Construction of engineered strain for high-yield production of insect-resistant immune cyclic peptide GXP-A based on metabolic engineering and synthetic biology strategy

Abstract

The application provides an engineering strain for constructing high-yield anti-insect immunity cyclic peptide GXP-A based on metabolic engineering and synthetic biology strategies, and the yield of GXP-A is significantly improved from 0.92 mg / L of the wild type to 587.5 mg / L, which is more than 600 times, and has the industrial production potential. The genetic stability of the application is high: through genome simplification and chassis optimization, the genetic background of the strain is clear, and is suitable for long-term fermentation production. The strategy of the application can be popularized: the integrated strategies such as promoter engineering, precursor engineering and efflux engineering can provide reference for the high-yield construction of other microbial natural products, and solve the problems of low yield, high production cost and difficult industrial application of GXP-A in the prior art. Through systematic metabolic engineering modification, the synthesis capacity of GXP-A is significantly improved, and key raw materials are provided for the development of efficient and environmentally friendly biological insecticide synergist.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology, metabolic engineering, and agricultural biological control. Specifically, it relates to an engineered strain that produces a high yield of the insect-resistant immune cyclic peptide GXP-A based on metabolic engineering and synthetic biology strategies. Background Technology

[0002] Agricultural pests pose a serious threat to global food security. Currently, chemical pesticides remain the primary means of control, but their long-term use has led to increasingly prominent environmental pollution, ecological imbalance, pest resistance, and food safety issues. Entomopathogenic fungi (such as Metarhizium anisopliae and Beauveria bassiana) serve as biological insecticides, possessing advantages such as high specificity and environmental friendliness. However, their infection efficiency is limited by the insect's immune system, resulting in limited insecticidal effects.

[0003] GameXPeptides (GXPs) are a class of cyclic polypeptides produced by symbiotic bacteria of entomopathogenic nematodes. GXP-A, in particular, has been shown to significantly inhibit insect immune responses (such as hemocyte nodule formation and antimicrobial peptide expression) and enhance the insecticidal activity of biopesticides such as *Metarhizium anisopliae*, demonstrating promising application prospects. However, the yield of GXP-A in natural strains is extremely low (wild-type). Xenorhabdus budapestensis The yield of XBD8 is approximately 0.92 mg / L, which is insufficient to meet the needs of industrialization and restricts its promotion and application in agricultural pest control.

[0004] Currently, modifying microbial cell factories through metabolic engineering and synthetic biology strategies has become an effective way to increase the yield of natural products. While various metabolic engineering strategies (such as promoter engineering, precursor supply enhancement, and genome simplification) have been reported in existing technologies, there is still no systematic construction of a high-yield engineered strain of GXP-A and its integrated application in enhancing the efficacy of biopesticides in China. Therefore, this paper proposes to construct an engineered strain that produces a high yield of the insect-resistant immune cyclic peptide GXP-A based on metabolic engineering and synthetic biology strategies. Summary of the Invention

[0005] This invention belongs to the fields of synthetic biology, metabolic engineering, and agricultural biological control technology, specifically relating to a method for modifying symbiotic bacteria of entomopathogenic nematodes using systematic metabolic engineering and synthetic biology techniques. Xenorhabdus budapestensis XBD8, an engineered strain for constructing high-yield insect-resistant immune cyclic peptide GameXPeptide-A (GXP-A), and its construction method and application.

[0006] The purpose of this invention is to provide an engineered strain that produces a high-yield insect-resistant immune cyclic peptide GXP-A and its construction method, thereby addressing the problems of low GXP-A yield, high production cost, and difficulty in industrial application in existing technologies. Through systematic metabolic engineering, the synthesis capacity of GXP-A is significantly enhanced, providing a key raw material for the development of efficient and environmentally friendly biopesticides.

[0007] To achieve the aforementioned objectives, this invention provides the following technical solution: an engineered strain that produces a high yield of the insect-resistant immune cyclic peptide GXP-A, wherein the strain is metabolically engineered. Xenorhabdus budapestensis The strains produced GXP-A yields of no less than 580 mg / L in shake flasks using LB liquid medium and no less than 2500 mg / L in 5 L fermenters using the fermentation medium optimized in this experiment.

[0008] As a preferred embodiment of the present invention, the metabolic engineering modification includes the modification of the enzyme encoding GXP-A nonribosomal peptide synthase. gxpS The transcriptional regulatory sequence of the gene is modified, wherein... gxpS The gene promoter is replaced with an endogenous promoter P. Xb13 .

[0009] As a preferred technical solution of the present invention, the gxpS The ribosome binding site (RBS) of the gene was replaced with RBS2.

[0010] As a preferred embodiment of the present invention, the metabolic engineering modification further includes deleting one or more natural product synthesis gene clusters that compete with GXP-A biosynthesis precursors from the genome of the strain, wherein the deleted gene clusters include synthesis gene clusters encoding NRPS synthase.

[0011] As a preferred embodiment of the present invention, the metabolic engineering modification further includes overexpression of a protein selected from the phenylalanine synthesis pathway. aroG , aroE , tyrB Genes, and cyclic peptide efflux-related gene3607 and gene3862 One or more genes.

[0012] As a preferred technical solution of the present invention, the strain is Xenorhabdus budapestensis G3, with a GXP-A yield of no less than 580 mg / L.

[0013] A method for constructing a bacterial strain includes the following steps: (a) In the starting strain, gxpS The gene promoter is replaced with an endogenous strong promoter P. Xb13 ; (b) Optionally, gxpS The RBS gene is replaced with RBS2; (c) Optionally, delete one or more competing natural product synthesis gene clusters in the genome; (d) Optionally, overexpressing genes that enhance precursor supply and / or genes that enhance product efflux; wherein step (a) is required, and one or more of steps (b)-(e) are performed in combination with (a).

[0014] Application of G3 strain in the fermentation production of GXP-A, or in the preparation of compositions for controlling agricultural pests.

[0015] As a preferred technical solution of the present invention, promoter-ribosome binding site (RBS) engineering: based on transcriptome data mining, endogenously highly expressed promoters are replaced with the original ones. gxpS Gene promoter; translation efficiency was further optimized through RBS engineering. The preferred promoter was P. Xb13 RBS2 was selected as the preferred RBS, and the engineered strain G1 was constructed, with the GXP-A yield increased to 388.2 mg / L. (2) Genome simplification: Non-essential transposase genes and natural product synthesis gene clusters that compete with GXP-A synthesis precursors (such as NRPS / PKS gene clusters) were deleted from the genome, and the genome-simplified chassis strain G2 was constructed, with the GXP-A yield increased to 498.2 mg / L. (3) Precursor supply enhancement: Based on the G1 strain, key genes of the phenylalanine synthesis pathway were overexpressed. aroG , aroE With tyrB, the supply of precursor phenylalanine was increased, and the engineered strain QT3 was constructed, increasing the GXP-A yield to 442.5 mg / L. (4) Enhanced product efflux: Based on the G1 strain, the efflux protein gene gene3607 was overexpressed and gene3862 (Belonging to the MFS superfamily), the extracellular transport capacity of GXP-A was enhanced, the product accumulation inhibition was alleviated, and the engineered strain WP3 was constructed, increasing the GXP-A yield to 435.4 mg / L. (5) Based on the G2 strain, the key genes of the phenylalanine synthesis pathway were overexpressed. aroG , aroE With tyrB and overexpression of efflux protein genes gene3607 and gene3862 The engineered strain G3 was constructed, and the GXP-A yield was further increased to 587.5 mg / L.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The yield of GXP-A has been significantly increased: from 0.92 mg / L in wild type to 587.5 mg / L, an increase of more than 600 times, which has the potential for industrial production.

[0017] (2) High genetic stability: Through genome simplification and chassis optimization, the genetic background of the strain is clear and suitable for long-term fermentation production.

[0018] (3) The strategy can be promoted: The promoter engineering, precursor engineering and efflux engineering strategies integrated in this invention can provide a reference for the high-yield construction of other microbial natural products. Attached Figure Description

[0019] Figure 1 A schematic diagram of the engineered strain for constructing the insect immune cyclic peptide GameXPeptide-A (GXP-A) based on systematic metabolic engineering and synthetic biology strategies provided by this invention; Figure 2 This is a data diagram provided for the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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.

[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0022] Example 1: Construction of engineered strain G1 by Xenorhabdus budapestensis XBD8 was the starting strain, and the endogenously highly expressed promoter P was obtained through transcriptome data screening. Xb13 The original promoter of the gxpS gene was replaced; further optimization of translation efficiency using the RBS2 sequence yielded engineered strain G1. After fermentation in LB medium for 48 hours, the GXP-A yield reached 388.2 mg / L.

[0023] Example 2: Construction of a genome-simplified strain G2 Based on strain G1, 10 transposase units and 10 competitive NRPS / PKS gene clusters were deleted sequentially to obtain strain G2 with a simplified genome, and the GXP-A yield was increased to 498.2 mg / L.

[0024] Example 3: Construction of precursor engineered strain QT3 Based on the G1 strain, overexpression of the phenylalanine synthesis pathway gene aroG , aroE and tyrB A combined overexpression strain QT3 was constructed. After fermentation in LB medium for 48 hours, the GXP-A yield reached 442.5 mg / L.

[0025] Example 4: Construction of efflux engineered strain WP3 Based on G1, overexpression of efflux protein genes gene3862 and gene3607 An engineered strain, WP3, was constructed. After fermentation in LB medium for 48 hours, the yield of GXP-A reached 435.4 mg / L.

[0026] Example 5: Construction of engineered strain G3 Based on the G2 strain, genes superimposed on the epiphenylalanine synthesis pathway in precursor and efflux engineering were combined. aroG , aroE and tyrB and efflux protein genes gene3862 and gene3607 A high-yielding strain, G3, was obtained. The GXP-A yield reached 587.5 mg / L, an increase of 17.9% compared to strain G2.

[0027] Terminology Explanation: GXP-A: GameXPeptide-A, an anti-insect immune cyclic peptide.

[0028] NRPS: Non-Ribosomal Peptide Synthetase.

[0029] RBS: Ribosome Binding Site.

[0030] Example 7: Promoter-ribosome binding site (RBS) engineering: Mining endogenously highly expressed promoters based on transcriptome data and replacing the original ones. gxpS Gene promoter; translation efficiency was further optimized through RBS engineering. The preferred promoter was P. Xb13 RBS2 was selected as the preferred RBS, and the engineered strain G1 was constructed, with the GXP-A yield increased to 388.2 mg / L. (2) Genome simplification: Non-essential transposase genes and natural product synthesis gene clusters that compete with GXP-A synthesis precursors (such as NRPS / PKS gene clusters) were deleted from the genome, and the genome-simplified chassis strain G2 was constructed, with the GXP-A yield increased to 498.2 mg / L. (3) Precursor supply enhancement: Key genes of the phenylalanine synthesis pathway were overexpressed. aroG , aroE and tyrB To enhance the supply of precursor phenylalanine, the engineered strain QT3 was constructed, and the GXP-A yield was increased to 442.5 mg / L. (4) Enhanced product efflux: Overexpression of efflux protein gene gene3607 and gene3862 (Belonging to the MFS superfamily), the extracellular transport capacity of GXP-A was enhanced, the product accumulation inhibition was alleviated, and the engineered strain WP3 was constructed, increasing the GXP-A yield to 435.4 mg / L. (5) Based on the G2 strain, the genes of the superimposed phenylalanine synthesis pathway in the precursor and efflux engineering were combined. aroG , aroE and tyrB and efflux protein genes gene3862 and gene3607 A high-yielding strain, G3, was obtained. The GXP-A yield reached 587.5 mg / L, a 17.9% increase compared to strain G2. Example 8: An engineered strain producing high levels of the insect-resistant immune cyclic peptide GXP-A, the strain being metabolically engineered. Xenorhabdus budapestensis The strain has a GXP-A yield of not less than 580 mg / L.

[0031] A genetically engineered bacterium that produces the insect-resistant immune cyclic peptide GXP-A, strain numbered CGMCC No. 22056. Xenorhabdus budapestensis XBD8 is the starting strain, which was obtained through metabolic engineering, and its fermentation production of GXP-A is not less than 580 mg / L.

[0032] Metabolic engineering modifications include the modification of enzymes encoding GXP-A nonribosomal peptide synthase. gxpS The transcriptional regulatory sequences of genes are modified, among which... gxpS The gene promoter is replaced with an endogenous promoter P. Xb13 .

[0033] gxpS The ribosome binding site (RBS) of the gene was replaced with RBS2.

[0034] Metabolic engineering also includes deleting one or more natural product synthesis gene clusters from the strain's genome that compete with GXP-A biosynthesis precursors. The deleted gene clusters include synthesis gene clusters encoding NRPS synthase.

[0035] Metabolic engineering also includes overexpression of molecules selected from the phenylalanine synthesis pathway. aroG , aroE , tyrB Genes, and cyclic peptide efflux-related gene3607 and gene3862 One or more genes.

[0036] strain is Xenorhabdus budapestensis G3, with a GXP-A yield of no less than 580 mg / L.

[0037] A method for constructing a bacterial strain includes the following steps: (a) In the starting strain, gxpS The gene promoter is replaced with an endogenous strong promoter P. Xb13 ; (b) Optionally, replace RBS in the gxpS gene with RBS2; (c) Optionally, delete one or more competing natural product synthesis gene clusters in the genome; (d) Optionally, overexpress genes that enhance precursor supply and / or genes that enhance product efflux. Step (a) is required, and one or more of steps (b)-(e) are performed in combination with (a).

[0038] Application of the strain in the fermentation production of GXP-A, or in the preparation of compositions for controlling agricultural pests.

[0039] Promoter and ribosome binding site (RBS) engineering: Mining endogenously highly expressed promoters based on transcriptome data and replacing original ones. gxpS Gene promoter; translation efficiency was further optimized through RBS engineering. The preferred promoter was P. Xb13 RBS2 was selected as the preferred RBS, and the engineered strain G1 was constructed, with the GXP-A yield increased to 388.2 mg / L. (2) Genome simplification: Non-essential transposase genes and natural product synthesis gene clusters that compete with GXP-A synthesis precursors (such as NRPS / PKS gene clusters) were deleted from the genome, and the genome-simplified chassis strain G2 was constructed, with the GXP-A yield increased to 498.2 mg / L. (3) Precursor supply enhancement: Key genes of the phenylalanine synthesis pathway were overexpressed. aroG , aroE and tyrB To enhance the supply of precursor phenylalanine, the engineered strain QT3 was constructed, and the GXP-A yield was increased to 442.5 mg / L. (4) Enhanced product efflux: Overexpression of efflux protein gene gene3607 and gene3862 (Belonging to the MFS superfamily), the extracellular transport capacity of GXP-A was enhanced, the product accumulation inhibition was alleviated, and the engineered strain WP3 was constructed, increasing the GXP-A yield to 435.4 mg / L. (5) Based on the G2 strain, the genes of the superimposed phenylalanine synthesis pathway in the precursor and efflux engineering were combined. aroG , aroE and tyrB and efflux protein genes gene3862 and gene3607A high-yielding strain, G3, was obtained. The GXP-A yield reached 587.5 mg / L, a 17.9% increase compared to strain G2. The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. An engineered strain that produces a high yield of the insect-resistant immune cyclic peptide GXP-A, characterized in that, The strain was metabolically engineered. Xenorhabdus budapestensis The strains produced GXP-A yields of no less than 580 mg / L in shake flasks using LB liquid medium and no less than 2500 mg / L in 5 L fermenters using the fermentation medium optimized in this experiment.

2. The engineered bacteria producing high-yield insect-resistant immune cyclic peptide GXP-A according to claim 1, characterized in that, The metabolic engineering modifications include the modification of the enzyme encoding GXP-A nonribosomal peptide synthase. gxpS The transcriptional regulatory sequence of the gene is modified, wherein... gxpS The gene promoter is replaced with an endogenous promoter P. Xb13 .

3. The genetically engineered bacterium for producing the insect-resistant immune cyclic peptide GXP-A according to claim 2, characterized in that, The gxpS The ribosome binding site (RBS) of the gene was replaced with RBS2.

4. The strain according to any one of claims 1-3, characterized in that, The metabolic engineering modification also includes deleting 10 natural product synthesis gene clusters from the genome of the strain that compete with GXP-A biosynthesis precursors, the deleted gene clusters containing encoding NRPS synthase.

5. The strain according to any one of claims 1-3, characterized in that, The metabolic engineering modification also includes overexpression of substances selected from the phenylalanine synthesis pathway. aroG , aroE , tyrB Genes, and efflux-related factors gene3607 and gene3862 One or more genes.

6. The strain according to any one of claims 1-3, characterized in that, The strain is Xenorhabdus budapestensis G3, with a GXP-A yield of no less than 580 mg / L.

7. A method for constructing the strain according to any one of claims 1-3, characterized in that, Includes the following steps: (a) In the starting strain, gxpS The gene promoter is replaced with an endogenous strong promoter P. Xb13 ; (b) Optionally, gxpS The RBS gene is replaced with RBS2; (c) Optionally, delete one or more competing natural product synthesis gene clusters in the genome; (d) Optionally, overexpressing genes that enhance precursor supply and / or genes that enhance product efflux; wherein step (a) is required, and one or more of steps (b)-(e) are performed in combination with (a).

8. The use of the strain according to any one of claims 1-3 in the fermentation production of GXP-A, or in the preparation of a composition for controlling agricultural pests.

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