Transgenic yeast engineering bacteria expressing cytochrome oxidase HcCYP81Q58 and its application

Abstract

The invention provides a cytochrome oxidase HcCYP81Q58 gene, and the sequence of the gene is shown in SEQ NO: 1. The invention constructs a cytochrome oxidase HcCYP81Q58 transgenic yeast engineering bacterium, which can produce a variety of cucurbitacin intermediates in high yield, including 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one, thereby providing a source for cucurbitacin production.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and specifically relates to an application of a cytochrome oxidase HcCYP81Q58 gene and a transgenic engineering bacterium thereof in the preparation of a cucurbitacin intermediate. Background Art

[0002] Cucurbitacin is a highly oxidized tetracyclic triterpenoid compound with high medicinal value, especially in anti-tumor. Due to the toxic and side effects of cucurbitacin, its clinical application is limited, so only a small number of cucurbitacin has been developed as drugs (CuB, cucurbitacin A, etc.). Cucurbitacin is rich in cucurbitacin components in the tubers of cucurbitacin plants, especially cucurbitacin (a mixture of cucurbitacin A and cucurbitacin B) extracted from the tubers of this genus of plants is the raw material for producing the Chinese patent medicine cucurbitacin tablets (protected Chinese medicine variety) and cucurbitacin capsules. The drug has the functions of clearing away heat and detoxifying, antibacterial and anti-inflammatory, and is mainly used clinically to treat bacillary dysentery, enteritis, bronchitis, acute tonsillitis and other diseases. Unlike other cucurbitacin compounds, researchers have found that cucurbitacin IIa (cucurbitacin A) has obvious cytotoxic effects, and inhibits the proliferation of tumor cells by inhibiting survivin (Survivin) downstream of JAK2 / STAT3, making it a new type of anticancer drug.

[0003] Cytochrome oxidase CYP450 is a class of heme-iron-sulfur proteins encoded by a supergene family. In eukaryotes, CYP450 oxidase is a membrane-anchored protein that can bind to organelles such as the endoplasmic reticulum, mitochondria, plastids, and Golgi bodies. It is mainly located in the endoplasmic reticulum and participates in the metabolism and oxidation of a variety of important substances in the body. In biochemical reactions, it can insert an oxygen atom into a hydrophobic molecule to obtain higher activity or hydrophilicity, and is also called monooxygenase (Mixed-function oxidase, MFO).

[0004] Studies have shown that the biosynthesis of cucurbitacin starts from the cyclization of 2,3-oxidosqualene to form cucurbitacinol (Cuol), which is catalyzed by cucurbitacinol synthase (CBS) of the oxidosqualene cyclase (OSCs) family. Under the catalysis of cytochrome P450, cucurbitacinol undergoes oxidation modification at specific sites by introducing hydroxyl, carboxyl or epoxy groups, thereby producing a variety of tetracyclic triterpenoids. CYP69 in the traditional Chinese medicine snow gall can catalyze the hydroxylation reaction of C24 and 25 positions of cucurbitacin skeleton cucurbitacinol, and the hydroxyl configuration of C24 position has two configurations: α and β.

[0005] Compounds such as cucurbitacin are highly oxidized, with low content in plants, and their utilization is limited. The extraction and purification process is complex and time-consuming, and requires a lot of manpower, material resources and other resources. At present, the development of related drugs is mainly prepared by extraction from plants. There are still many challenges to the chemical synthesis of cucurbitacin today. Especially the key intermediate compounds of cucurbitacin. With the development of synthetic biology and metabolic engineering, new opportunities have been provided for the efficient heterologous synthesis of tetracyclic triterpenoids. Saccharomyces cerevisiae, as a eukaryotic expression system, has a clear genetic background and mature genetic modification strategies. Its endogenous mevalonate (MVA) pathway can provide the required precursors for the synthesis of more terpenoids, which is conducive to the synthesis of terpenoids. Therefore, in the study of cucurbitacin biosynthesis pathway and possible future production, it is very important to establish and optimize yeast engineering bacteria for the efficient production of cucurbitacin intermediates 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one, and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a cytochrome oxidase HcCYP81Q58 gene which can produce cucurbitacin intermediates at a high level.

[0007] To achieve the purpose of the present invention, the present invention adopts the following technical solutions

[0008] In a first aspect, the present invention provides a snow gall cytochrome oxidase HcCYP81Q58 gene, the sequence of which is shown in SEQ NO:1.

[0009] Preferably, the cytochrome oxidase HcCYP81Q58 gene is used in the preparation of cucurbitacin intermediates.

[0010] Preferably, the cucurbitacin intermediate comprises

[0011] 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol、

[0012] 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol、

[0013] 11-Carbonyl-cucurbita-5, 23-diene-20, 25-diol-3β-one and

[0014] 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one.

[0015] In a second aspect, the present invention provides an expression vector, wherein the expression vector is a gene expression cassette composed of a promoter and genes HCCYP81Q58, HcSDR34, LEU and a terminator connected in series in sequence.

[0016] Preferably, the promoter is composed of promoter UP, GPMp and TEF1p connected in series in sequence; and the terminator is composed of IDP1t, PGK1t and Down connected in series in sequence.

[0017] In a third aspect, the present invention provides a method for constructing a transgenic yeast engineering bacterium expressing cytochrome oxidase HcCYP81Q58, comprising the following steps:

[0018] The constitutive promoter GPMp and the constitutive terminator IDp1t were used to control the expression of the HCCYP81Q58 gene. The constitutive promoter TEF1p and the constitutive terminator PGk1t were used to control the expression of the HcSDR34 gene, and LEU2 was used as the screening tag. Gene expression cassettes were constructed to become downstream modules for the production of cucurbitacin intermediate synthesis pathways, and the gene expression cassettes were inserted into the γDNA site in Saccharomyces cerevisiae by co-transformation. The yeast homologous recombination ability was used to assemble and form an engineered Saccharomyces cerevisiae strain containing a complete cucurbitacin intermediate biosynthetic pathway.

[0019] Preferably, the constitutive promoter is a combination of promoters UP, GPMp and TEF1p.

[0020] Preferably, the constitutive terminator is a combination of terminators IDP1t, PGK1t and Down.

[0021] Preferably, the gene expression cassette further comprises a LEU gene, and the LEU gene is a screening gene.

[0022] Preferably, the cucurbitacin intermediate comprises

[0023] 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol、

[0024] 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol、

[0025] 11-Carbonyl-cucurbita-5, 23-diene-20, 25-diol-3β-one and

[0026] 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one.

[0027] The beneficial effect of the present invention is that the present invention successfully excavated the gene HcCYP81Q58 through the genome and transcriptome analysis of the traditional Chinese medicine snow gall, and found that the gene can catalyze the C25 hydroxylation of 11-carbonyl-20β-hydroxy-Cuol to form 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, and then can be catalyzed by HcSDR34 to convert C3-ketone into 11-Carbonyl-cucurbita-5,23-diene-20, At the same time, HcCYP81Q58 can also catalyze the C23 hydroxylation of 11-carbonyl-20β-hydroxy-Cuol to form 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, which can then be catalyzed by HcSDR34 to convert C3-ketone into 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one, generating multiple cucurbitacin intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 Schematic diagram of the construction of the yeast engineering strain Cuol-04-1 (where HC81Q58 is the HcCYP81Q58 gene);

[0030] Figure 2 The figure is a schematic diagram of the deduced synthesis pathway of the HcCYP81Q58 gene (wherein OSC6 represents the cucurbitacinol synthase derived from the snow thorn plant; HCCYP87D20 represents the cucurbitacinol C11 keto C20 β-hydroxylase derived from the snow thorn plant);

[0031] Figure 3 Schematic diagram of TLC and LCMS detection results of HcYPHCCYP81Q58 gene products;

[0032] FIG4 is the NMR spectrum of the HcYPHCCYP81Q58 gene product 4a (wherein, Figure 4-1 For product 4a 13 C NMR spectrum in CDCl3 at 600MHz; 4-2 is the product 4a 1 H NMR spectrum in CDCl3 at 600MHz chart;

[0033] FIG5 is the NMR spectrum of HcYPHCCYP81Q58 gene product 5 (wherein, Figure 5-1 For product 5 13 C NMR spectrum in CDCl3 at 600MHz; Figure 5-2 is the product 4a 1 H NMR spectrum in CDCl3 at 600MHz picture);

[0034] FIG6 is the NMR spectrum of the HcYPHCCYP81Q58 gene product 5a (wherein, Figure 6-1 For product 5a 13 C NMR spectrum in CDCl3 at 600MHz; Figure 6-2 is the product of 4a 1 H NMR spectrum in CDCl3 at 600MHz (Figure). DETAILED DESCRIPTION

[0035] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0036] Example 1: This example provides a method for constructing a recombinant yeast strain Coul-04-1

[0037] 1. Optimization and cloning of HcCYP81Q58 gene:

[0038] (1) Yeast codon optimization of HcCYP81Q58 gene: The HcCYP81Q58 gene was delivered to Wuhan Jinkairui Bioengineering Co., Ltd. for gene synthesis and codon optimization;

[0039] (2) Amplification and recovery of optimized HcCYP81Q58 gene: Wuhan Jinkairui Biotechnology Co., Ltd. synthesized and optimized HcCYP81Q58 gene in Y33 plasmid, performed gene amplification PCR, and then performed electrophoresis. After confirming successful amplification, the target band was recovered. The HcCYP81Q58 gene was identified as shown in SEQ NO: 1;

[0040] The PCR reaction program was 95°C, 3 min; 95°C, 15S, 58°C, 45S, 72°C, 1 min, 35 cycles; 72°C, 10 min.

[0041] 2. The activation and cultivation method of the transgenic engineering host bacteria includes the following steps:

[0042] (1) transferring the host bacteria of Saccharomyces cerevisiae to YPD liquid medium for activation to obtain yeast liquid, and culturing the activation conditions at 30° C. and 220 rpm for 24 h;

[0043] (2) transferring the yeast solution to YPD liquid medium for cultivation and collecting yeast cells;

[0044] (3) Resuspend the yeast cells in deionized water, centrifuge to obtain yeast precipitate, and culture the yeast precipitate by the following method: the initial OD600 is 0.4, and culture the yeast precipitate at 30° C. and 220 rpm in a shaker until the OD600 reaches 0.8 to 0.9.

[0045] 3. The method for constructing the recombinant yeast Coul-04-1 comprises the following steps:

[0046] (1) Construction of a yeast engineered strain Cuol-04-1 capable of producing 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one, and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one at high levels:

[0047] The mevalonate pathway-related genes ERG20, ERG9, ERG1 and tHMG1 from Saccharomyces cerevisiae BY4742 and the cucurbitadienol synthase genes synHcOSC6 and synHcCYP87D20, as well as the cytochrome P450 reductase gene synHcCPR1 from the plant Saccharomyces cerevisiae were constructed to form gene expression cassettes, which became modules for the production of 11-carbonyl-20β-hydroxy-Cuol (cucurbitadienol) synthesis pathway. Then, on this basis, the constitutive promoter GPMp and the constitutive terminator IDp1t were used to control the expression of the HCCYP81Q58 gene, and the constitutive promoter TEF1p and the constitutive terminator PGk1t were used to control the expression of the HcSDR34 gene (the sequence of the HcSDR34 gene is shown in SEQ ID NO: 1). NO: 2), LEU2 was used as a screening tag, and gene expression cassettes were constructed to become downstream modules of the synthetic pathway for producing cucurbitacin intermediates. The gene expression cassettes were inserted into the γDNA site of Saccharomyces cerevisiae by co-transformation, and the key enzyme gene HcCYP81Q58 in the cucurbitacin pathway was overexpressed to construct the yeast engineering bacterium Cuol-04-1. The HcCYP81Q58 gene provided the hydroxylation of cucurbitacin at the C25 and C23 positions in the yeast, and the HcSDR34 gene provided the enzyme catalysis reaction of the carbonylation of cucurbitacin at the C3 position in the yeast.

[0048] (2) The gene expression cassette was co-transformed into the γDNA site of Saccharomyces cerevisiae Cuol-04-1 using the lithium acetate transformation method, and the yeast homologous recombination ability was used to assemble and contain the selection marker gene LEU2 to form the Saccharomyces cerevisiae engineered strain Cuol-04-1 containing the complete 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one, and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one biosynthetic pathway;

[0049] Among them, the construction method of the gene expression cassette provided by the brewer's yeast Cuol-04-1 is to use the constitutive promoter UP, GPMp and terminator IDP1t to control the expression of the HCCYP81Q5 gene to form the HCCYP81Q5 gene expression cassette: UP-GPMp-HCCYP81Q58-IDp1t; use the promoter TEF1p and terminator PGK1t to control the expression of the HcSDR34 gene to form the HcSDR34 gene expression cassette: TEF1p-HcSDR34-IPGK1t; and use the terminator Down to control the expression of the screening gene LEU ( Figure 1 ).

[0050] The above gene expression cassette is used to produce the recombinant engineering bacteria

[0051] 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol、

[0052] 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol、

[0053] 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one,

[0054] The module of 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one pathway, LEU2 gene is a selection marker. The gene expression cassette construction method is as follows Figure 1 shown.

[0055] The yeast transformation system includes the following components: PEG4000 solution, LiAc solution, sSDNA, recombinant plasmid and sterilized deionized water. The standing time is 20 minutes, and the heat shock condition is maintained at 42° C. for 40 minutes.

[0056] Table 1: Primers used to construct the engineered bacteria Cuol-04-1

[0057]

[0058]

[0059] 4. Identify products synthesized by genetically engineered bacteria

[0060] The brewer's yeast Cuol-04-1 was fermented and its fermentation products were collected. The fermentation and product collection methods were as follows: Activate the high-yield

[0061] 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol、

[0062] 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol、

[0063] 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one,

[0064] The chassis engineered bacteria Cuol-04-1 of 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one was used to prepare the fermentation seed solution in YPD liquid medium (30°C, 200rpm, 16 hours); transferred to a 250ml Erlenmeyer flask containing 50ml YPD liquid medium (30°C, 200rpm, 6 hours), and then expanded and fermented and transferred to a 3L Erlenmeyer flask containing 1L YPD liquid medium, 30°C, 200rpm / min. The culture was shaken and grown for 8 days to obtain the fermentation product.

[0065] Product extraction conditions: The fermentation product was centrifuged at 8000 rpm for 10 min to collect the cell bodies, soaked in 300 ml of ethyl acetate for 30 min, ultrasonically extracted for 30 min, and shaken every 10 min; the cell bodies were collected by centrifugation at 8000 rpm for 10 min, and the supernatant was taken to detect the product.

[0066] LC-MS and NMR were used to identify the fermentation products of Saccharomyces cerevisiae engineered strain Cuol-04-1. The results are shown in Tables 2, 3 and Figure 3 ~6 (compound 4 was not produced in large quantities and was only detected by LC-MS, so it could not be purified for NMR identification):

[0067] Table 2: Compound abbreviations and chemical names

[0068]

[0069]

[0070] Table 3: CYPHCCYP81Q58 product 13C & 1H NMR (800 MHz, solvent: CDCl3) data (J in Hz, δ in ppm)

[0071]

[0072]

[0073]

[0074] From the above table and Figure 3 From Figure 6, it can be seen that the brewer's yeast Cuol-04-1 constructed by the present invention has a high yield, wherein the yields of 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol and 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one are 10.58 mg / L and 18.02 mg / L, respectively; the yields of 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol and HcSDR34 catalyzing the conversion of C3-ketone into 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one are 12.58 mg / L and 16.02 mg / L, respectively.

[0075] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A snow gall cytochrome oxidase HcCYP81Q58 A gene characterized by The sequence of the gene is shown in SEQ NO:

1.

2. The snow gall cytochrome oxidase according to claim 1 HcCYP81Q58 The application of the gene in the preparation of cucurbitacin intermediates is characterized in that: The cucurbitacin intermediates include: 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one.

3. An expression vector, characterized in that: The expression vector consists of a promoter and a gene HCCYP81Q58, HcSDR34, LEU and a gene expression cassette formed by sequentially connecting terminators in series, HCCYP81Q58 The sequence of the gene is shown in SEQ NO:

1. HcSDR34 The sequence of the gene is shown in SEQ NO:

2.

4. A method for constructing snow gall cytochrome oxidase HcCYP81Q58 The method of genetically modified yeast engineering bacteria is characterized in that: The following steps are involved: The constitutive promoter GPMp and the constitutive terminator IDp1t control HCCYP81Q58 Gene expression; controlled by the constitutive promoter TEF1p and the constitutive terminator PGk1t HcSDR34 The expression of the gene is carried out, with LEU2 as the screening tag; the gene expression cassettes are respectively constructed to become the downstream modules of the synthesis pathway for producing cucurbitacin intermediates, the gene expression cassettes are inserted into the γDNA site of Saccharomyces cerevisiae by co-transformation, and the homologous recombination ability of yeast is used to assemble to form an engineered Saccharomyces cerevisiae strain containing a complete biosynthetic pathway for cucurbitacin intermediates, the HCCYP81Q58 The sequence of the gene is shown in SEQ NO:

1. HcSDR34 The sequence of the gene is shown in SEQ NO: 2; The cucurbitacin intermediates include 11-Carbonyl-cucurbita-5,23-diene-3β,20,25-triol, 11-Carbonyl-cucurbita-5,24-diene-3β,20,23-triol, 11-Carbonyl-cucurbita-5,23-diene-20,25-diol-3β-one and 11-Carbonyl-cucurbita-5,24-diene-20,23-diol-3β-one.

5. The construction method according to claim 4, characterized in that: The gene expression cassette further comprises the LEU gene, which is a screening gene.

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