Mutant tolr and its use in aromatic amino acid production strains

Abstract

The application relates to the field of genetic engineering, and discloses a mutant of TolR and application of the mutant in an L-phenylalanine production strain. The mutant comprises a TolR mutant obtained by respectively mutating an amino acid residue R85 in the 85th position of a TolR enzyme into G, A, S, T, C, V, I, L, M and P through site-directed mutation. The L-phenylalanine genetically engineered bacteria comprising the mutant can promote the ability of Escherichia coli to produce L-phenylalanine, and the yield is obviously improved by 20.74%, and the mutant has specific and large application prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to mutants of TolR and their application in aromatic amino acid-producing strains. Background Technology

[0002] Aromatic amino acids are amino acids whose molecular structure contains one or more aromatic rings, mainly including phenylalanine, tyrosine, and tryptophan. These three amino acids not only participate in protein synthesis in organisms but also have important physiological functions. L-phenylalanine is an α-amino acid with a benzene ring side chain, with the molecular formula C9H10. 11 NO2 is one of the eight essential amino acids for the human body—it cannot be synthesized de novo by the body itself and must be obtained from exogenous food or supplements. Its benzene ring structure gives it unique hydrophobicity, making it play a "structural backbone" role in protein folding, enzyme active site construction, and membrane protein anchoring. At the same time, L-phenylalanine is a precursor to key neurotransmitters such as tyrosine, dopamine, and norepinephrine, and is directly related to mood regulation, cognitive function, and stress response. Therefore, it is regarded as a "molecular switch" connecting nutrient intake and neuroendocrine homeostasis.

[0003] Due to its combination of nutritional value and reactivity, L-phenylalanine has transcended its traditional role as a "protein raw material" and become a core intermediate in multiple scenarios, including food, medicine, agriculture, and biomaterials. In the food industry, it is one of the two building blocks of the high-intensity sweetener aspartame, supporting the global market for sugar-free beverages and low-calorie baked goods. In the pharmaceutical industry, it is not only a basic component of compound amino acid infusions but also participates in the synthesis of antiviral, antitumor, and antidepressant drugs and is used in special medical foods for patients with phenylketonuria (PKU). In green agriculture, exogenous spraying can activate the phenylpropanoid metabolic pathway in plants, promoting the accumulation of lignin and flavonoids, thereby enhancing the lodging resistance and disease resistance of crops. In the emerging field of biomanufacturing, its aromatic ring side chains provide rigid units for biodegradable polymers, hydrogels, and biomimetic materials, and it is being developed as a high-end medical carrier and electronic skin raw material.

[0004] Early methods relied on soybean acid hydrolysis or chemical asymmetric synthesis, which suffered from bottlenecks such as low yield, high cost of racemic mixture resolution, and large emissions of waste. In the past two decades, systems metabolic engineering and synthetic biology have driven the production paradigm towards "microbial cell factories": using *E. coli* and *Corynebacterium glutamicum* as the substrate, by knocking out degradation pathways, introducing feedback-resistant mutations, optimizing the phenylpyruvate transaminase / dehydratase system, and enhancing the supply of precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP), the concentration of L-phenylalanine in fermenters has increased from <20 g / L. -1 Increased to 100 g L -1The optical purity is ≥99.5%, and the overall production cost is reduced by approximately 40%. The green downstream process, which couples continuous centrifugation, membrane separation, and simulated moving bed chromatography, reduces carbon emissions per ton of product by 60% compared to the chemical route, providing an environmentally friendly and economically viable supply solution for the rapidly growing functional food, special medical, and pharmaceutical markets. Summary of the Invention

[0005] The purpose of this invention is to provide a TolR mutant.

[0006] Another objective of this invention is to provide an application of a TolR mutant in L-phenylalanine and L-tryptophan producing strains, which significantly increases the yield of L-phenylalanine and L-tryptophan by improving the ability of Escherichia coli to produce L-phenylalanine and L-tryptophan.

[0007] The technical solution adopted to achieve the purpose of this invention is:

[0008] By modifying the TolR motor protein of the energy-transferring Tol complex in Escherichia coli using genetic engineering methods, strains that can expand the production of L-phenylalanine and L-tryptophan were obtained.

[0009] In a first aspect, the present invention provides a TolR mutant.

[0010] The TolR enzyme is an energy-transferring Tol complex motor protein, and the TolR mutant is a site-directed mutation of the energy-transferring Tol complex motor protein.

[0011] Further, the TolR mutant is a mutant obtained by site-directed mutagenesis of the 85th amino acid residue R85 of the wild-type TolR enzyme to G, A, S, T, C, V, I, L, M or P. In this application, the TolR mutants are represented by TolR(R85G), TolR(R85A), TolR(R85S), TolR(R85T), TolR(R85C), TolR(R85V), TolR(R85I), TolR(R85L), TolR(R85M) and TolR(R85P), respectively, wherein the amino acid sequence of the wild-type TolR enzyme is shown in SEQ ID NO: 1.

[0012] Secondly, the present invention provides the coding gene of the TolR mutant. The nucleotide sequence of the wild-type TolR is shown in SEQ ID NO: 28.

[0013] The TolR (R85G) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to ggt; the TolR (R85A) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to gct; the TolR (R85S) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to agt; the TolR (R85T) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to acg; and the TolR (R85C) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to tgt. The TolR (R85V) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to gtt; the TolR (R85I) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to atc; the TolR (R85L) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to ctg; the TolR (R85M) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to atg; and the TolR (R85P) is formed by mutating the nucleotide sequence encoding the 85th amino acid in TolR from cgt to cct.

[0014] Thirdly, the present invention provides L-phenylalanine and L-tryptophan genetically engineered bacteria containing the TolR mutant encoding gene.

[0015] The L-phenylalanine and L-tryptophan genetically engineered bacteria containing TolR mutants are obtained by mutating the 85th amino acid residue R85 of the TolR enzyme to G, A, S, T, C, V, I, L, M and P, respectively.

[0016] Furthermore, the host bacterium of the genetically engineered bacteria is Escherichia coli.

[0017] Fourthly, the present invention provides a method for constructing the L-phenylalanine and L-tryptophan genetically engineered bacteria, comprising the following steps:

[0018] S1. In L-phenylalanine-producing and L-tryptophan-producing Escherichia coli strains, the 85th amino acid residue R85 of the energy-transferring Tol complex motor protein was mutated to G, A, S, T, C, V, I, L, M, and P respectively through site-directed mutagenesis, resulting in mutants TolR (R85G), TolR (R85A), TolR (R85S), TolR (R85T), TolR (R85C), TolR (R85V), TolR (R85I), TolR (R85L), TolR (R85M), and TolR (R85P), thereby constructing L-phenylalanine-producing strains and L-tryptophan-producing strains. In this application, the L-phenylalanine genetically engineered bacteria constructed are represented by HTolR-1, HTolR-2, HTolR-3, HTolR-4, HTolR-5, HTolR-6, HTolR-7, HTolR-8, HTolR-9, and HTolR-10, and the L-tryptophan genetically engineered bacteria constructed are represented by KTolR-1, KTolR-2, KTolR-3, KTolR-4, KTolR-5, KTolR-6, KTolR-7, KTolR-8, KTolR-9, and KTolR-10.

[0019] S2. Further includes verifying the mutant, specifically by shake-flask fermentation;

[0020] Fourthly, the present invention provides the application of the TolR mutant or the L-phenylalanine and L-tryptophan genetically engineered bacteria in the production of aromatic amino acids such as L-phenylalanine and L-tryptophan.

[0021] Furthermore, the present invention also provides the application of the TolR mutant or the L-phenylalanine and L-tryptophan genetically engineered bacteria in the food or pharmaceutical fields.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The following mutants, TolR(R85G), TolR(R85A), TolR(R85S), TolR(R85T), TolR(R85C), TolR(R85V), TolR(R85I), TolR(R85L), TolR(R85M), and TolR(R85P), were successfully obtained through site-directed mutagenesis of TolR. This innovative modification has been applied to Escherichia coli, primarily targeting the energy transduction type TolR in E. coli. By modifying the complex motor protein and introducing mutants of TolR (R85G), TolR (R85A), TolR (R85S), TolR (R85T), TolR (R85C), TolR (R85V), TolR (R85I), TolR (R85L), TolR (R85M), and TolR (R85P), the biosynthesis and accumulation of L-phenylalanine and L-tryptophan were successfully promoted. This not only effectively enhanced the ability of *E. coli* to produce L-phenylalanine and L-tryptophan, achieving significant increases in yield of up to 20.74% and 61%, respectively, but also laid a solid foundation for the industrial production of L-phenylalanine and L-tryptophan, indicating enormous potential and value in promoting the development of related industries. Attached Figure Description

[0024] Figure 1 The results of shake-flask fermentation of the L-phenylalanine producing strain in Example 3 of this invention.

[0025] Figure 2 The results of shake-flask fermentation of the L-tryptophan-producing strain in Example 6 of this invention are shown. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0030] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0031] The amino acid sequences of the mutants TolR (R85G), TolR (R85A), TolR (R85S), TolR (R85T), TolR (R85C), TolR (R85V), TolR (R85I), TolR (R85L), TolR (R85M), and TolR (R85P) are all based on the wild-type TolR sequence shown in SEQ ID NO: 1, with substitution mutations.

[0032] Escherichia coli strain HDH6-D12 is described in the following literature: Yongfei Liu, Yinyin Zhuang, Dongqin Ding, Yiran Xu, Jibin Sun, Dawei Zhang . Biosensor-based evolution and elucidation of a biosynthetic pathway in Escherichia coli[J]. ACSSynthetic Biology, 2017, 6(5), 837-848.

[0033] Example 1: Construction of L-phenylalanine producing strains HTolR-1, HTolR-2, HTolR-3, HTolR-4, HTolR-5, HTolR-6, HTolR-7, HTolR-8, HTolR-9 and HTolR-10

[0034] TolR is the inner membrane component of the Escherichia coli K-12 Tol-Pal system. The amino acid sequence of wild-type TolR is shown in SEQ ID No: 1: MARARGRGRRDLKSEINIVPLLDVLLVLLLIFMATAPIITQSVEVDLPDATESQAVSSNDNPPVIVEVSGIGQYTVVVEKDRLERLPPEQVVAEVSSRFKANPKTVFLIGGAKDVPYDEIIKALNLLHSAGVKSVGLMTQPI.

[0035] Its nucleotide sequence is as SEQ ID No: 28 shown: atggccagagcgcgtggacgaggtcgtcgcgatctcaagtccgaaatcaacattgtaccgttgctggacgtactgctggtgctgttgctgatctttatggc gacagcgcccatcatcacccagcgtggaggtcgatctgccagacgctactgaatcacaggcggtgagcagtaacgataatccgccagtgattgttgaagtgtctggta ttggtcagtacaccgtggtggttgagaaagatcgcctggagcgtttaccaccagagcaggtggtggcggaagtgtccagccgtttcaaggccaacccgaaaacggtctt tctgatcggtggcgcaaaagatgtgccttacgatgaaataattaaagcactgaacttgttacatagtgcgggtgtgaaatcggttggtttaatgacgcagcctatctaa.

[0036] The Tol-Pal system is a group of interoperating proteins that cross the cell membrane, participate in outer membrane invagination and septum peptidoglycan processing during cell division, and maintain outer membrane integrity through lipid homeostasis. The transmembrane helices of TolR and TolQ are tightly locked together by an "R85-D23" salt bridge (Arg85 to Asp23), forming a proton-sealed ring of the Tol energy transduction complex. This salt bridge not only transmits the proton dynamic potential (PMF) but also acts as a mechanical switch for outer membrane contraction.

[0037] To increase L-phenylalanine production, we attempted to mutate the 85th amino acid of TolR by altering glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), cysteine ​​(Cys), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), and proline (Pro), respectively. The aim was to weaken or disrupt the "R85-D23" salt bridge, thereby loosening the outer membrane and allowing intracellular phenylalanine to leak out more easily, alleviating intracellular product inhibition, and ultimately increasing L-phenylalanine production.

[0038] Construction of the Cas9-tolR(R85G) plasmid: Using wild-type Escherichia coli MG1655 (commercially available) as a template, the R85G-UP fragment with adapter was amplified using primers tolR-up-F and R85G-up-R, and the R85G-Down fragment with adapter was amplified using primers R85G-down-F and tolR-down-R. The R85G-UP and R85G-Down fragments were then assembled into the R85G-UD fragment. Using the Cas9 plasmid as a template, the plasmid backbone tolR-ver1 fragment was amplified using primers tolR-N20-F and tolR-ver-R, and the plasmid backbone tolR-ver2 fragment was amplified using primers tolR-ver-F and tolR-N20-R. The plasmid backbones tolR-ver1 and tolR-ver2, along with the fragment R85G-UD above, were assembled using Gibson assembly (the Gibson assembly method, invented by Gibson et al., is a method for achieving intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the cas9-tolR(R85G) plasmid.

[0039] Construction of the Cas9-tolR(R85A) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85A-UP fragment with the adapter was amplified using primers tolR-up-F and R85A-up-R, and the R85A-Down fragment with the adapter was amplified using primers R85A-down-F and tolR-down-R. The R85A-UP and R85A-Down fragments were assembled into the R85A-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85A-UD using Gibson assembly to obtain the Cas9-tolR(R85A) plasmid.

[0040] Construction of the Cas9-tolR(R85S) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85S-UP fragment with the adapter was amplified using primers tolR-up-F and R85S-up-R, and the R85S-Down fragment with the adapter was amplified using primers R85S-down-F and tolR-down-R. The R85S-UP and R85S-Down fragments were assembled into the R85S-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85S-UD using Gibson assembly to obtain the Cas9-tolR(R85S) plasmid.

[0041] Construction of the Cas9-tolR(R85T) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85T-UP fragment with the adapter was amplified using primers tolR-up-F and R85T-up-R, and the R85T-Down fragment with the adapter was amplified using primers R85T-down-F and tolR-down-R. The R85T-UP and R85T-Down fragments were assembled into the R85T-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85T-UD using Gibson assembly to obtain the Cas9-tolR(R85T) plasmid.

[0042] Construction of the Cas9-tolR(R85C) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85C-UP fragment with the adapter was amplified using primers tolR-up-F and R85C-up-R, and the R85C-Down fragment with the adapter was amplified using primers R85C-down-F and tolR-down-R. The R85C-UP and R85C-Down fragments were assembled into the R85C-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85C-UD using Gibson assembly to obtain the Cas9-tolR(R85C) plasmid.

[0043] Construction of the Cas9-tolR(R85V) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85V-UP fragment with the adapter was amplified using primers tolR-up-F and R85V-up-R, and the R85V-Down fragment with the adapter was amplified using primers R85V-down-F and tolR-down-R. The R85V-UP and R85V-Down fragments were assembled into the R85V-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85V-UD using Gibson assembly to obtain the Cas9-tolR(R85V) plasmid.

[0044] Construction of the Cas9-tolR(R85I) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85I-UP fragment with the adapter was amplified using primers tolR-up-F and R85I-up-R, and the R85I-Down fragment with the adapter was amplified using primers R85I-down-F and tolR-down-R. The R85I-UP and R85I-Down fragments were assembled into the R85I-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were assembled with the above fragment R85I-UD using Gibson assembly to obtain the Cas9-tolR(R85I) plasmid.

[0045] Construction of the Cas9-tolR(R85L) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85L-UP fragment with the adapter was amplified using primers tolR-up-F and R85L-up-R, and the R85L-Down fragment with the adapter was amplified using primers R85L-down-F and tolR-down-R. The R85L-UP and R85L-Down fragments were assembled into the R85L-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were then assembled with the above fragment R85L-UD using Gibson assembly to obtain the Cas9-tolR(R85L) plasmid.

[0046] Construction of the Cas9-tolR(R85M) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85M-UP fragment with the adapter was amplified using primers tolR-up-F and R85M-up-R, and the R85M-Down fragment with the adapter was amplified using primers R85M-down-F and tolR-down-R. The R85M-UP and R85M-Down fragments were assembled into the R85M-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were assembled with the above fragment R85M-UD using Gibson assembly to obtain the Cas9-tolR(R85M) plasmid.

[0047] Construction of the Cas9-tolR(R85P) plasmid: Using wild-type Escherichia coli MG1655 as a template, the R85P-UP fragment with the adapter was amplified using primers tolR-up-F and R85P-up-R, and the R85P-Down fragment with the adapter was amplified using primers R85P-down-F and tolR-down-R. The R85P-UP and R85P-Down fragments were assembled into the R85P-UD fragment. The plasmid backbones tolR-ver1 and tolR-ver2 were assembled with the above fragment R85P-UD using Gibson assembly to obtain the Cas9-tolR(R85P) plasmid.

[0048] The primers used in this section are as follows:

[0049] Table 1. Primers used to construct the L-phenylalanine-producing strain

[0050]

[0051] Plasmids Cas9-tolR(R85G), Cas9-tolR(R85A), Cas9-tolR(R85S), Cas9-tolR(R85T), Cas9-tolR(R85C), Cas9-tolR(R85V), Cas9-tolR(R85I), Cas9-tolR(R85L), Cas9-tolR(R85M), and Cas9-tolR(R85P) were transformed into strain HDH6-D12. After arabinose induction, TolR(R85G), TolR(R85A), and TolR(R85P) were transformed into plasmids Cas9-tolR(R85G), Cas9-tolR(R85A), Cas9-tolR(R85S), Cas9-tolR(R85T), Cas9-tolR(R85C), Cas9-tolR(R85V), Cas9-tolR(R85I), Cas9-tolR(R85L), Cas9-tolR(R85M), and Cas9-tolR(R85P) respectively. The mutants R(R85A), TolR(R85S), TolR(R85T), TolR(R85C), TolR(R85V), TolR(R85I), TolR(R85L), TolR(R85M), and TolR(R85P) were used to construct phenylalanine-producing strains HTolR-1, HTolR-2, HTolR-3, HTolR-4, HTolR-5, HTolR-6, HTolR-7, HTolR-8, HTolR-9, and HTolR-10, respectively, by substituting substitutions at the original sites in the genome. The strains and plasmids used in this section are as follows:

[0052] Table 2. Strains and plasmids used to construct L-phenylalanine production strains

[0053]

[0054] Example 2: Fermentation of L-phenylalanine producing strains

[0055] The shake-flask fermentation process for Escherichia coli L-phenylalanine producing strains HDH6-D12 and HTolR-1, HTolR-2, HTolR-3, HTolR-4, HTolR-5, HTolR-6, HTolR-7, HTolR-8, HTolR-9, and HTolR-10 is as follows:

[0056] (1) Slant activation culture: Take out the preserved strain from the -80℃ freezer and streak it on a solid medium containing kanamycin resistance (5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride, 15g / L agar), and incubate at 37℃ for 10-14h.

[0057] (2) Seed culture: Use an inoculation loop to pick a single colony from the fresh activated slant and place it in seed basal medium (50 mL LB medium, kanamycin resistant, in a 500 mL Erlenmeyer flask), and incubate at 37℃ and 220 r / min for 6-8 h until OD. 600 Approximately 3-5.

[0058] (3) Shake-flask batch fermentation: The seed culture was inoculated into the basic fermentation medium (500mL Erlenmeyer flask, 50mL liquid volume, kanamycin resistant) at a 10% inoculation rate, and L-phenylalanine was fermented in batches at 37℃ and 220r / min for 38-50h with shaking. Table 3 shows the shake-flask fermentation medium.

[0059] Table 3. Fermentation medium formulation for L-phenylalanine

[0060]

[0061] Example 3: High-performance liquid chromatography (HPLC) detection and result analysis of L-phenylalanine fermentation strains

[0062] The fermentation broth was centrifuged at 5500 rpm / min for 15-20 min in a refrigerated centrifuge, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter membrane and analyzed by HPLC.

[0063] The HPLC conditions were as follows: ZORBAX Eclipse AAA (amino acid analysis) column; mobile phase A: 40 mM Na₂HPO₄, pH 7.8; mobile phase B: methanol:acetonitrile:water = 45:45:10, v / v / v. The elution gradient was: 0-1 min, 100% A; 9.8 min: 43% A + 57% B; 10 min: 100% B; 12 min: 100% B; 12.5 min: 100% A. The flow rate was 2.0 mL / min. A RID and VWD detector were connected in series. The detection cell temperature was controlled at 40℃. The injection volume was 10 μL. The analysis time was 26 min. The UV detection wavelength was 338 nm.

[0064] In the later stages of L-phenylalanine fermentation, the intracellular product concentration rapidly increases, triggering a feedback inhibition mechanism for the final product: the active sites of DAHP synthase (AroF / AroG / AroH) and the bifunctional enzyme PheA are occupied by phenylalanine, resulting in a sharp drop in catalytic efficiency. Carbon flow is forced to redirect to the central carbon metabolism bypass, causing the synthesis stage to plateau prematurely and significantly slowing the yield growth rate. The TolR-R85 mutation precisely breaks this "rate ceiling." This site is located at the core of the Arg-Asp salt bridge at the TolR transmembrane helix and TolQ docking interface; replacing arginine with glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), cysteine ​​(Cys), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), or proline (Pro) breaks the salt bridge, reducing the radial binding force of the Tol-Pal complex on the outer cell membrane and forming a reversible "nanoscale microleakage." Phenylalanine is continuously effluxed through this channel, resulting in a decrease in intracellular concentration and thus mitigating product inhibition. Carbon flow is then rapidly advanced along the aromatic pathway. Simultaneously, impaired Tol-Pal function delays the recruitment time of the septal contractile protein FtsN, prolonging the cell division cycle and causing the cell structure to shift from short rods to filamentous elongation. In a constant-volume fermentation system, the total amount of "effective cytoplasm" per unit volume increases, effectively creating a continuous production line by connecting multiple phenylalanine synthesis workshops without increasing OD. The combined effect of the efflux and spatial expansion ultimately leads to an increase in the final titer of the fermentation broth.

[0065] like Figure 1As shown, the introduction of the TolR mutant TolR (R85G) increased the phenylalanine production capacity of strain HTolR-1 by 6.89% compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 9.84 g / L. The introduction of the TolR mutant TolR (R85A) increased the phenylalanine production capacity of strain HTolR-2 by 12.46% compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 10.36 g / L. The introduction of the TolR mutant TolR (R85S) increased the phenylalanine production capacity of strain HTolR-3 by 12.92% compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 10.4 g / L. Introducing the TolR mutant TolR (R85T), strain HTolR-4 showed a 3.48% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 9.53 g / L. Introducing the TolR mutant TolR (R85C), strain HTolR-5 showed a 20.74% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 11.12 g / L. Introducing the TolR mutant TolR (R85V), strain HTolR-6 showed a 10.52% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 10.18 g / L. Introducing the TolR mutant TolR (R85I), strain HTolR-7 showed a 6.28% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 9.79 g / L. Introducing the TolR mutant TolR (R85L), strain HTolR-8 showed a 12.48% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 10.36 g / L. Introducing the TolR mutant TolR (R85M), strain HTolR-9 showed a 20.19% increase in phenylalanine production compared to strain HDH6-D12 (9.2 g / L), achieving an L-phenylalanine yield of 11.07 g / L. The TolR mutant TolR (R85P) was introduced, and the ability of strain HTolR-10 to produce phenylalanine was increased by 8.25% compared with strain HDH6-D12 (9.2 g / L), with the L-phenylalanine yield reaching 9.97 g / L.

[0066] Example 4: Construction of L-tryptophan-producing strain

[0067] To further verify the effect of the TolR mutant on the production of other aromatic amino acids, plasmids Cas9-tolR(R85G), Cas9-tolR(R85A), Cas9-tolR(R85S), Cas9-tolR(R85T), Cas9-tolR(R85C), Cas9-tolR(R85V), Cas9-tolR(R85I), Cas9-tolR(R85L), Cas9-tolR(R85M), and Cas9-tolR(R85P) were transformed into L-tryptophan-producing strain KW018, and induced by arabinose. The mutants TolR(R85G), TolR(R85A), TolR(R85S), TolR(R85T), TolR(R85C), TolR(R85V), TolR(R85I), TolR(R85L), TolR(R85M), and TolR(R85P) were replaced at their original sites in the genome to construct tryptophan-producing strains KTolR-1, KTolR-2, KTolR-3, KTolR-4, KTolR-5, KTolR-6, KTolR-7, KTolR-8, KTolR-9, and KTolR-10, respectively.

[0068] The KW018 strain is described in the following literature: Chen, Y. et al. Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production. Journal of Industrial Microbiol & Biotechnology, 45(5), 357-367 (2018).

[0069] Example 5: Fermentation of L-tryptophan-producing strains

[0070] The shake-flask fermentation process for Escherichia coli L-tryptophan producing strains KW018, KTolR-1, KTolR-2, KTolR-3, KTolR-4, KTolR-5, KTolR-6, KTolR-7, KTolR-8, KTolR-9 and KTolR-10 is as follows:

[0071] (1) Slant activation culture: Take out the preserved strain from the -80℃ freezer and streak it on a solid medium containing tetracycline resistance, and incubate at 37℃ for 10-14h.

[0072] (2) Seed culture: Use an inoculation loop to pick a single colony from the fresh activated slant and place it in seed basal medium (50 mL LB medium in a 500 mL Erlenmeyer flask, sealed with sealing film), and incubate at 37℃ and 220 r / min for 6-8 h until OD. 600 Approximately 2-3.

[0073] (3) Shake-flask batch fermentation: The seed culture was inoculated at a rate of 10% into a tetracycline-resistant fermentation medium (50 mL Erlenmeyer flask, 50 mL inoculation volume, sealed with sealing film), and L-tryptophan was fermented in batches at 37℃ and 220 r / min for 36-42 h with shaking. Table 4 shows the shake-flask fermentation medium.

[0074] Table 4. L-Tryptophan Fermentation Medium Formulation

[0075]

[0076] Example 6: Analysis of Fermentation Results of L-Tryptophan-Producing Strains

[0077] After fermentation by the tryptophan strain, the sample processing method and HPLC determination method were the same as in Example 3.

[0078] like Figure 2As shown, the introduction of the TolR mutant TolR (R85G) increased the tryptophan production capacity of strain KTolR-1 by 4.31% compared to strain KW018 (2.2 g / L), achieving an L-tryptophan production of 2.3 g / L. The introduction of the TolR mutant TolR (R85A) increased the tryptophan production capacity of strain KTolR-2 by 47.39% compared to strain KW018 (2.2 g / L), achieving an L-tryptophan production of 3.25 g / L. The introduction of the TolR mutant TolR (R85S) increased the tryptophan production capacity of strain KTolR-3 by 47.39% compared to strain KW018 (2.2 g / L), achieving an L-tryptophan production of 3.25 g / L. Introducing the TolR mutant TolR (R85T), strain KTolR-4 showed a 4.08% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 2.3 g / L. Introducing the TolR mutant TolR (R85C), strain KTolR-5 showed a 61% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 3.55 g / L. Introducing the TolR mutant TolR (R85V), strain KTolR-6 showed a 47.4% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 3.25 g / L. Introducing the TolR mutant TolR (R85I), strain KTolR-7 showed an 8.84% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 2.4 g / L. Introducing the TolR mutant TolR (R85L), strain KTolR-8 showed a 47.4% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 3.25 g / L. Introducing the TolR mutant TolR (R85M), strain KTolR-9 showed a 56.46% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 3.45 g / L. Introducing the TolR mutant TolR (R85P), strain KTolR-10 showed a 17.91% increase in tryptophan production compared to strain KW018 (2.2 g / L), achieving an L-tryptophan yield of 2.6 g / L.

[0079] 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, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a genetically engineered bacterium in the production of aromatic amino acids, wherein the aromatic amino acid is L-phenylalanine or L-tryptophan; wherein the genetically engineered bacterium is constructed by site-directed mutagenesis of the 85th amino acid residue R85 to C of the endogenous TolR enzyme in an L-phenylalanine-producing or L-tryptophan-producing Escherichia coli strain; the amino acid sequence of the endogenous TolR enzyme is shown in SEQ ID NO:

1.

2. A method for producing aromatic amino acids using genetically engineered bacteria, characterized in that, It includes fermenting the genetically engineered bacteria to produce aromatic amino acids; the aromatic amino acids are L-phenylalanine or L-tryptophan; The genetically engineered bacteria were constructed by mutating the 85th amino acid residue R85 of the endogenous TolR enzyme to C in either an L-phenylalanine-producing or L-tryptophan-producing Escherichia coli strain through site-directed mutagenesis; the amino acid sequence of the endogenous TolR enzyme is shown in SEQ ID NO:

1.

3. The method as described in claim 2, characterized in that, It also includes the step of separating the aromatic amino acids produced.

Images