Engineering strain for producing beta-caryophyllene by transforming eutrophic alcaligenes Rosei through regulatory factor metabolism

Abstract

The invention relates to the technical field of bioengineering, in particular to a method for producing beta-caryophyllene by transforming alcaligenes rosea through metabolic engineering. According to the present invention, the Alcaligenes Rosei is adopted as the starting strain, the exogenous gene QHSopt is introduced to construct the recombinant strain of the Alcaligenes Rosei so as to produce the beta-caryophyllene, the heterologous genes IDI and GPPS are introduced so as to improve the beta-caryophyllene production ability of the strain, and the MEP pathway key genes dxs and dxr are overexpressed to further improve the yield of the beta-caryophyllene; finally, the regulatory factor capable of improving the yield of beta-caryophyllene is obtained through overexpression of a global regulatory factor gene and screening. Therefore, the eutrophy alcaligenes Rosei engineering strain for efficiently producing the beta-caryophyllene is obtained.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for producing beta-caryophyllene by using metabolic engineering alcaligenes eutrophus rosellae. Background Art

[0002] Terpenes are the largest class of natural products, with more than 40,000 known structures, many of which are essential for plant growth, development and general metabolism. Terpenes can generally be classified according to the number of carbon atoms they contain: hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Sesquiterpenes are the most diverse class of terpenes and are often used in the pharmaceutical industry because of their wide range of activities, including antioxidant, antibacterial and anti-inflammatory properties. Because their structures often contain rings and methyl branches, they have high energy density, making them attractive candidates for sustainably produced biofuels.

[0003] β-Caryophyllene is a bicyclic sesquiterpenoid compound widely found in essential oils extracted from various plants. It was first discovered in 1834 and has three conformations: α-, β- and γ-. Since the content of β-caryophyllene in plants is relatively low and plant growth is greatly affected by factors such as seasons and regional climate, it is challenging to extract β-caryophyllene from plants for the mass market. The chemical synthesis method of caryophyllene has inevitable problems such as high production cost, low production efficiency and environmental pollution. With the development of synthetic biology, metabolic engineering, as a new technology that enables microorganisms to efficiently produce isoprenoids, has represented a promising technology to replace plant extraction and chemical synthesis in recent years. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the yield of β-caryophyllene by utilizing global regulatory factors and metabolic engineering, and optimizing the β-caryophyllene production pathway of the recombinant strain of Alcaligenes ruckeri by expressing exogenous genes and endogenous genes.

[0005] To achieve the above-mentioned object of the invention, the present invention provides the following solutions:

[0006] The invention provides a method for producing beta-caryophyllene by utilizing a recombinant strain of Alcaligenes eutrophus.

[0007] The invention provides a recombinant alcaligenes eutrophus of rosella, wherein the genome of the recombinant bacterium comprises exogenous genes, and the exogenous genes include: IDI, GPPS and QHSopt.

[0008] The present invention provides a recombinant alcaligenes eutrophus of Roche, wherein the genome of the recombinant bacterium includes endogenous genes, and the endogenous genes include: dxs, dxr, ispD, ispH and QHSopt. The caryophyllene synthase gene (QHSopt) is synthesized by a gene company (Qingke Biotechnology Co., Ltd.) through codon optimization, and the synthesized gene is amplified and purified and recovered by PCR, and connected with the amplified linearized plasmid p2MCBAD-lac at a molar ratio of 2-5:1, and the connection product is transformed into Escherichia coli competent cells, and positive clones are screened on an LB solid plate containing chloramphenicol antibiotics, and the recombinant plasmid p2MCBAD-QHSopt-lac is obtained after verification and sequencing.

[0009] In the future, the gene GPPS of Saccharomyces cerevisiae will be purified and recovered after PCR amplification, and then connected with the plasmid p2MCBAD-QHSopt-lac after linearization at a molar ratio of 2-5:1. The ligation product will be transformed into Escherichia coli competent cells, and positive clones will be screened on LB solid plates containing chloramphenicol antibiotics. After verification and sequencing, the recombinant plasmid p2MCBAD-QHSopt-GPPS-lac will be obtained;

[0010] The gene IDI from Abies grandis was amplified by PCR, purified, recovered and linearized, and then connected with the plasmid p2MCBAD-QHSopt-lac at a molar ratio of 2-5:1. The ligation product was transformed into competent E. coli cells, and positive clones were screened on LB solid plates containing chloramphenicol antibiotics. After verification and sequencing, the recombinant plasmid p2MCBAD-QHSopt-IDI-lac was obtained.

[0011] In the future, the endogenous genes dxs, dxr and endogenous regulatory factor genes will be purified and recovered after PCR amplification, and then connected with the amplified linearized plasmid p2MCBAD-QHSopt-lac at a molar ratio of 2-5:1. The connection product will be transformed into Escherichia coli competent cells, and positive clones will be screened on LB solid plates containing chloramphenicol antibiotics. After verification and sequencing, the recombinant plasmids p2MCBAD-QHSopt-dxs-lac, p2MCBAD-QHSopt-dxr-lac, p2MCBAD-QHSopt-ispD-lac, p2MCBAD-QHSopt-ispH-lac, and p2MCBAD-QHSopt-lac-rpoS / Fnr5 / uspA will be obtained.

[0012] E. coli DH5α containing the recombinant plasmid was used as the donor bacterium, E. coli T1 containing the plasmid pRK2013 was used as the helper bacterium, and the C. necatorH16 gene-deficient strain H16ΔCAB was used as the recipient bacterium for combination and PCR verification to obtain a positive recombinant strain

[0013] According to the recombinant bacteria, the exogenous genes GPPS and IDI are expressed under an inducible promoter.

[0014] According to the recombinant bacteria, the exogenous gene is expressed on a plasmid.

[0015] According to the recombinant bacteria, the codon-optimized caryophyllene synthase gene QHSopt is from Artemisia annua

[0016] The nucleotide sequence of (Artemisia caruifolia Buch.-Ham. ex Roxb.) is shown in SEQ ID NO.1

[0017] shown.

[0018] And / or, the nucleotide sequence of said dxs is as shown in SEQ ID NO.2;

[0019] And / or, the nucleotide sequence of dxr is shown in SEQ ID NO.3;

[0020] And / or, the nucleotide sequence of ispD is shown in SEQ ID NO.4;

[0021] And / or, the nucleotide sequence of ispD is shown in SEQ ID NO.5;

[0022] And / or, the nucleotide sequence of rpoS is shown in SEQ ID NO.6;

[0023] And / or, the nucleotide sequence of Fnr5 is shown in SEQ ID NO.7;

[0024] And / or, the nucleotide sequence of uspA is shown in SEQ ID NO.8;

[0025] And / or, the GPPS is from Saccharomyces cerevisiae (SEQ ID NO.9);

[0026] And / or, the IDI is from Abiesgrandis as shown in SEQ ID NO.10;

[0027] The present invention discloses the following technical effects:

[0028] The present invention uses p2MCBAD-lac as a basic plasmid, connects a codon-optimized caryophyllene synthase gene QHSopt capable of catalyzing a substrate into β-caryophyllene, and transfers the obtained recombinant plasmid into Alcaligenes eutrophus of Rothschild to obtain a recombinant strain capable of producing β-caryophyllene. In the fermentation process, 0.1 g / L arabinose is used for induction, and the fermentation liquid induced for 72 hours is detected. The β-caryophyllene yield is the largest and tends to be stable, and the optimal caryophyllene yield is 1.6 mg / L. The expression intensity of the caryophyllene synthase gene is further increased by 1.7 times to 2.748 mg / L by the arabinose induction concentration.

[0029] The present invention uses p2MCBAD-QHSopt-lac as a basic plasmid, connects the key exogenous genes IDI and GPPS of the MEP pathway, and the endogenous genes dxs, dxr, ispD and ispH, and the obtained recombinant plasmid is transferred into Alcaligenes eutrophus Roche to obtain a recombinant strain with an optimized MEP pathway, and obtains an optimal caryophyllene yield of 3.6 mg / L by overexpressing the dxr gene in fermentation.

[0030] The present invention uses p2MCBAD-QHSopt-lac as the basic plasmid through P lac The endogenous global regulatory factor genes rpoS, Fnr5, and uspA were constitutively connected to obtain the recombinant plasmid, which was transferred into Alcaligenes eutrophus of Rochester to obtain a recombinant strain expressing the regulatory factor. The fermentation yield results showed that Fnr5 and rpoS increased the caryophyllene production, among which Fnr5 had the greatest yield increase of 1.4 times and 1.5 times the basic yield of 4.1 mg / L, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-lac.

[0033] Figure 2 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-dxs-lac.

[0034] Figure 3 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-dxr-lac.

[0035] Figure 4This is the plasmid map of the constructed vector p2MCBAD-QHSopt-ispD-lac.

[0036] Figure 5 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-ispH-lac.

[0037] Figure 6 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-lac-rpoS.

[0038] Figure 7 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-lac-Fnr5.

[0039] Figure 8 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-lac-uspA.

[0040] Fig. 9 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-GPPS-lac.

[0041] Fig.10 This is the plasmid map of the constructed vector p2MCBAD-QHSopt-IDI-lac.

[0042] Fig.11 This is a diagram of the fermentation results of the recombinant strain that optimized β-caryophyllene.

[0043] Fig.12 This is a diagram of the fermentation results of the recombinant strain with the optimized induction concentration.

[0044] Fig.13 This is a diagram of the fermentation results of the recombinant strain that constructed the optimized MEP pathway.

[0045] Fig.14 This is a diagram of the fermentation results of the constructed recombinant strain that overexpresses the regulatory factor. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] Without departing from the scope or spirit of the present invention, various modifications and variations may be made to the specific embodiments of the present invention description, which will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. The present invention description and examples are merely exemplary. As used herein, "comprising", "including", "having", "containing", etc. are all open-ended terms, i.e., meaning including but not limited to.

[0050] In the process of microbial synthesis of β-caryophyllene, the naturally existing 2-C-methyl-D-erythritol-4-phosphate pathway of microorganisms is utilized. In the current study, overexpression of key genes of the 2-C-methyl-D-erythritol-4-phosphate pathway has achieved good results. At the same time, global transcription factors refer to transcription factors required for most gene transcription, which can simultaneously activate or inhibit the expression of multiple genes in a specific pathway. Based on this characteristic, global transcription factors are often used to improve the synthesis of specific metabolites and improve strain tolerance.

[0051] Therefore, the production of β-caryophyllene was increased by overexpressing the exogenous genes IDI and GPPS, and overexpressing endogenous key genes to optimize the 2-C-methyl-D-erythritol-4-phosphate pathway. At the same time, the stimulation of carbon flux by global regulatory factors was utilized to increase the production of β-caryophyllene by overexpressing the global regulatory factors rpoS, Fnr5, and uspA.

[0052] Example 1 Gene acquisition and vector construction

[0053] QHS from Artemisia caruifolia Buch.-Ham. ex Roxb., whose nucleotide sequence is shown in SEQ ID No. 1, was chemically synthesized by BGI Corporation onto a pUC-57 vector to obtain a pUC-QHS vector.

[0054] The dxs gene from (Cupriavidus necatorH16), whose nucleotide sequence is shown in SEQ ID No.2, was amplified from the genome of Cupriavidus necatorH16.

[0055] The dxr gene from (Cupriavidus necatorH16), whose nucleotide sequence is shown in SEQ ID No.3, was amplified from the Cupriavidus necatorH16 genome.

[0056] The ispD gene from (Cupriavidus necatorH16), whose nucleotide sequence is shown in SEQ ID No. 4, was amplified from the (Cupriavidus necatorH16) genome.

[0057] The ispH gene from (Cupriavidus necatorH16), whose nucleotide sequence is shown in SEQ ID No. 5, was amplified from the (Cupriavidus necatorH16) genome.

[0058] The rpoS gene from (Cupriavidus necator H16), whose nucleotide sequence is shown in SEQ ID No. 6, was amplified from the Cupriavidus necator H16 genome.

[0059] The Fnr5 gene from (Cupriavidus necator H16), whose nucleotide sequence is shown in SEQ ID No. 7, was amplified from the Cupriavidus necator H16 genome.

[0060] The uspA gene from (Cupriavidus necatorH16), whose nucleotide sequence is shown in SEQ ID No. 8, was amplified from the Cupriavidus necatorH16 genome.

[0061] The GPPS gene from Saccharomyces cerevisiae, whose nucleotide sequence is shown in SEQ ID No. 9, was chemically synthesized by BGI Corporation onto the pUC-57 vector to obtain the pUC-GPPS vector.

[0062] The GPPS gene, whose nucleotide sequence is shown in SEQ ID No. 10, was chemically synthesized by BGI Corporation into a pUC-57 vector to obtain a pUC-IDI vector.

Claims

1. The genome of a recombinant alcaligenes rosenbergii producing β-caryophyllene is phaA, phaB1, phaC1 Gene-deficient strains ( H16∆CAB ).

2. A recombinant alcaligenes eutrophus for producing β-caryophyllene comprises an exogenous gene in its genome, wherein the exogenous gene comprises: IDI , GPPS and QHSopt .

3. A recombinant alcaligenes eutrophus for producing β-caryophyllene comprises an endogenous gene in its genome, wherein the exogenous gene comprises: dxs , dxr .

4. The method according to claim 2 and 3, characterized in that: The foreign gene IDI and GPPS Expression is driven by an inducible promoter, endogenous global regulatory factor gene rPb , Fnr5 , uspA is expressed through a constitutive promoter, and the endogenous gene dxs , dxr Expression is driven by a constitutive promoter.

5. The recombinant bacterium according to claim 2 or 3, characterized in that: Said QHSopt The nucleotide sequence is shown in SEQ ID No.

1.

6. And / or, dxs The nucleotide sequence is shown in SEQ ID NO.

2.

7. and / or, dxr The nucleotide sequence is shown in SEQ ID NO.

3.

8. and / or, iD The nucleotide sequence is shown in SEQ ID NO.

4.

9. and / or, i The nucleotide sequence is shown in SEQ ID NO.

5.

10. and / or, rPb The nucleotide sequence is shown in SEQ ID NO.

6.

11. And / or, Fnr5 The nucleotide sequence is shown in SEQ ID NO.

7.

12. And / or, the nucleotide sequence of uspA is shown in SEQ ID NO.

8.

13. and / or, GPPS The nucleotide sequence is shown in SEQ ID NO.

9.

14. and / or, IDI The nucleotide sequence is shown in SEQ ID NO.10.

Images