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CN-121991189-A - Aromatic compound transporter mutant and application thereof

CN121991189ACN 121991189 ACN121991189 ACN 121991189ACN-121991189-A

Abstract

The invention discloses an aromatic compound transport protein mutant and application thereof, and belongs to the technical field of bioengineering. The mutant is obtained by carrying out fixed-point substitution on key residues of a gating loop of the outer membrane transport protein based on pseudomonas putida (Pseudomonas putida), specifically comprises one or more of substitution of phenylalanine at position 120 to glycine, leucine at position 121 to glycine and glutamine at position 122 to glycine, preferably comprises single mutant F120G, double mutant F120G/L121G or triple mutant F120G/L121G/Q122G, and can also be subjected to conservative substitution derivatization on the basis of the single mutant F120G, the double mutant F120G/L121G/Q122G. The mutant can be used for efficient production of microorganisms and cell tolerance regulation of aromatic compounds according to physical and chemical characteristics of target products, and has important application values in green biological manufacturing, fermentation engineering and bioremediation.

Inventors

  • XIN FENGJIAO
  • WANG YULU
  • LIU BOLIN
  • LI ZHEN

Assignees

  • 中国农业科学院农产品加工研究所

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. An aromatic compound transporter mutant, characterized in that the mutant comprises one or more combinations selected from the group consisting of substitution at position 120 with glycine, substitution at position 121 with glycine, and substitution at position 122 with glycine based on the amino acid sequence shown in SEQ ID NO. 1.
  2. 2. The aromatic compound transporter mutant according to claim 1, wherein the mutant is a protein having an amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.
  3. 3. A nucleic acid molecule capable of encoding the aromatic compound transporter mutant according to claim 1 or 2, said nucleic acid molecule comprising the sequences shown in SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12.
  4. 4. A recombinant vector comprising the nucleic acid molecule of claim 3.
  5. 5. A recombinant cell comprising the aromatic compound transporter mutant of claim 1 or 2 or the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.
  6. 6. Use of the aromatic compound transporter mutant of claim 1 or 2 or the nucleic acid molecule of claim 3 or the recombinant vector of claim 4 or the recombinant cell of claim 5 for the production of small molecule aromatic compounds.
  7. 7. Use of the aromatic compound transporter mutant of claim 1 or 2 or the nucleic acid molecule of claim 3 or the recombinant vector of claim 4 or the recombinant cell of claim 5 for increasing tolerance of a microorganism to a small molecule aromatic compound.
  8. 8. A method for producing a small molecule aromatic comprising the steps of: Providing an engineered bacterium genetically engineered to express the aromatic compound transporter mutant of claim 1 or 2, and comprising a metabolic pathway gene that synthesizes the small molecule aromatic; Fermenting and culturing the engineering bacteria under a proper culture medium and culture conditions to grow and synthesize the small molecular aromatic compound, wherein in the fermentation and culture process, the aromatic compound transport protein mutant expressed by the engineering bacteria discharges the synthesized small molecular aromatic compound from inside and outside the cell into fermentation liquor; separating and harvesting the small molecule aromatic compounds from the culture ending the fermentation; wherein the small molecule aromatic compound is vanillin, p-hydroxybenzoic acid and cinnamaldehyde.
  9. 9. The method for producing a small molecule aromatic according to claim 8, wherein the fermentation culture process is specifically; Inoculating engineering bacteria into a liquid culture medium, and culturing at 30-37 ℃, when the OD 600 value of the culture reaches 0.4-0.8, adding isopropyl-beta-D-thiogalactoside with the final concentration of 0.2 mM-1.0 mM into a fermentation system to induce the expression of vanillin anabolic pathway genes and aromatic compound transporter mutant genes simultaneously; Adding ferulic acid with final concentration of 5 mM-20 mM as precursor for vanillin synthesis into fermentation system after adding isopropyl-beta-D-thiogalactoside, adjusting culture temperature to 25-30 ℃, and fermenting and culturing 16 h-120 h; when the concentration of the small molecular aromatic compound in the fermentation liquid is higher than 800 mg/L, the fermentation culture process is stopped.
  10. 10. The method for producing small molecule aromatic of claim 9, wherein the addition of ferulic acid employs a feedback fed-batch strategy based on product synthesis rate, specifically: feeding ferulic acid into the fermentation system at an initial rate; monitoring the concentration of small molecular aromatic compounds in the fermentation liquor in real time or at fixed time, and calculating the synthesis rate vp, mg.h/L; Dynamically associating a flow rate FA of ferulic acid with a vanillin synthesis rate vp, controlling fa=k×vp, wherein an association coefficient k is 0.05mM ·l/mg to 0.2mm·l/mg; By this feedback control, the concentration of ferulic acid in the fermentation system is maintained in the range of 5mM to 10 mM.

Description

Aromatic compound transporter mutant and application thereof Technical Field The invention relates to the technical field of bioengineering. More particularly, the present invention relates to an aromatic compound transporter mutant and its use. Background Aromatic compounds are important raw materials in the fields of medicine, food, daily chemicals, materials and the like, and are produced by mainly relying on plant extraction and chemical synthesis in the traditional process. Microbial cell factories provide a promising alternative platform for sustainable synthesis of aromatic compounds, but the production efficiency is often limited by toxicity of the product to host cells, resulting in low conversion efficiency and difficulty in meeting the requirements of industrial scale. To alleviate product toxicity, transporter engineering has become one of the key strategies to promote host tolerance. Efflux pumps are effective in enhancing host tolerance and productivity by transporting intracellular accumulated deleterious compounds out of the cell. In gram-negative bacteria (e.g., E.coli, pseudomonas putida, etc.), outer membrane efflux proteins are key components to performing this function, mediating the efflux of a variety of substances including antibiotics, organic solvents, and metabolites. Among them, the OMF family of outer membrane efflux proteins represented by TolC has been widely studied. Typically, the structure is a trimer, which is capable of forming channels across the outer membrane. However, such channels typically exhibit a semi-open extracellular outlet conformation in the resting state, while favoring substrate efflux, may also result in influx of external compounds or reflux of transported substrates. Research shows that the nonselective bidirectional diffusion weakens the detoxification efficiency of cells and induces toxicity accumulation under the condition of high concentration products, thereby restricting the practical application effect in microbial production. Therefore, the development of the outer membrane efflux protein with the accurate one-way valve function enables the outer membrane efflux protein to effectively block harmful backflow while efficiently excreting substrates, and the engineering transformation enables the outer membrane efflux protein to be adapted according to the physical and chemical characteristics of a target product, so that the efficient microbial production of aromatic compounds and the improvement of host cell tolerance are realized, and the technology difficulty in the current field is realized. Disclosure of Invention Aiming at the problems in the prior art, the invention provides an application of outer membrane efflux protein with a one-way valve function, which can effectively block the backflow of hydrophobic macromolecular aromatic compounds and improve the tolerance of host bacteria. The invention further provides the outer membrane efflux protein mutant, which is used for improving the tolerance of host bacteria to small molecular aromatic compounds, and the mutant gene is applied to the microbial synthesis of target aromatic compounds, so that the product efflux can be efficiently promoted, and the efficient production of the aromatic compounds and the cooperative regulation and control of cell tolerance are realized. 1. Aromatic compound transporter mutant proteins and nucleic acids expressing same To achieve these objects and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, there is provided an aromatic compound transporter mutant comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, which is a protein of a) or b) as follows: a) Comprises one or more of substitution of phenylalanine at position 120 corresponding to the amino acid sequence shown in SEQ ID NO. 1 with glycine (F120G), substitution of leucine at position 121 with glycine (L121G) of the amino acid sequence shown in SEQ ID NO. 1, or substitution of glutamine at position 122 with glycine (Q122G) of the amino acid sequence shown in SEQ ID NO. 1, preferably single mutant F120G, double mutant F120G/L121G or triple mutant 120G/L121G/Q122G; b) A protein derived from (a) wherein the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and still retains the efflux activity of an aromatic compound. The invention also provides a coding nucleic acid of the aromatic compound transporter mutant. For example, the base sequence of one DNA molecule of the F120G mutant shown in SEQ ID NO.2 is shown in SEQ ID NO. 8, the base sequence of one DNA molecule of the L121G mutant shown in SEQ ID NO. 3 is shown in SEQ ID NO. 9, the base sequence of one DNA molecule of the Q122G mutant shown in SEQ ID NO.4 is shown in SEQ ID NO. 10, the base sequence of one DNA molecule of the F120G/L121G mutant shown in SEQ ID NO.5 is shown in SEQ ID NO. 11, and the base sequence of one D