Aromatic compound transporter mutants and uses thereof

Abstract

This invention discloses an aromatic compound transporter protein mutant and its applications, belonging to the field of bioengineering technology. The mutant is based on *Pseudomonas putida* (…). Pseudomonas putida The outer membrane transport protein derived from [a specific source] is obtained by site-directed substitution of key residues in its gating loop. Specifically, this includes replacing phenylalanine at position 120 with glycine, leucine at position 121 with glycine, and glutamine at position 122 with glycine, or one or more combinations thereof. Preferred mutants include single mutant F120G, double mutant F120G / L121G, or triple mutant F120G / L121G / Q122G, and conserved substitution derivatization can also be performed based on these mutants. These mutants can be used for the efficient microbial production and cell tolerance regulation of aromatic compounds, based on the physicochemical properties of the target product, and have significant application value in green biomanufacturing, fermentation engineering, and bioremediation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology. More specifically, this invention relates to an aromatic compound transporter mutant and its applications. Background Technology

[0002] Aromatic compounds are important raw materials in the fields of pharmaceuticals, food, daily chemicals, and materials, and have traditionally been produced mainly through plant extraction and chemical synthesis. Microbial cell factories offer a promising alternative platform for the sustainable synthesis of aromatic compounds, but their production efficiency is often limited by the toxicity of the products to host cells, resulting in low conversion efficiency and making it difficult to meet the needs of industrial-scale production.

[0003] To mitigate product toxicity, transporter protein engineering has become a key strategy for enhancing host tolerance. Efflux pumps effectively enhance host tolerance and productivity by transporting harmful compounds accumulated intracellularly to the extracellular space. In Gram-negative bacteria (such as *Escherichia coli* and *Pseudomonas putida*), extracellular efflux proteins are crucial components in performing this function, mediating the efflux of various substances, including antibiotics, organic solvents, and metabolites. Among them, the OMF family of extracellular efflux proteins, represented by TolC, has been extensively studied. Their typical structure is a trimer, capable of forming channels across the outer membrane. However, these channels typically exhibit a semi-open extracellular outlet conformation in the resting state. While this facilitates substrate efflux, it can also lead to influx of external compounds or backflow of transported substrates. Studies have shown that this non-selective bidirectional diffusion weakens the cell's detoxification efficiency and induces toxicity accumulation under high product concentration conditions, thus limiting its practical application in microbial production.

[0004] Therefore, developing extracellular efflux proteins with precise "one-way valve" functions to efficiently efflux substrates while effectively blocking harmful backflow, and through engineering modifications to adapt them to the physicochemical properties of target products, thereby achieving efficient microbial production of aromatic compounds and enhancing host cell tolerance, are current technical challenges in this field. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides an application of an efflux protein with a "one-way valve" function, which can effectively block the backflow of hydrophobic macromolecular aromatic compounds and improve host bacterial tolerance. This invention also provides a mutant of the aforementioned efflux protein to enhance host bacterial tolerance to small molecule aromatic compounds. Applying this mutant gene to the microbial synthesis of target aromatic compounds can efficiently promote product efflux, thereby achieving synergistic regulation of efficient aromatic compound production and cell tolerance.

[0006] I. Aromatic compound transporter mutant proteins and their expressed nucleic acids

[0007] To achieve these objectives and other advantages according to the invention, the invention provides an aromatic compound transporter mutant comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1, and is a protein as follows a) or b):

[0008] a) Contains one or more combinations of the following: phenylalanine at position 120 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine (F120G); leucine at position 121 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine (L121G); or glutamine at position 122 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine (Q122G), preferably a single mutant F120G, a double mutant F120G / L121G or a triple mutant 120G / L121G / Q122G;

[0009] b) Proteins derived from (a) whose amino acid sequence in a) has been substituted, deleted, or added with one or more amino acids, but which still retain the efflux activity of aromatic compounds.

[0010] This invention also provides a nucleic acid encoding an aromatic compound transporter mutant. For example, the base sequence of a DNA molecule encoding the F120G mutant shown in SEQ ID NO: 2 is shown in SEQ ID NO: 8; the base sequence of a DNA molecule encoding the L121G mutant shown in SEQ ID NO: 3 is shown in SEQ ID NO: 9; the base sequence of a DNA molecule encoding the Q122G mutant shown in SEQ ID NO: 4 is shown in SEQ ID NO: 10; the base sequence of a DNA molecule encoding the F120G / L121G mutant shown in SEQ ID NO: 5 is shown in SEQ ID NO: 11; and the base sequence of a DNA molecule encoding the F120G / L121G / Q122G mutant shown in SEQ ID NO: 6 is shown in SEQ ID NO: 12. Of course, due to the degeneracy of codons, any DNA molecule capable of encoding the above amino acid sequences is acceptable.

[0011] II. Expression Box

[0012] The present invention also provides a recombinant expression vector containing the coding gene (such as plasmid pBD-24, pETuet, etc.).

[0013] The present invention also provides a recombinant cell comprising an aromatic compound transporter mutant protein or a recombinant expression vector encoding nucleic acid. In some embodiments, the recombinant cell is an engineered bacterium, such as Escherichia coli (e.g., E. coli C41(DE3), BL21(DE3)) or other microbial cells.

[0014] III. Uses

[0015] This invention provides the use of aromatic compound transporter mutants in improving the tolerance of bacterial strains to aromatic compounds, and their use in promoting the efflux of target products and reducing intracellular product accumulation to increase the yield of target products during microbial fermentation for the production of small molecule aromatic compounds. The products include, but are not limited to, monocyclic aromatic compounds, preferably including p-hydroxybenzoic acid, cinnamaldehyde, vanillin, etc.

[0016] This invention further relates to the use of wild-type PP_0179 protein (SEQ ID NO: 1) in the processing or production of specific types of compounds. In one specific embodiment, it is used to construct engineered strains with stronger reflux blocking and cell-protective effects against macromolecular hydrophobic aromatic compounds. In one specific embodiment, the macromolecular hydrophobic aromatic compound is a bicyclic aromatic compound; in a more specific embodiment, the bicyclic aromatic compound may be resveratrol or naringenin.

[0017] IV. Methods and Applications

[0018] The present invention also provides a method for improving the tolerance of microorganisms to small molecule aromatic compounds, the method comprising introducing and expressing any aromatic compound transporter mutant into the microorganisms. Preferably, the small molecule aromatic compound is vanillin, p-hydroxybenzoic acid, or cinnamaldehyde.

[0019] This invention also provides a method for producing small molecule aromatic compounds, comprising the following steps:

[0020] An engineered bacterium is provided, which is genetically modified to express the aromatic compound transporter mutant and contains genes for the metabolic pathway of synthesizing the small molecule aromatic compound;

[0021] The engineered bacteria are fermented under suitable culture medium and culture conditions to grow and synthesize the small molecule aromatic compounds; during the fermentation process, the aromatic compound transporter protein mutant expressed by the engineered bacteria releases the synthesized small molecule aromatic compounds from inside and outside the cell into the fermentation broth;

[0022] The small molecule aromatic compounds were isolated and harvested from the culture after fermentation was completed;

[0023] The small molecule aromatic compounds are vanillin, p-hydroxybenzoic acid, and cinnamaldehyde.

[0024] Preferably, the fermentation and cultivation process is specifically as follows:

[0025] The engineered bacteria were inoculated into liquid culture medium and cultured at 30-37℃; when the OD of the culture... 600 When the value reaches 0.4 to 0.8, isopropyl-β-D-thiogalactoside with a final concentration of 0.2 mM to 1.0 mM is added to the fermentation system to simultaneously induce the expression of the vanillin biosynthesis pathway gene and the mutant gene of the aromatic compound transporter protein.

[0026] After adding isopropyl-β-D-thiogalactoside, ferulic acid with a final concentration of 5 mM to 20 mM was added to the fermentation system as a precursor for vanillin synthesis; the culture temperature was adjusted to 25℃ to 30℃, and fermentation was continued for 16 h to 120 h.

[0027] The fermentation process is terminated when the concentration of small molecule aromatic compounds in the fermentation broth exceeds 800 mg / L.

[0028] Preferably, the addition of ferulic acid employs a feedback feeding strategy based on the product synthesis rate, specifically:

[0029] Ferulic acid is added to the fermentation system at an initial rate;

[0030] The concentration of small molecule aromatic compounds in the fermentation broth was monitored in real time or at regular intervals, and their synthesis rate vp, mg·h / L was calculated.

[0031] The flow rate of ferulic acid (FA) was dynamically correlated with the vanillin synthesis rate (vp), with FA = k × vp, where the correlation coefficient k ranged from 0.05 mM·L / mg to 0.2 mM·L / mg.

[0032] This feedback control maintains the concentration of ferulic acid in the fermentation system within the range of 5 mM to 10 mM.

[0033] Preferably, the fermentation culture process further includes an induction and enhancement phase based on cell metabolic state:

[0034] After adding isopropyl-β-D-thiogalactoside and ferulic acid, the dissolved oxygen (DO) concentration in the fermentation broth was continuously monitored; when the DO concentration decreased from the saturation value and stabilized in the range of 10%-30%, the cells were determined to have entered the metabolically vigorous production period.

[0035] At this critical moment, isopropyl-β-D-thiogalactoside was added to the fermentation system once, instantly increasing its concentration by 0.1 mM to 0.5 mM, in order to enhance the expression of the aromatic compound transporter mutant.

[0036] The present invention also provides a method for improving the tolerance of microorganisms to macromolecular aromatic compounds, the method comprising introducing and expressing wild-type aromatic compound transporters into the microorganisms. Preferably, the hydrophobic macromolecular aromatic compound is selected from naringenin or resveratrol.

[0037] The present invention also provides a method for screening the function of aromatic compound transport proteins, the method comprising using microorganisms expressing aromatic compound transport proteins or any mutants to determine colony formation efficiency on plates containing said aromatic compounds.

[0038] The present invention has at least the following beneficial effects:

[0039] Based on the resolved cryo-electron microscopy structure, we discovered that the aromatic compound transporter PP_0179 possesses a unique gate valve structure at the extracellular exit of its transmembrane channel. This structure, composed of steric hindrance formed by three key residues (phenylalanine at position 120, leucine at position 121, and glutamine at position 122), effectively blocks the backflow of hydrophobic macromolecular aromatic compounds (such as naringenin and resveratrol), thus providing crucial support for the efficient production of these compounds. However, while this gate valve structure provides a blocking effect, it also limits the efficient efflux of small molecule substrates. To improve its transport performance for small molecule substrates, this invention, based on high-resolution structural information, rationally designed and performed site-directed mutagenesis on key components of this gate valve structure. By disrupting the gate valve structure or reducing its steric hindrance, we obtained a series of mutants. The selected mutants, while retaining channel transduction function, effectively reduced steric hindrance to smaller substrates, thus significantly improving the efflux efficiency of small molecule aromatic compounds and effectively reducing their intracellular toxicity accumulation. This resulted in a synergistic improvement in target product titer and production intensity while maintaining high cell activity. Furthermore, this study demonstrated that substrate selectivity and transport direction can be fine-tuned by precisely controlling the opening and closing of the outer membrane protein gate structure. This provides a new universal strategy and core protein element for the customized design of "one-way valve" transport proteins targeting products with different physicochemical properties (such as molecular size and hydrophobicity), with broad application prospects.

[0040] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0041] Figure 1 This is a cryo-electron microscopy structure and a schematic diagram of the extracellular gate valve structure of the aromatic compound transporter PP_0179.

[0042] Figure 2 This is a diagram showing the tolerance of recombinant Escherichia coli overexpressing PP_0179 wild-type and different mutants to vanillin in the embodiments of the present invention.

[0043] Figure 3 This is a graph showing the tolerance of recombinant Escherichia coli overexpressing PP_0179 wild-type and F120G to different aromatic compounds in an embodiment of the present invention. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can implement it based on the description.

[0045] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0046] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0047] This invention utilizes conventional techniques and methods found in the fields of genetic engineering and molecular biology, and these general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can, based on the technical solutions described in this invention, employ other conventional methods, experimental protocols, and reagents, without being limited to the specific embodiments of this invention. For example, the following experimental materials and reagents may be used in this invention:

[0048] Strains and vectors: Escherichia coli DH5α Trans 1-T1 (TransGold, Beijing, China) was used for gene cloning; Escherichia coli C41 (DE3) (TransGold, Beijing, China) was used for protein expression; Escherichia coli BL21 (DE3) (TransGold, Beijing, China) was used for fermentation production; and vector pBD-24 (Novagen) was used for recombinant plasmid construction.

[0049] Enzymes and kits: DNA polymerase, ligase, and DpnI enzyme were purchased from Takara; restriction endonucleases were purchased from NEB; plasmid extraction kit and gel purification and recovery kit were purchased from Tiangen Biotech; Gibson ligation kit was purchased from Lamborghini; vanillin, p-hydroxybenzoic acid, cinnamaldehyde, resveratrol, and naringenin were purchased from Macklin; L-arabinose, the protein expression inducer, was purchased from Lamborghini; and other reagents were available from general biochemical reagent companies.

[0050] Culture medium formulations: Liquid LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; Solid LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 1.5% agar powder; All the above culture media were autoclaved at 103 kPa and 121℃ for 20 min. For the solid culture medium, after cooling to about 50℃, 100 mg / mL ampicillin was added and the medium was poured into plates in a clean bench for later use.

[0051] Aromatic compound plate tolerance test method: Inoculate a single colony into 10 mL LB liquid medium containing ampicillin and incubate at 37°C with shaking at 220 rpm for 14–16 h. Collect the bacterial cells and resuspend them in PBS to OD200. 600 ~1.0, perform 10-fold serial dilutions. Take 5 μL of the diluted bacterial culture and spot it onto a plate containing 0.2% L-arabinose and the corresponding aromatic compounds. After absorption, incubate at 30℃ upside down for 48 h and observe the results.

[0052] Product detection method: The content of the target product was determined by high-performance liquid chromatography (HPLC). Chromatographic column: ZORBAX StableBond C18, 4.6 × 250 nm. 2 5 μm; UV absorption wavelength: 308 nm; flow rate: 0.5 mL / min; injection volume: 10 μL / 20 μL. Mobile phase: Mobile phase A was an aqueous solution of 0.1% formic acid; mobile phase B was a methanol solution of 0.1% formic acid. Gradient elution was used, with the following elution program: 0–8.5 min 60% mobile phase A; 9.5–18 min 15% mobile phase A; 19–25 min 60% mobile phase A. The yield of aromatic compounds was determined using the external standard method based on retention time and peak area.

[0053] SEQ ID NO: 1 PP_0179 WT protein sequence:

[0054] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGE AFLQPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYYARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAEL PRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTN GTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEXAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0055] SEQ ID NO:2 PP_0179 F120G Report:

[0056] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGE AGLQPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYYARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAEL PRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTN GTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEXAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0057] SEQ ID NO:3 PP_0179 L121G:

[0058] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGE AFGQPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAEL PRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTN GTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEXAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0059] SEQ ID NO:4 PP_0179 Q122G:

[0060] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGE AFLGPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYHIARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAEL PRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTN GTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEXAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0061] SEQ ID NO:5 PP_0179 F120G / L121G Contact:

[0062] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGE AGGQPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAEL PRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTN GTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEXAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0063] SEQ ID NO:6PP_0179 F120G / L121G / Q122G

[0064] MKQLILAGLCLSLGACMMVGPDYEVPGDAAVQRNDLNGPLRQDADSVVSAPVPEDWWQLYQDQRLNELVRQALSANTELRVAAANIAKARAQVEVAESQGGFNGGVKLGAQRLQESGEAGGGPEKVPVANIGEAIISASYQFDLWGTFKRGTEAAKANADAVQAAADTARITLVADVVKAYTQVCSANEEYHIARESLDLQEQSVKLNQRLRDAGRGDETQVTRSQTQFKSLRAELPRFKAERETGMYTLAALLAKPVDQLPAGTADCAELPHLNQLVPVGDGAALLKRRPDVRQAERQLAAATAYIGVATGALYPDISIGAQVGTIGILENLGEPSTNRWGFGPQISWSIPTNGTRARIRMAEASTQAALAHFDGVVLNAIRETQTRLAQYSALLDRRDALAEAEKSAKEAADQTHRYYQAGRESFLADLQATRTYTDMRAQLAAANSQVAQGQIAVFLALGGGWKGTAKP

[0065] SEQ ID NO: 7 PP_0179 WT DNA sequence

[0066]

[0067] SEQ ID NO: 8PP_0179 F120G DNA sequence

[0068]

[0069] SEQ ID NO: 9PP_0179L121G DNA sequence

[0070]

[0071] SEQ ID NO: 10PP_0179Q122G DNA sequence

[0072]

[0073] SEQ ID NO: 11PP_0179 F120G / L121G DNA sequence

[0074]

[0075] SEQ ID NO: 12PP_0179 F120G / L121G / Q122G DNA sequence

[0076]

[0077] Cryo-electron microscopy analysis of the aromatic compound transporter PP_0179 from *Pseudomonas putida* revealed a unique gate valve structure at the extracellular exit of the transmembrane channel (e.g., ...). Figure 1 As shown, the structure is composed of steric hindrance formed by three key residues (phenylalanine at position 120, leucine at position 121, and glutamine at position 122), which can effectively block the backflow of macromolecular aromatic compounds.

[0078] Example 1:

[0079] Construction of aromatic compound transporter mutants: Using recombinant plasmid pBD-24 carrying the encoding gene of wild-type aromatic compound transporter PP_0179 as a template, corresponding primers were designed:

[0080] F (PP_0179F120G) CGAAGCCGGCCTGGCAGCCCGAGAAGGTG

[0081] R (PP_0179 F120G) TGCAGGCCGGCTTCGCCCGACTCCTG

[0082] F (PP_0179L121G) AGCCTTCGGCCAGCCCGAGAAGGTGCCG

[0083] R (PP_0179L121G) GGCTGGCCGAAGGCTTCGCCCGACTCC

[0084] F (PP_0179 Q122G) CTTCCTGGGCCCCGAGAAGGTGCCGGTA

[0085] R (PP_0179 Q122G) TCGGGGCCCAGGAAGGCTTCGCCCGA

[0086] F (PP_0179F120G / L121G) AAGCCGGCGGCCAGCCCGAGAAGGTGCCG

[0087] R (PP_0179F120G / L121G) GCTGGCCGCCGGCTTCGCCCGACTCCTGC

[0088] F (PP_0179 F120G / L121G / Q122G)AGCCGCGGCGGCCCCGAGAAGGTGCCGGTAG

[0089] R (PP_0179F120G / L121G / Q122G) GGGCCGCCGCCGGCTTCGCCCGACTCCTG

[0090] In vitro site-directed mutagenesis was performed using the Quickchange PCR method. The PCR products were treated overnight with Dpn I and then transformed into E. coli DH5α. Trans The 1-T1 competent cells were used, and the resulting plasmids were confirmed by DNA sequencing.

[0091] Example 2:

[0092] Tolerance analysis of PP_0179 wild-type or mutant strains to vanillin: Wild-type PP_0179 and mutants modified for the gate valve structure (F120G, L121G, Q122G, F120G / L121G, F120G / L121G / Q122G) were transformed into E. coli C41(DE3) competent cells, and tolerance was analyzed on plates supplemented with 0.3 g / L vanillin. The results showed that all mutant strains exhibited superior growth compared to the wild-type control, with the F120G, F120G / L121G, and F120G / L121G / Q122G mutants showing the most significant growth advantages (e.g., ...). Figure 2 As shown in the figure, this indicates that the above mutants have effectively improved tolerance to vanillin and their ability to efflux it.

[0093] Example 3:

[0094] Tolerance analysis of wild-type or mutant PP_0179 strains to different aromatic compounds: Wild-type PP_0179 and F120G mutant were transformed into Escherichia coli C41(DE3) competent cells, and tolerance tests were performed on plates containing different aromatic compounds, including: 0.2 g / L p-hydroxybenzoic acid, 0.1 g / L cinnamaldehyde, 0.1 g / L resveratrol, and 0.1 g / L naringenin. The results showed that for monocyclic aromatic compounds (p-hydroxybenzoic acid and cinnamaldehyde), the F120G mutant exhibited significantly better growth performance than the wild-type; while for bicyclic aromatic compounds (resveratrol and naringenin), the wild-type strain showed better growth performance than the F120G mutant (e.g., 0.2 g / L p-hydroxybenzoic acid, 0.1 g / L cinnamaldehyde, 0.1 g / L resveratrol, and 0.1 g / L naringenin). Figure 3 (As shown in the image). The above results indicate that the *E. coli* engineered strain expressing the mutant exhibits higher tolerance and efflux efficiency to small-molecule aromatic compounds such as vanillin, p-hydroxybenzoic acid, and cinnamaldehyde; while the wild-type protein shows stronger reflux blocking and cell protection effects against large-molecule hydrophobic aromatic compounds such as naringenin and resveratrol. Based on this, the mutant and wild-type strains can be used for the efficient microbial production and cell tolerance regulation of different types of aromatic compounds, depending on the molecular size and hydrophobicity of the target products.

[0095] Example 4:

[0096] Determination of intracellular vanillin content in wild-type or mutant strain PP_0179: Single colonies were inoculated into liquid LB medium containing 0.2% L-arabinose and 0.3 g / L vanillin and cultured at 30°C for 48 h. After fermentation, the sample was centrifuged at 13,000×g for 2 min at 4°C to separate the supernatant from the bacterial cells. The bacterial precipitate was washed twice with 1 mL of 1×PBS buffer (pH 7.0), resuspended in 400 μL of 1×PBS, and mixed with an equal volume of anhydrous ethanol and an appropriate amount of quartz sand. The cells were vortexed (2000×g, 30 min) to disrupt the mixture, followed by filtration through a 0.22 μm filter to obtain the intracellular extract. The intracellular vanillin concentration was determined by high-performance liquid chromatography (HPLC). The results showed that under vanillin stress, the intracellular vanillin content of the wild-type strain was 19.3 mg / L / OD. 600 It is 1.58 times higher than the F120G mutant.

[0097] Example 5:

[0098] Intracellular naringenin content determination of wild-type or mutant strain PP_0179: The same strategy as in Example 4 was used, except that 0.1 g / L naringenin was used as a stress agent for fermentation culture and sample processing. High-performance liquid chromatography analysis showed that the intracellular naringenin accumulation in the F120G mutant was 16.4 mg / L / OD. 600 It is 1.2 times that of the wild-type strain.

[0099] Example 6:

[0100] (1) Construction of a mutant aromatic compound transporter for vanillin biosynthesis: Using the previously constructed recombinant plasmid pETuet-Ech-Fcs-adh7-PP_0179 as a template, this plasmid contains the vanillin synthesis gene Ech from *Amylopectinus*. R37M Fcs and the gene encoding the aromatic compound transporter PP_0179, regulated by the vanillin self-inducible promoter. Specific primers used:

[0101] F (PP_0179 F120G) CGAAGCCGGCCTGGCAGCCCGAGAAGGTG

[0102] R (PP_0179 F120G) TGCAGGCCGGCTTCGCCCGACTCCTG

[0103] In vitro site-directed mutagenesis was performed using the Quickchange PCR method to obtain the plasmid pETuet-Ech-Fcs-adh7-PP_0179F120G. The PCR product was treated overnight with Dpn I and transformed into E. coli DH5αTrans1-T1 competent cells. The obtained plasmid was confirmed by DNA sequencing.

[0104] (2) Fermentation culture and process control of engineered bacteria: The recombinant plasmid of the PP_0179 F120G mutant constructed above was transformed into Escherichia coli BL21(DE3). A single colony was inoculated into 10 mL LB liquid medium containing 100 µg / mL ampicillin and cultured overnight at 37℃ and 220 rpm to obtain the seed culture. 1 mL of the seed culture was transferred to a 500 mL shake flask containing 200 mL LM9 medium at a 1% inoculation rate. The initial OD was... 600 Approximately 0.1. Cultured at 30℃ and 220 rpm.

[0105] Induction and initial feeding: When the OD600 of the culture reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM to simultaneously induce the expression of the vanillin synthesis pathway and transport protein mutant genes. At the same time, ferulic acid was added to the fermentation system to a final concentration of 10 mM as a precursor for vanillin synthesis.

[0106] Adjustment of culture conditions: The culture temperature was adjusted to 28℃ and the rotation speed was adjusted to 180 rpm to reduce metabolic stress and promote product synthesis and accumulation.

[0107] (3) Implementation of the ferulic acid feedback feeding strategy: In order to optimize the precursor supply and avoid waste, approximately 4 hours after the initial feeding (when vanillin begins to be synthesized in large quantities), the ferulic acid feedback feeding based on the product synthesis rate is initiated:

[0108] Monitoring and calculation: Samples were taken every 2 hours, and the concentration of vanillin in the fermentation broth was determined by high performance liquid chromatography (HPLC), and its synthesis rate vp was calculated.

[0109] Feeding control: The feeding rate of ferulic acid (FA, added as a stock solution via a peristaltic pump) is dynamically correlated with vp. The feeding rate is adjusted in real time according to the formula FA = k × vp (k is 0.1 mM·L / mg in this example). The correlation coefficient k = 0.10 mM⋅L / mg is determined based on the theoretical stoichiometric ratio of ferulic acid to vanillin (1:1, molar ratio), and adjusted according to the actual conversion efficiency and safety factor observed in previous shake-flask experiments. This coefficient ensures that the concentration of ferulic acid in the fermentation broth can be stably maintained within the optimized window of 2-8 mM when vp changes dynamically.

[0110] Through this feedback control, the concentration of ferulic acid in the fermentation system was successfully maintained stably within the range of 2-8 mM, which not only met the requirements for efficient synthesis but also avoided the excessive accumulation of precursors and potential inhibition.

[0111] (4) Induction enhancement based on dissolved oxygen signal: During fermentation, the metabolic state of the culture is monitored using a fermenter system (or a large shaker) with an online dissolved oxygen (DO) probe.

[0112] Signal monitoring: DO concentration was continuously monitored. Approximately 12 hours after initial induction, DO levels were observed to decrease rapidly from near saturation levels (approximately 80%) and stabilize at approximately 20%.

[0113] State determination and operation: This DO inflection point is determined to be the production period in which the cells enter a period of vigorous metabolism and rapid product synthesis. At this moment, IPTG is added to the fermentation system once, causing its concentration to increase instantaneously by 0.3 mM.

[0114] This enhanced induction aims to refresh and enhance the activity of some expressed transport proteins in order to cope with the efflux pressure caused by the increased concentration of products in the later stages of fermentation.

[0115] (5) Fermentation endpoint: The culture was terminated when the total fermentation time reached 96 hours. At this time, online monitoring showed that the vanillin concentration had reached 1020 mg / L, which met the termination condition (>800 mg / L).

[0116] (6) Vanillin yield determination of wild-type or mutant strain PP_0179: Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min at 4℃, and separate the supernatant and precipitate. Transfer the supernatant to a new EP tube, add an equal volume of anhydrous ethanol, and filter through a 0.22 μm organic filter membrane into an HPLC sample vial. Add 1 mL of 1×PBS buffer to the precipitate sample, gently mix by pipetting, centrifuge at 12000 rpm for 2 min, discard the supernatant to remove residual compounds on the cell surface, repeat the washing twice, add 200 μL of 1×PBS to dissolve the cells, add an equal volume of anhydrous ethanol and an appropriate amount of quartz sand, vortex, and filter through a 0.22 μm organic filter membrane into an HPLC sample vial with an inner tube. After processing, the yield of vanillin in the fermentation broth is determined by high performance liquid chromatography. The results showed that the vanillin yield of the strain expressing the F120G mutant was 122 mg / L, which was 1.22 times higher than that of the control strain expressing wild-type PP_0179, further confirming that the mutant plays a role in promoting product efflux and increasing yield in the process of vanillin biosynthesis.

[0117] In Example 6, the correlation coefficient k = 0.1 mM·L / mg used in the ferulic acid feeding strategy was determined based on previous shake-flask experiments and chemometric analysis. The specific basis and derivation process are as follows:

[0118] 1. Fundamentals of Chemometrics

[0119] Vanillin has a molecular weight of 152.15 g / mol, and ferulic acid has a molecular weight of 194.19 g / mol. Theoretically, 1 mol of ferulic acid can be converted into 1 mol of vanillin, therefore the theoretical molar conversion ratio is 1:1.

[0120] Therefore, the theoretical amount of ferulic acid required to generate 1 mg of vanillin is: 194.19 / 152.15≈1.28 mg;

[0121] Converted to molar concentration (based on a 1 L fermentation system):

[0122] 1 mg / L vanillin ≈ 1 / 152.15 mmol ≈ 0.00657 mmol;

[0123] The required amount of ferulic acid is:

[0124] 0.00657 mmol × 194.19 mg / mmol ≈ 1.28 mg or 0.00657 mmol;

[0125] That is: 1 mg / L vanillin ↔ 0.00657 mM ferulic acid

[0126] Therefore, the theoretical coefficient k 理论 ≈0.00657 mM⋅L / mg.

[0127] 2. Preliminary shake-flask experiments to determine the actual conversion efficiency.

[0128] Under shake-flask conditions, fed-batch fermentation was performed on engineered bacteria expressing the F120G mutant, and the vanillin synthesis rate (vp) was monitored under different ferulic acid addition rates. The experiment revealed:

[0129] When ferulic acid supply is insufficient (k<0.05), vanillin synthesis is limited by precursors;

[0130] When ferulic acid is in excess (k>0.15), cell growth is inhibited and the accumulation of byproducts increases;

[0131] Within the range of k=0.08~0.12, vanillin yield was the highest and cell viability was well maintained.

[0132] Analysis shows that the conversion of ferulic acid to vanillin during actual fermentation is not entirely stoichiometric. Some ferulic acid is used for cell metabolism or forms byproducts (such as vanillyl alcohol and vanillic acid). Therefore, the actual amount of ferulic acid required is higher than the theoretical value.

[0133] 3. Dynamic process considerations and safety factor

[0134] In the feedback flow plus strategy, the following practical factors need to be considered:

[0135] Degradation or non-target consumption of ferulic acid during fermentation;

[0136] Dynamic fluctuations in the product synthesis rate vp;

[0137] The need to maintain the homeostasis of the intracellular precursor pool.

[0138] To ensure sufficient ferulic acid supply and avoid inhibition, a safety factor of 1.5-2.0 was introduced based on the theoretical value, and combined with previous experimental data, the final determination was: k = 0.10 mM⋅L / mg;

[0139] This value balances precursor utilization efficiency and process robustness, and can dynamically maintain the ferulic acid concentration within the optimized range of 2-8 mM when the vanillin synthesis rate vp changes.

[0140] Comparative Example 1: Conventional one-time fed-batch fermentation process

[0141] The same engineered strain as in Example 6 (expressing the PP_0179 F120G mutant) was used.

[0142] Fermentation process: After inoculation, culture at 30℃ until OD reaches [the desired fermentation temperature]. 600 =0.6, add IPTG to a final concentration of 0.5 mM for induction. Simultaneously, add the entire 20 mM ferulic acid solution at once (this is standard practice to provide sufficient precursors). Continue fermentation at 30°C and 220 rpm without temperature adjustment.

[0143] Endpoint: Fermentation was terminated after 24 hours (this is a common fermentation duration in similar studies) because cell growth was observed to enter the late stationary phase, and the increase in product concentration almost stopped.

[0144] Comparative Example 2: Fixed-rate flow process for ferulic acid

[0145] Using the same engineered strain, the basic fermentation steps were exactly the same as in Example 6 (i.e., induction at the same OD point, addition of initial ferulic acid, and adjustment of temperature).

[0146] Feeding strategy: Four hours after the initial feeding, ferulic acid was fed at a fixed rate of 0.15 mM / h (this rate is approximately the estimated average feeding rate from Example 6).

[0147] Other conditions remained the same as in Example 6, with a total fermentation time of 96 hours.

[0148] Comparative Example 3: Secondary Induction Process Based on Fixed Time Points

[0149] Using the same engineered strain, the basic fermentation steps were exactly the same as in Example 6 (including the feedback strategy).

[0150] Secondary induction strategy: Instead of monitoring dissolved oxygen (DO), a second IPTG supplement was performed at a fixed time point (24 h) during fermentation, increasing its concentration by 0.3 mM. The total fermentation time was 96 h.

[0151] For Example 6 and Comparative Examples 1, 2, and 3, the final yield of vanillin, intracellular vanillin accumulation, and production intensity were measured respectively. The test results are shown in Table 1.

[0152] Table 1

[0153]

[0154] The results show that the process in Example 6 of this invention significantly outperformed all comparative examples in terms of vanillin final yield, verifying the superiority of its overall scheme. The lowest intracellular accumulation and highest production intensity demonstrate the advantages of this invention in product efflux efficiency and process efficiency, which is precisely achieved through the combination of transporter protein mutants and optimized processes.

[0155] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A mutant of an aromatic compound transporter protein, characterized in that, The mutant is a protein with the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

2. A nucleic acid molecule, characterized in that, It can encode the aromatic compound transporter mutant as described in claim 1; the nucleic acid molecule is the sequence shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the aromatic compound transporter mutant of claim 1, the nucleic acid molecule of claim 2, or the recombinant vector of claim 3.

5. The application of the aromatic compound transporter mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in the production of small molecule aromatic compounds, wherein the small molecule aromatic compounds are vanillin, p-hydroxybenzoic acid, and cinnamaldehyde.

6. The application of the aromatic compound transporter mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in improving the tolerance of microorganisms to small molecule aromatic compounds, wherein the small molecule aromatic compounds are vanillin, p-hydroxybenzoic acid, and cinnamaldehyde.

7. A method for producing small molecule aromatic compounds, characterized in that, Includes the following steps: An engineered bacterium is provided, which is genetically modified to express the aromatic compound transporter mutant as described in claim 1, and contains genes for the metabolic pathways that synthesize the small molecule aromatic compounds; The engineered bacteria were fermented and cultured under suitable culture medium and conditions to grow and synthesize the small molecule aromatic compounds. During the fermentation process, the engineered bacteria expressed an aromatic compound transporter mutant that releases the synthesized small molecule aromatic compounds from inside and outside the cell into the fermentation broth. The small molecule aromatic compounds were isolated and harvested from the culture after fermentation was completed; The small molecule aromatic compounds are vanillin, p-hydroxybenzoic acid, and cinnamaldehyde.

8. The method for producing small molecule aromatic compounds as described in claim 7, characterized in that, The fermentation and culture process is specifically as follows: The engineered bacteria were inoculated into liquid culture medium and cultured at 30-37℃; when the OD of the culture... 600 When the value reaches 0.4 to 0.8, isopropyl-β-D-thiogalactoside with a final concentration of 0.2 mM to 1.0 mM is added to the fermentation system to simultaneously induce the expression of the vanillin biosynthesis pathway gene and the mutant gene of the aromatic compound transporter protein. After adding isopropyl-β-D-thiogalactoside, ferulic acid with a final concentration of 5 mM to 20 mM was added to the fermentation system as a precursor for vanillin synthesis; the culture temperature was adjusted to 25℃ to 30℃, and fermentation was continued for 16 h to 120 h. The fermentation process is terminated when the concentration of small molecule aromatic compounds in the fermentation broth exceeds 800 mg / L.

9. The method for producing small molecule aromatic compounds as described in claim 8, characterized in that, The addition of ferulic acid employs a feedback feeding strategy based on the product synthesis rate, specifically: Ferulic acid is added to the fermentation system at an initial rate; The concentration of small molecule aromatic compounds in the fermentation broth was monitored in real time or at regular intervals, and their synthesis rate vp, mg·h / L was calculated. The flow rate of ferulic acid, FA, was dynamically correlated with the vanillin synthesis rate, vp, with FA = k × vp, where the correlation coefficient k ranged from 0.05 mM·L / mg to 0.2 mM·L / mg. This feedback control maintains the concentration of ferulic acid in the fermentation system within the range of 5 mM to 10 mM.

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