阅读说明：本技术 紫杉二烯合酶TcTS2、编码核苷酸序列及其应用 (Taxadiene synthase TcTS2, coding nucleotide sequence and application thereof ) 是由 闫建斌 苟君波 廖庆刚 梁海菲 李冲 杜然 熊兴耀 黄三文 于 2020-07-07 设计创作，主要内容包括：本发明提供了一种紫杉二烯合酶TcTS2,编码核苷酸序列及其应用,涉及植物基因工程技术领域。TcTS2的氨基酸序列包含或由如下序列组成：a)SEQ ID NO.1所示的氨基酸序列；或,b)与SEQ ID NO.1所示的氨基酸序列具有至少80％序列相似性的功能性同源序列；或c)在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个氨基酸且具有TcTS2活性的氨基酸序列。TcTS2和编码TcTS2的核苷酸序列,为提高紫杉醇的产量提供了新的基因资源,可用于植物基因工程和代谢工程策略改造底盘宿主,生产紫杉醇及其中间体等,具有显著经济和社会价值。(The invention provides a taxadiene synthase TcTS2, a coding nucleotide sequence and application thereof, relating to the technical field of plant genetic engineering. The amino acid sequence of TcTS2 comprises or consists of the sequence: a) an amino acid sequence shown as SEQ ID NO. 1; or, b) a functional homologous sequence having at least 80% sequence similarity to the amino acid sequence depicted in SEQ ID No. 1; or c) an amino acid sequence with TcTS2 activity, wherein one or more amino acids are added, deleted or replaced in the amino acid sequence shown in SEQ ID NO. 1. The nucleotide sequences of TcTS2 and TcTS2 provide new gene resources for increasing the yield of paclitaxel, can be used for plant genetic engineering and metabolic engineering strategy to modify chassis hosts and produce paclitaxel and intermediates thereof, and has obvious economic and social values.)

1. A taxadiene synthase TcTS2, wherein the amino acid sequence of said taxadiene synthase TcTS2 comprises or consists of:

a) an amino acid sequence shown as SEQ ID NO. 1; or the like, or, alternatively,

b) a functional homologous sequence having at least 80% sequence similarity to the amino acid sequence shown in SEQ ID No. 1; or

c) An amino acid sequence which is obtained by adding, deleting and replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 and has TcTS2 activity.

2. A nucleotide sequence encoding the taxadiene synthase TcTS2 of claim 1.

3. The nucleotide sequence encoding taxadiene synthase TcTS2 of claim 2, wherein said nucleotide sequence comprises or consists of:

a) a nucleotide sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

b) a complementary sequence, a degenerate sequence or a homologous sequence (preferably having 70% or more homology) of the nucleotide sequence shown in SEQ ID NO. 2; or the like, or, alternatively,

c) a nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.2 under strict conditions and can code taxadiene synthase TcTS 2; or the like, or, alternatively,

d) a cDNA sequence of the nucleotide sequence of any one of the sequences a) to c).

4. The nucleotide sequence of claim 3, wherein the cDNA sequence comprises or consists of:

a) a nucleotide sequence shown as SEQ ID NO. 3; or the like, or, alternatively,

b) a degenerate or homologous sequence of the nucleotide sequence shown in SEQ ID NO.3 (preferably having a homology of 80% or more).

5. Use of the taxadiene synthase TcTS2 according to claim 1 or the nucleotide sequence encoding taxadiene synthase TcTS2 according to any one of claims 2 to 4 for the synthesis of baccatin III or paclitaxel.

6. A primer for detecting the nucleotide sequence of any one of claims 2 to 4;

preferably, the primers comprise an upstream primer and/or a downstream primer;

preferably, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 4; and/or the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5.

7. A vector comprising the nucleotide sequence of any one of claims 2 to 4.

8. A host cell into which the nucleotide sequence of any one of claims 2 to 4 has been introduced, or into which the vector of claim 7 has been introduced;

preferably, the host cell comprises a plant cell and/or a microbial cell;

preferably, the plant cell comprises a taxus chinensis cell and/or a tobacco cell;

preferably, the microbial cells comprise at least one of streptomyces, pseudomonas, bacillus, yeast cells, and escherichia coli.

9. A method for expressing a taxadiene synthase TcTS2 in a plant, comprising transferring a nucleotide sequence according to any one of claims 2 to 4, or a vector according to claim 7, or a host cell according to claim 8 into a plant, thereby expressing taxadiene synthase TcTS2 in the plant;

preferably, the plant body is taxus chinensis and/or tobacco.

10. A method for producing paclitaxel and an intermediate thereof, characterized in that a plant body is allowed to express the taxadiene synthase TcTS2 of claim 1;

preferably, the method comprises: transferring the nucleotide sequence of any one of claims 2 to 4, or the vector of claim 7, or the host cell of claim 8 into a plant, and expressing the taxadiene synthase TcTS2 to obtain paclitaxel and its intermediates.

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a taxadiene synthase TcTS2, a coding nucleotide sequence and application thereof in synthesizing baccatin III or paclitaxel.

Background

Paclitaxel is a diterpenoid alkaloid, is separated from Taxus brevifolia bark at first, is a star anticancer drug, and is widely used for clinical treatment of various cancers. Cancer is one of ten causes of death of human, at present, the number of cancer diseases is still over 1000 thousands (WHO) in the world, and paclitaxel and a preparation thereof are important first-line anticancer drugs. Currently, the main production mode of paclitaxel is semi-synthesis, i.e. firstly by extracting natural precursors baccatin III and 10-deacetylbaccatin III (10-DAB), and then chemically synthesizing paclitaxel (Li et al, 2015; Liu et al, 2016), but the precursor used in the method still depends on plant extraction, is subject to plant or cell resources, and cannot completely solve the supply problem.

Taxadiene Synthase (TS) was first cloned from taxus brevifolia, and catalyzes the cyclization reaction of geranyl Diphosphate (GGPP) to Taxadiene, which reacts through a series of functional groups to form baccatin iii, which has an essential role in paclitaxel synthesis (willung et al, 1996).

The taxadiene synthase is a first step enzyme for catalyzing the formation of taxane skeleton of paclitaxel parent, the efficiency of catalyzing GGPP cyclization is very low compared with the efficiency of cyclase of other gymnosperms, the step is a rate-limiting step of a paclitaxel synthesis pathway, and TS is proved to be a slow enzyme. Therefore, it is very important to discover a taxol biosynthesis gene having higher enzymatic activity and an enzyme encoded by the gene.

Disclosure of Invention

In view of the above, the present invention aims to provide a taxadiene synthase TcTS2, which improves the efficiency of catalyzing the cyclization of GGPP, greatly improves the yields of taxadiene, baccatin iii, and paclitaxel, which are intermediates in the synthesis process of paclitaxel, significantly reduces the production cost of paclitaxel drugs, and effectively solves the current situations of high price and short supply and demand of paclitaxel in the market.

In a first aspect, the invention provides a taxadiene synthase TcTS2, the amino acid sequence of said taxadiene synthase TcTS2 comprises or consists of the sequence:

a) an amino acid sequence shown as SEQ ID NO. 1; or the like, or, alternatively,

b) a functional homologous sequence having at least 80% sequence similarity to the amino acid sequence shown in SEQ ID No. 1; or

c) An amino acid sequence which is obtained by adding, deleting and replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 and has TcTS2 activity.

The inventor of the application found a novel taxadiene synthase gene in Taxus chinensis var.main of the southern yew, named TcTS2, and successfully obtained the encoded protein of the gene (namely taxadiene synthase TcTS2, also named TcTS2 in the invention). Functional analysis finds that the efficiency of catalyzing a substrate GGPP by TcTS2 is remarkably higher than that of another type of taxadiene synthase gene TcTS1, and the gene is expressed in an inducible expression mode under the stress of plant hormone methyl jasmonate (MeJA).

In one embodiment of the invention, the amino acid sequence of taxadiene synthase TcTS2 is the amino acid sequence shown in SEQ id No. 1.

In one embodiment of the invention, the amino acid sequence of taxadiene synthase TcTS2 is a functional homologous sequence having at least 80% sequence similarity to the amino acid sequence depicted in SEQ id No. 1. The functional homologous sequence of the identity includes, but is not limited to, an amino acid sequence of which the amino acids shown in SEQ ID No.1 have about 80% or more, 82% or more, 84% or more, 85% or more, 88% or more, 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more identity.

In one embodiment of the invention, the amino acid sequence of taxadiene synthase TcTS2 is an amino acid sequence having TcTS2 activity, wherein one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ id No. 1.

The efficiency of catalyzing GGPP cyclization by the taxadiene synthase TcTS2 is obviously improved, the influence of speed limit is reduced, the yields of intermediate taxadiene, baccatin III and product taxol in the process of synthesizing the taxol are greatly improved, the production cost of taxol medicaments is obviously reduced, and the current situations of high price and short supply and demand of the taxol in the market are effectively solved.

In addition, the expression of the taxadiene synthase TcTS2 in plant cells is obviously induced and regulated by MeJA, which is beneficial to the application of synthetic biology technology, and the expression condition of the taxadiene synthase TcTS2 can be better regulated and controlled by combining micromolecule MeJA induction and other synthetic biology technical means, so that the taxadiene synthase TcTS2 can be used for plant genetic engineering and metabolic engineering modification to produce taxol and intermediates thereof in the future.

In a second aspect, the invention provides a nucleotide sequence encoding said taxadiene synthase TcTS 2.

Further, on the basis of the technical scheme provided by the invention, the nucleotide sequence for coding the taxadiene synthase TcTS2 comprises or consists of the following sequences:

a) a nucleotide sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

b) a complementary sequence, a degenerate sequence or a homologous sequence (preferably having 70% or more homology) of the nucleotide sequence shown in SEQ ID NO. 2; or the like, or, alternatively,

c) a nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.2 under strict conditions and can code taxadiene synthase TcTS 2; or the like, or, alternatively,

d) a cDNA sequence of the nucleotide sequence of any one of the sequences a) to c).

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 is the nucleotide sequence shown as SEQ ID NO. 2.

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 is a complementary sequence formed by the nucleotide sequence shown in SEQ ID NO.2 according to the base complementary pairing principle.

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 is degenerate as the nucleotide sequence shown in SEQ ID NO. 2. The degenerate sequence means that after one or more nucleotide sequences of SEQ ID NO.2 are changed, the corresponding coded amino acid types of the changed nucleotide sequences are not changed, and the function of the taxadiene synthase TcTS2 gene and the expression level of the taxadiene synthase TcTS2 are not influenced.

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 is a homologous sequence of the nucleotide sequence shown as SEQ ID NO. 2.

The homologous nucleotide sequence comprises a mutant gene, an allele or a derivative which is generated by adding and/or substituting and/or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2 and can code the activity of the taxadiene synthase.

More preferably, the homologous sequence is about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, identical.

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 hybridizes under stringent conditions with the nucleotide sequence of SEQ ID No.2 and is capable of encoding taxadiene synthase TcTS 2.

Illustratively, the "stringent conditions" refer to conditions under which a probe will hybridize to a detectable degree to its target sequence over to other sequences (e.g., at least 2 times background). Stringent conditions are sequence dependent and will vary from one environment to another. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the probe. Alternatively, stringency conditions can be adjusted to allow some sequence mismatches so that a lower degree of similarity is detected.

In one embodiment of the invention, the nucleotide sequence encoding taxadiene synthase TcTS2 is a cDNA sequence of the nucleotide sequence shown as SEQ ID NO. 2.

Further, the sequence of the cDNA comprises or consists of:

a) a nucleotide sequence shown as SEQ ID NO. 3; or the like, or, alternatively,

b) a degenerate or homologous sequence of the nucleotide sequence shown in SEQ ID No. 3; preferably, the homologous sequence is a polynucleotide that is about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more identical to the nucleotide sequence shown in SEQ ID No. 3.

In a third aspect, the invention provides the use of said taxadiene synthase TcTS2, or said nucleotide sequence encoding taxadiene synthase TcTS2, in the synthesis of baccatin III or paclitaxel.

Wherein, the application of the taxadiene synthase TcTS2 in synthesizing baccatin III or paclitaxel comprises the following aspects: (1) the polypeptides of the amino acid sequence TcTS2 or at least partial sequence provided by the invention may still have biological activity or even new biological activity after removing or replacing some amino acids, or improve the yield or optimize the protein kinetic characteristics or other properties aimed at; (2) to the biosynthesis of taxadienes; (3) relates to the biosynthesis of taxol and its intermediates (e.g., baccatin III).

The application of the nucleotide sequence for coding the taxadiene synthase TcTS2 in synthesizing baccatin III or paclitaxel comprises the following aspects: (1) the nucleotide sequence or at least part of the nucleotide sequence provided by the invention is modified or mutated, and the modification or mutation way comprises insertion, deletion, Polymerase Chain Reaction (PCR), error-prone PCR, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences with other sources, or mutagenesis by chemical reagents and the like. (2) The nucleotide sequence provided by the invention or at least partial nucleotide sequence cloning gene is expressed in an exogenous host by a suitable expression system to obtain corresponding enzyme or other higher biological activity or yield. (3) The nucleotide sequence or at least partial nucleotide sequence gene or gene cluster provided by the invention can construct recombinant plasmid through genetic recombination to obtain a novel biosynthesis pathway, and can also obtain the novel biosynthesis pathway through insertion, replacement, deletion or inactivation.

In a fourth aspect, the invention provides a primer for detecting said nucleotide sequence.

The nucleotide sequence of the PCR product for amplifying the nucleotide sequence encoding the taxadiene synthase TcTS2 is not particularly limited, and the primer capable of specifically amplifying or specifically detecting the nucleotide sequence encoding the taxadiene synthase TcTS2 can be used.

In a preferred embodiment of the present invention, the primer comprises an upstream primer and/or a downstream primer; wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 4: 5'-CGAGGCTTGCAAGTTACACA-3', respectively; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5: 5'-CAGGGCATTTGAAACCTCAT-3' are provided.

In a fifth aspect, the present invention provides a vector into which the nucleotide sequence is introduced.

The kind of the vector is not particularly limited, and examples of the vector include, but are not limited to, pET28b, pIJ702, pUCP19, pYMB03 or pHT43, and pET28b is preferable.

In a sixth aspect, the present invention provides a host cell into which the nucleotide sequence is introduced, or into which the vector is introduced.

Further, the host cell comprises a plant cell and/or a microbial cell;

further, the plant cells include, but are not limited to, taxus chinensis cells, tobacco cells, white bean cedar cells, artemisia apiacea cells;

further, the microbial cells include but are not limited to streptomycete, pseudomonas, bacillus, yeast cells, escherichia coli.

Further, methods for introducing the nucleotide sequence encoding taxadiene synthase TcTS2, or said recombinant plasmid, or said expression vector into host cells in vivo and in vitro include, but are not limited to, electroporation, polyethylene glycol (PEG) transformation, lipofection, heat shock, calcium phosphate precipitation, viral mediation and microinjection.

The seventh aspect of the present invention provides a method for expressing taxadiene synthase TcTS2 in plants, wherein the nucleotide sequence, or the vector, or the host cell is transferred into plants to obtain taxadiene synthase TcTS 2.

Further, the plants include, but are not limited to: taxus chinensis, Douglas fir, tobacco, and herba Artemisiae Annuae.

Further, the plant body also includes plant parts such as explants, including but not limited to: cuttings, tissue cultures, cell suspensions, and calli.

Further, the plant is more preferably taxus chinensis and/or tobacco.

The eighth aspect of the present invention provides a method for producing paclitaxel and intermediates thereof, comprising allowing a plant to express the taxadiene synthase TcTS 2.

Further, the method comprises: transferring the nucleotide sequence, the carrier or the host cell into a plant body to express the taxadiene synthase TcTS2 and obtain the taxol and the intermediate thereof.

The invention adopts the technical scheme and has the following beneficial effects:

(1) the invention provides a taxadiene synthase TcTS2, which remarkably improves the efficiency of catalyzing GGPP cyclization, greatly improves the yields of intermediate taxadiene, baccatin III and product taxol in the process of synthesizing taxol, remarkably reduces the production cost of taxol medicaments, and effectively solves the current situations of high price and short supply and demand of taxol in the market.

(2) The expression of the taxadiene synthase TcTS2 provided by the invention in plant cells is obviously induced and regulated by MeJA, and can be used for producing taxol and intermediates thereof in plant genetic engineering and metabolic engineering modification in the future by combining small molecule MeJA induction and other synthetic biological technical means.

(3) The nucleotide sequence for coding the taxadiene synthase TcTS2 provided by the invention provides a new gene resource for improving the yield of paclitaxel, and the nucleotide sequence and TcTS2 can be used for modifying a chassis host by plant genetic engineering and metabolic engineering strategies, producing paclitaxel and intermediates thereof and the like, and have obvious economic and social values.

Drawings

FIG. 1 shows the in vitro biochemical activity assay of TcTSs. Wherein A is GC-MS detection of TcTSs enzymatic reaction products, and extraction of taxadiene characteristic ion m/z 122 chromatogram; b is a reaction schematic diagram of the GGPP catalyzed by TcTSs to generate taxadiene; c is the mass spectrum of the product of TcTSs enzymatic reaction.

FIG. 2 is a graph showing the content change of metabolites under MeJA stress and the expression pattern of TcTSs gene. Wherein A is a graph of relative change of baccatin III and taxol yield under MeJA stress; b is an expression pattern diagram of a TcTS1 gene under MeJA stress; c is an expression pattern diagram of a TcTS2 gene under MeJA stress; MeJA + represents 100. mu.M MeJA treatment group; MeJA-refers to solvent control.

Detailed Description

In the present invention, the term "TcTS 2" may refer to taxadiene synthase, or taxadiene synthase gene, or a nucleotide sequence encoding taxadiene synthase, and the meaning may be determined in combination with the context.

In the present invention, the term "gene" is defined as a genetic unit (usually represented by a DNA sequence) that occupies a specific position in a chromosome and contains genetic instructions that contribute to a potential phenotypic characteristic or trait of a plant. "

In the present invention, the term "nucleotide" is used in its ordinary sense as understood by those skilled in the art.

In the present invention, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids), including but not limited to alpha-amino acids, beta-amino acids, gamma-amino acids, and-amino acids. Examples of suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention is described in detail below with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.