; ackA The nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing; poxB The nucleotide sequence of the gene is shown in SEQ ID NO.17 of the sequence listing; ldhA The nucleotide sequence of the gene is shown in SEQ ID NO.18 of the sequence listing; adhE The nucleotide sequence of the gene is shown in SEQ ID NO.19 of the sequence listing.

6. The method for constructing the L-tryptophan-engineered bacteria according to any one of claims 1-5, characterized in that: In the originating strain E. coli The following are the specific steps for targeted modification based on the W3110: (1) Knockout of the enzyme encoding tryptophanase tnaA Gene; (2) Knockout of the protein encoding repressor trpR Gene, and insert at that site to relieve feedback inhibition aroG Genes are started using the trc promoter; (3) Knockout of the protein encoding repressor tyrR Gene, and insert at that site tktA Genes are started using the trc promoter; (4) Knockout of the precursor peptide trpL Genes and Genome Origins trpE and at that site, the feedback inhibition was relieved. trpE Gene replacement trpLE Genes, and start them using the trc promoter; (5) Overexpressed on the genome talB Gene; (6) Overexpressed on the genome yddG Gene; (7) Knockout ackA Gene, poxB Gene, ldhA Gene, adhE Gene; (8) Overexpression of artificial operons on the genome aroED ; (9) Overexpression of the tryptophan operon in the genome trpBA Gene; (10) Overexpression of Aspergillus niger-derived gene on the genome trpC Gene.

7. The use of the L-tryptophan genetically engineered bacteria according to any one of claims 1-5 in the production of L-tryptophan.

8. The application according to claim 7, characterized in that: L-tryptophan is produced by fermentation in shake flasks or fermenters.

9. The application according to claim 7 or 8, characterized in that: Fermentation is carried out using a fermentation tank. The specific steps are as follows: (1) Slant culture: The genetically engineered strain was inoculated on LB solid medium as the first generation slant culture and cultured at 32-34℃ for 10-14h; the colonies of the first generation slant culture were inoculated on another LB slant medium as the second generation slant culture and cultured at 32-34℃ for 10-12h. (2) Seed culture: The second generation slant was washed with sterile water into the seed culture medium. The culture temperature was 36℃. The pH of the seed culture was maintained at 6.7±0.2 and the dissolved oxygen value was 35-45% by automatically adding ammonia solution through the bioreactor. When the OD600nm of the seed culture reached 18-20, the next fermentation culture was carried out. (3) Fermentation culture: The inoculum amount is 20-30%, the culture temperature is 34℃, and the pH of the seed culture is maintained at 7.0±0.2 and the dissolved oxygen value is 45%-55% by automatically adding ammonia solution through the bioreactor.

10. The application according to claim 9, characterized in that: The seed culture medium used in step (2) is: glucose 30-40 g / L, yeast extract powder 4-6 g / L, ammonium sulfate 1-3 g / L, potassium dihydrogen phosphate 1-6 g / L, anhydrous magnesium sulfate 1-4 g / L, V H 0.5-2 mg / L, V B1 0.5-2 mg / L, the remainder being water; the fermentation medium used in step (3) is: glucose 20-30 g / L, yeast extract powder 4-8 g / L, ammonium sulfate 2-6 g / L, potassium dihydrogen phosphate 6-10 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 40-80 mg / L, manganese sulfate monohydrate 10-20 mg / L, V H 0.1-1 mg / L, V B1 1-2 mg / L, the remainder being water; in step (3), after the glucose in the culture medium is consumed, glucose solution is added to maintain the glucose concentration in the fermentation culture medium at 0.1-1 g / L.

Images