Yeast engineering bacteria for producing cordycepin and application thereof
By integrating the cordycepin biosynthesis module and purine metabolic pathway into the yeast genome, constructing an efficient yeast engineered bacterium, and using sugarcane molasses as a carbon source, the problems of long production cycle, high cost and environmental pollution in the cordycepin production process were solved, and efficient, green and safe cordycepin production was achieved.
Patent Information
- Application Number
- CN202210873787.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-25
- Publication Date
- 2025-10-10
- Estimated Expiration
- 2042-07-25
AI Technical Summary
Existing cordycepin production methods have problems such as long fermentation cycle, low production intensity, high cost, complicated process and serious environmental pollution. Both biological and chemical methods have shortcomings.
The cordycepin biosynthesis module, purine metabolism pathway overexpression module and ATP and precursor supply module were inserted into the yeast genome to construct an efficient yeast engineered bacterium, which uses sugarcane molasses, a cheap carbon source, for fermentation production.
The efficient, green and safe production of cordycepin has been achieved, with an output of more than 1500 mg/L, and has prospects for industrial application.
Abstract
Description
Technical Field
[0001] The invention belongs to the field of bioengineering, and particularly relates to an engineered yeast for producing cordycepin and its application. Background Art
[0002] Cordycepin, also known as cordycepin and cordycepin, chemically known as 3'-deoxyadenosine, is the first nucleoside antibiotic isolated from fungi. Research results have shown that cordycepin possesses multiple important physiological activities, including antibacterial, anti-tumor, anti-inflammatory, antiviral, and immunomodulatory. It has been widely used in the pharmaceutical and food industries and has broad market prospects.
[0003] Cordycepin is primarily produced through biological and chemical methods. The biological method, which primarily utilizes liquid fermentation of Cordyceps militaris, suffers from long fermentation cycles, low production efficiency, and high costs. Chemical methods for synthesizing cordycepin, on the other hand, suffer from high costs, complex processes, low product yields, and significant environmental pollution. Therefore, there is an urgent need to develop efficient, environmentally friendly, and safe methods for producing cordycepin.
[0004] With the advancement of synthetic biology and metabolic engineering, the construction of microbial cell factories for heterologous synthesis of natural products has become an effective green production method. Success has been achieved in the production of a range of natural products, including artemisinic acid, ginsenosides, and morphine. Yeast (Saccharomyces), a unicellular fungus, is an important model organism that combines advantages such as a clear genetic background, short fermentation cycles, and high biosafety. Therefore, constructing and optimizing the cordycepin biosynthetic pathway in yeast to achieve high cordycepin production through microbial cell engineering is a key approach to achieving efficient, green, and safe cordycepin production. Summary of the Invention
[0005] The present invention aims to overcome the deficiencies in the cordycepin production process and provides an engineered yeast for producing cordycepin and its application.
[0006] In order to achieve the above object, the technical solution provided by the present invention is:
[0007] The cordycepin-producing yeast engineered bacteria has a cordycepin biosynthesis module and a purine metabolic pathway overexpression module or an ATP and precursor supply module inserted into the yeast genome; the cordycepin biosynthesis module is composed of the Cns1 gene and the Cns2 gene; the purine metabolic pathway overexpression module is composed of the ADE4 gene and the Cns3-NK gene; and the ATP and precursor supply module is composed of the PGK gene and the Cns3-NK gene.
[0008] Preferably, the nucleotide sequence of the Cns1 gene is identical to or has a homology greater than 80% to the sequence shown in Genbank accession number XM_006669584 and has an equivalent function for cordycepin synthesis; the nucleotide sequence of the Cns2 gene is identical to or has a homology greater than 80% to the sequence shown in Genbank accession number XM_006669585 and has an equivalent function for cordycepin synthesis; the nucleotide sequence of the Cns3-NK gene is identical to or has a homology greater than 80% to the sequence shown in Genbank accession number XM_ 006669586 or a nucleotide sequence with a homology greater than 80% and having an equivalent function for cordycepin synthesis; the nucleotide sequence of the ADE4 gene is a nucleotide sequence that is identical to or has a homology greater than 80% with the sequence shown in Genbank Accession No. XM_500924 and has an equivalent function for cordycepin synthesis; the nucleotide sequence of the PGK gene is a nucleotide sequence that is identical to or has a homology greater than 80% with the sequence shown in Genbank Accession No. XM_502742 and has an equivalent function for cordycepin synthesis.
[0009] More preferably, the amino acid sequence of the protein encoded by the Cns1 gene is an amino acid sequence that is identical to or has a homology greater than 80% to the sequence shown by Genbank Accession No. XP_006669647 and has equivalent function; the amino acid sequence of the protein encoded by the Cns2 gene is an amino acid sequence that is identical to or has a homology greater than 80% to the sequence shown by Genbank Accession No. XP_006669648 and has equivalent function; the amino acid sequence of the protein encoded by the Cns3-NK gene is an amino acid sequence that is identical to or has a homology greater than 80% to the sequence shown by Genbank Accession No. XP_006669649 and has equivalent function; the amino acid sequence of the protein encoded by the ADE4 gene is an amino acid sequence that is identical to or has a homology greater than 80% to the sequence shown by Genbank Accession No. XP_500924 and has equivalent function; and the amino acid sequence of the protein encoded by the PGK gene is an amino acid sequence that is identical to or has a homology greater than 80% to the sequence shown by Genbank Accession No. XP_502742 and has equivalent function.
[0010] More preferably, the Genbank accession number of the Cns1 gene is XM_006669584, the Genbank accession number of the Cns2 gene is XM_006669585, the Genbank accession number of the ADE4 gene is XM_500924, the Genbank accession number of the Cns3-NK gene is XM_006669586, and the Genbank accession number of the PGK gene is XM_502742.
[0011] Preferably, the yeast is a Saccharomyces fungus such as Yarrowia lipolytica or Saccharomyces cerevisiae.
[0012] The yeast engineering bacteria for producing cordycepin can be used for preparing a cordycepin production preparation.
[0013] The method for producing cordycepin is to ferment the yeast engineering bacteria in the YYG medium or to replace 50 g / L of glucose in the YYG medium with 70 g / L of total sugar concentration of sulfuric acid hydrolyzed sugarcane molasses as a substrate, so as to obtain cordycepin.
[0014] The application is further described as follows:
[0015] In the application, the Cns1 and Cns2 genes for biosynthesis of cordycepin and at least one functional gene such as Cns3-NK gene for regulating biosynthesis of cordycepin are integrated into the genome of the yeast. The yeast chassis cell can be a eukaryote such as Yarrowia lipolytica and Saccharomyces cerevisiae. The yeast engineering bacteria can utilize cheap raw materials such as sugarcane molasses as a carbon source to produce cordycepin by fermentation.
[0016] Taking Yarrowia lipolytica as an example, first, the biosynthesis pathway of cordycepin in Cordyceps militaris is integrated into the chassis cell Yarrowia lipolytica, and the genes related to biosynthesis of cordycepin in Cordyceps militaris include Cns1 (Genbank accession number: XM_006669584) and Cns2 (Genbank accession number: XM_006669585).
[0017] On this basis, a purine metabolism overexpression module or an ATP and precursor supply module is integrated into the genome of Yarrowia lipolytica containing the cordycepin biosynthesis module, so as to obtain the Yarrowia lipolytica engineering bacteria capable of efficiently producing cordycepin. The purine metabolism overexpression module is composed of ADE4 (Genbank accession number: XM_500924) encoding phosphoribosylpyrophosphate amidotransferase (PPAT) and Cns3-NK (Genbank accession number: XM_006669586) encoding nucleoside / nucleotide kinase (NK); and the ATP and precursor supply module is composed of PGK (Genbank accession number: XM_502742) encoding phosphoglycerate kinase (PGK) in the glycolysis pathway and Cns3-NK encoding nucleoside / nucleotide kinase (NK).
[0018] Using YYG medium (9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids, and 50 g / L glucose) as a substrate or replacing 50 g / L glucose in YYG medium with 70 g / L total sugar concentration, the engineered Yarrowia lipolytica bacteria that hydrolyzes sugarcane molasses can effectively accumulate cordycepin in the fermentation broth, reaching more than 1500 mg / L, and has prospects for industrial application.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The technology of the present invention can achieve the adaptation of the cordycepin biosynthesis pathway in Cordyceps militaris to the chassis cell Yarrowia lipolytica, and realizes the efficient and stable synthesis of cordycepin in Yarrowia lipolytica by overexpressing the purine metabolism module or the ATP and precursor supply module. At the same time, the constructed recombinant engineered bacteria can use cheap raw materials such as sugarcane molasses as a carbon source to ferment and efficiently produce cordycepin, with the characteristics of high efficiency, greenness and safety.
[0021] In summary, based on the biosynthetic pathway of cordycepin and its metabolic regulation, the present invention constructs and optimizes the biosynthetic integration module required for cordycepin in yeast, creates a yeast cell factory, and establishes a technical system for producing cordycepin by microbial fermentation using sugarcane molasses, a cheap carbon source, as raw material, thereby achieving efficient, green, and safe production of cordycepin. DETAILED DESCRIPTION
[0022] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope defined by the claims appended hereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The present invention is described in detail below in conjunction with specific examples.
[0023] Example 1: Construction of a cordycepin biosynthesis module optimized by the purine metabolic pathway and green and efficient production of cordycepin by engineered bacteria
[0024] High-fidelity enzyme amplification was used to sequentially obtain the following elements: multi-copy upstream integration site homology arm rDNA up, multi-copy downstream integration site homology arm rDNA down, promoters TEFp and FBAp, gene Cns1 (Genbank accession number: XM_006669584), gene Cns2 (Genbank accession number: XM_006669585), gene Cns3-NK (Genbank accession number: XM_006669586), gene ADE4 (Genbank accession number: XM_500924), terminators XPR2t and LIP2t, and selection markers URA3 and LEU2. Integration vectors pUC19-RI-Cns2, pUC19-RI-Cns1, pUC19-RI-ADE4, pUC19-RI-Cns3-NK, pUC19-RI-URA3, and pUC19-RI-LEU2 were then constructed, respectively. The recovered functional module fragments RI-Cns2 and RI-Cns1 were co-transformed with the selection marker module fragment RI-URA3 into the yeast Po1f-Δku70. Furthermore, the ADE4 gene, encoding phosphoribosylpyrophosphate amidotransferase (PPAT), and the Cns3-NK gene, encoding nucleoside / nucleotide kinase (NK), in the purine metabolism pathway, were co-overexpressed. The engineered strain YL-C01 was constructed by co-transforming the recovered functional module fragments RI-ADE4, RI-Cns3-NK, and the selection marker module fragment RI-LEU2 into chassis cells. When fermented for 72 hours using YYG medium (9 g / L yeast extract, 8 g / L yeast nitrogen base without amino acids, and 50 g / L glucose) as substrate, YL-C01 produced 1621.41 mg / L of cordycepin. In order to achieve green and efficient production of cordycepin, the 50 g / L glucose in the YYG medium was replaced with sulfuric acid hydrolyzed sugarcane molasses with a total sugar concentration of 70 g / L. When YL-C01 was fermented with this as the substrate for 72 h, the cordycepin yield reached 1668.08 mg / L.
[0025] Example 2: Construction of a cordycepin biosynthesis module using a combination of ATP and precursors and efficient cordycepin production by engineered bacteria
[0026] High-fidelity enzyme amplification was used to sequentially obtain the following elements: multicopy integration site upstream homology arm rDNA up, multicopy integration site downstream homology arm rDNA down, promoters TEFp and FBAp, gene Cns1 (Genbank accession number: XM_006669584), gene Cns2 (Genbank accession number: XM_006669585), gene Cns3-NK (Genbank accession number: XM_006669586), gene PGK (Genbank accession number: XM_502742), terminators XPR2t and LIP2t, and selection markers URA3 and LEU2. Integration vectors pUC19-TCns1, pUC19-TCns2, pUC19-RI-PGK, pUC19-RI-Cns3-NK, pUC19-RI-URA3, and pUC19-RI-LEU2 were constructed, respectively. The recovered functional module fragments TCns1 and TCns2 were co-transformed with the selection marker module fragment RI-URA3 into the yeast Po1f-Δku70. Furthermore, the PGK gene, encoding the phosphoglycerate kinase (PGK) enzyme, and the Cns3-NK gene, encoding the nucleoside / nucleotide kinase (NK) enzyme, in the glycolytic pathway, were co-overexpressed to provide sufficient ATP and precursors. The engineered strain YL-C02 was constructed by co-transforming the recovered functional module fragments RI-PGK, RI-Cns3-NK, and the selection marker module fragment RI-LEU2 into chassis cells. When fermented for 72 hours using YYG medium (9 g / L yeast extract, 8 g / L yeast nitrogen base without amino acids, and 50 g / L glucose) as the substrate, YL-C02 produced 1585.32 mg / L of cordycepin. By optimizing the medium composition (9 g / L yeast extract, 8 g / L yeast nitrogen base without amino acids, 30 g / L glucose, and 1 g / L adenine), the cordycepin yield of YL-C02 increased to 2663.78 mg / L after 72 hours. Furthermore, through fed-batch feeding at the shake flask level, YL-C02 produced 4362.54 mg / L of cordycepin after consuming 90 g / L of glucose.
Claims
1. A yeast engineered bacterium for producing cordycepin, characterized in that: The engineered yeast strain has a cordycepin biosynthesis module and an ATP and precursor supply module inserted into the yeast genome; the cordycepin biosynthesis module consists of a Cns1 gene and a Cns2 gene; the ATP and precursor supply module consists of a PGK gene and a Cns3-NK gene; the Genbank accession number of the Cns1 gene is XM_006669584, the Genbank accession number of the Cns2 gene is XM_006669585, the Genbank accession number of the Cns3-NK gene is XM_006669586, and the Genbank accession number of the PGK gene is XM_502742; the yeast is Yarrowia lipolytica Po1f-Δku70.
2. Use of the engineered yeast strain for producing cordycepin as claimed in claim 1 in the preparation of cordycepin preparations.