CN117106807A - Streptomyces-derived sesquiterpene synthase encoding gene, genetically engineered bacterium and application thereof - Google Patents

Streptomyces-derived sesquiterpene synthase encoding gene, genetically engineered bacterium and application thereof Download PDF

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CN117106807A
CN117106807A CN202311109983.8A CN202311109983A CN117106807A CN 117106807 A CN117106807 A CN 117106807A CN 202311109983 A CN202311109983 A CN 202311109983A CN 117106807 A CN117106807 A CN 117106807A
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gene
streptomyces
sdts29
sesquiterpene
escherichia coli
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李爱英
郝进芳
李瑞娟
赵一名
李彩云
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Shandong University
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Abstract

The application belongs to the technical field of genetic engineering and biosynthesis, and particularly relates to a streptomycete-derived sesquiterpene synthase coding gene, genetically engineered bacteria and application thereof. Specifically, the application performs the function verification of a new gene cluster (BGC 1.29) which is derived from streptomyces and is responsible for synthesizing sesquiterpene alcohols, and the function verification of terpene synthase SdTS 29; discloses application of modifying gene clusters thereof by utilizing promoter engineering to construct heterologous expression engineering bacteria and promote and improve production of terpenoid; meanwhile, the E.coli platform is utilized to verify the gene function, and engineering bacteria for producing sesquiterpenes are constructed. The application enriches the biological sources of isobauc-8-en-11-ol and also helps to provide references for functional identification of homologous genes. The application is beneficial to the construction of the high-yield strain of the corresponding terpenoid, provides a new element for the combined biosynthesis of novel terpenoid, and has good practical application value.

Description

Streptomyces-derived sesquiterpene synthase encoding gene, genetically engineered bacterium and application thereof
Technical Field
The application belongs to the technical field of genetic engineering and biosynthesis, and particularly relates to a streptomycete-derived sesquiterpene synthase coding gene, genetically engineered bacteria and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The terpenoid is a family of small molecular natural products with extremely diverse structures, has various biological activities, and has wide application in the fields of food, agriculture, spice, medicine, industry and the like. Genome sequencing revealed an abundant cluster of terpenoid biosynthesis genes (biosynthetic gene cluster, BGC) in the genomes of various bacteria represented by streptomyces. The research on the gene clusters is helpful for revealing the structural and functional novelty of the terpenoid, expanding the cognition of the diversity of the enzymes related to the terpenoid synthesis, and has important research value.
Terpenoid is obtained by mixing isoprene (C) 5 ) As a structural unit, by repeating the connection of side chains. Two activated forms of isoprene precursor are isopentenyl pyrophosphate (isopentenyl diphosphate, IPP) and allyl pyrophosphate (dimethylallyl diphosphate, DMAPP), which can be obtained in eukaryotic cellsThe Mevalonate pathway (MVA), which is more common in organisms, and the methylerythrose phosphate pathway (MEP), which is more common in bacteria. The universal precursors are polymerized end-to-end by the action of Prenyltransferases (PTs) to form a precursor containing a variable number C 5 Linear precursors of the units; terpene synthases (terpene synthases, TSs) catalyze their cyclization and rearrangement to form structurally diverse core skeletons of terpenoids; the catalysis of post-modification enzymes such as methylation, hydroxylation, glycosylation and the like further endows the complex structure and various biological functions of the final product of the terpenoid, thereby forming a rich terpenoid family.
Heterologous expression is an effective means for identifying gene and gene cluster functions and exploring biosynthetic pathways. The streptomycete has low homology of the whole amino acid sequence of the terpene synthase, but has higher similarity in higher structure, and is helpful for guiding the selection of the terpene gene cluster by means of bioinformatics analysis. By multiple sequence alignment, highly conserved amino acid motifs (motif) contained in terpene synthases can be determined, making preliminary predictions about the type of terpene synthase catalysis. Further amino acid sequence alignment and phylogenetic analysis with known terpene synthases can infer the type of terpene synthase product and the cyclization mode based on the branching positions.
Disclosure of Invention
Aiming at the prior art, the inventor provides a streptomycete-derived sesquiterpene synthase coding gene, genetic engineering bacteria and application thereof through long-term technical and practical exploration. The application identifies more terpenoid and elements participating in biosynthesis of natural terpenoid products from streptomyces, clones and identifies terpenoid gene clusters and terpene synthase and post-modification enzyme coding genes therein, constructs heterologous expression engineering strains and application thereof in promoting production of terpenoid in a heterologous system. Based on the above results, the present application has been completed.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect of the application, there is provided a sesquiterpene synthase encoding gene designated SdTS29, the sequence of which originates from Streptomyces DSM731 (Streptomyces tanashiensis Kala DSM 731).
Specifically, the sesquiterpene synthase encoding gene has the nucleotide sequence of any one of (a 1) to (a 4):
(a1) A nucleotide sequence shown as SEQ ID NO. 1; the nucleotide sequence consists of 1032 bases and codes for isodacu c-8-en-11-ol synthase;
(a2) A sequence formed by substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence as shown in (a 1);
(a3) A nucleic acid molecule having 50% or more identity to the nucleotide sequence defined in (a 1) or (a 2) and encoding said fusion protein;
(a4) A nucleotide sequence capable of hybridizing to the nucleotide sequence according to any one of (a 1) to (a 3) under stringent conditions and encoding an isobauc-8-en-11-ol synthase.
In a second aspect of the present application, there is provided a sesquiterpene synthesis gene cluster designated BGC1.29 comprising at least the above-mentioned sesquiterpene synthase-encoding gene, said gene cluster being derived from Streptomyces DSM731 (Streptomyces tanashiensis Kala DSM 731), said gene cluster encoding a total of 19 genes.
In a third aspect of the present application, there is provided a host bacterium comprising a gene encoding a sesquiterpene synthase, a sesquiterpene synthesis gene cluster or being capable of expressing isobauc-8-en-11-ol.
Further, the host bacterium may be Streptomyces.
Specifically, the construction method of the host bacteria comprises the following steps:
directly cloning the gene cluster BGC1.29 of Streptomyces tanashiensis Kala DSM731, adding a conjugation transfer and site-specific recombination element, and integrating the gene cluster BGC1.29 onto the genome of the streptomyces strain through indirect transfer between seeds;
the Streptomyces roseoflavus strain can be Streptomyces albus J1074;
at this time, the host bacterium obtained by construction is named Streptomyces albus J1074/BGC1.29; the genotype is Streptomyces albus J1074, p15A-apra carries BGC1.29 integrated on S.albus J1074 genome, and resistance to apramycin (apramycin resistance).
Alternatively, the host bacterium can be further obtained by constructing the following method:
directly cloning the gene cluster BGC1.29 from Streptomyces tanashiensis Kala DSM731, adding a conjugation transfer and site-specific recombination element, replacing a primary promoter with a strong promoter SP44 at the upstream of a terpene synthase gene SdTS29, and integrating the primary promoter into the genome of a streptomycete strain through indirect transfer between species;
the Streptomyces roseoflavus strain can be Streptomyces albus J1074;
at this time, the host bacterium obtained by construction is named Streptomyces albus J1074/P-BGC1.29; the genotype is Streptomyces albus J1074, the p15A-apra carries the BGC1.29 of the insertion promoter and is integrated on the S.albus J1074 genome, the apramycin resistance (apramycin resistance), the hygromycin resistance (hygromycin resistance) and the constitutive strong promoter SP44.
In a fourth aspect of the application, there is provided the use of a sesquiterpene synthase encoding gene, a sesquiterpene synthesis gene cluster as described above or of said host bacterium for biosynthesis and/or promotion of biosynthesis of terpenoids.
Wherein the terpenoid can be sesquiterpene alcohol isodauc-8-en-11-ol.
In a fifth aspect of the application, there is provided a method of biosynthesizing the sesquiterpene alcohol isodauc-8-en-11-ol, the method comprising: culturing the host bacteria, and separating and purifying to obtain the sesquiterpene alcohol isodauc-8-en-11-ol.
The sixth aspect of the application provides an engineering bacterium for detecting the function of terpene synthases in a gene cluster BGC1.29, wherein the engineering bacterium is obtained by transferring pMHI and pFZ81 and a plasmid pGB231-1 obtained by inserting a terpene synthase encoding gene SdTS29 into a pGB231 plasmid into a starting strain escherichia coli;
the escherichia coli of the initial strain can be Escherichia coli BL;
at this time, the obtained engineering bacterium was named Escherichia coli F1 SdTS29 (abbreviated as F1), genotypes of which were Escherichia coli BL, pMHI, pFZ81, pGB231-1 carrying SdTS29 were introduced into BL21, chloramphenicol resistance (chloramphenicol resistance), kanamycin resistance (kanamycin resistance), promoter T7, and inducible promoter Lac.
Or the engineering bacteria are obtained by transferring plasmid pGB231-2 obtained by inserting pMHI and pFZ81 and a cytochrome oxidase P450 coding gene SdP into pGB231-1 plasmid SdTS29 downstream of the genes into an original strain escherichia coli;
the escherichia coli of the initial strain can be Escherichia coli BL;
at this time, the obtained engineering bacterium was named Escherichia coli F2 SdTS29-SdP450 (abbreviated as F2), the genotypes of which are Escherichia coli BL, pMHI, pFZ81, pGB231-2 carrying SdTS29 and SdP were introduced into BL21, chloramphenicol resistance (chloramphenicol resistance), kanamycin resistance (kanamycin resistance), promoter T7, inducible promoter Lac.
In a seventh aspect, the application provides an application of the engineering bacterium in detecting related coding genes and functions of the related coding genes in a gene cluster BGC 1.29. Specifically, according to the application, sdTS29 is isodauc-8-en-11-ol synthase, and SdP450 does not function.
Compared with the prior art, the one or more technical schemes have the following beneficial technical effects:
the sesquiterpene isodauc-8-en-11-ol is separated and identified from the heterologous expression engineering strain of the streptomyces BGC1.29 disclosed in the technical scheme. The sesquiterpene alcohol source has a relatively limited report, and only terpene synthases from Streptomyces venezuelae ATCC and 10712 (CCA 53839) are reported to catalyze in vitro reactions, which are different from the sequence of the terpene synthase SdTS29 in the technical scheme.
The technical proposal enriches the biological sources of the isobauc-8-en-11-ol and is also helpful for providing reference for the functional identification of homologous genes. In addition, the E.coli platform based on the high-yield sesquiterpene precursor disclosed in the technical scheme establishes an in-vivo expression system of streptomyces source terpene synthase SdTS29 in escherichia coli for the first time, and detects the generation of sesquiterpene alcohol isoauc-8-en-11-ol, thereby identifying a new isoauc-8-en-11-ol synthase SdTS29. The application of the strain is beneficial to the construction of the strain with high yield of the corresponding terpenoid, and provides a new element for the combined biosynthesis of the novel terpenoid, so the strain has good practical application value.
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The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.
Fig. 1: composition of the gene cluster BGC 1.29.
Fig. 2: bioinformatic analysis of terpene synthases is assumed in BGC1.29;
a: the putative terpene synthase SdTS29 was subjected to multiple sequence alignment with known terpene synthases (SaTS 4 is a putative terpene synthase from other Streptomyces sources). SdTS29 contains highly conserved DDxxD/E and NSE/DTE aspartate enrichment motifs in type I terpene synthases; b: phylogenetic analysis of SdTS29 with other microbial sources known terpene synthases (where SaTS4, sdTS2 are putative terpene synthases of other Streptomyces sources). SdTS29 is clustered in the same group as sesquiterpene synthases.
Fig. 3: heterologous expression of BGC1.29 in Streptomyces albus J1074;
a: direct cloning of BGC1.29 using the p15A-cm-tetR-tetO-hyg-ccdB vector; b: adding conjugation transfer and site-specific recombination elements on the BGC1.29 cloning plasmid; c: enzyme digestion verification of BGC1.29 clone plasmid and colony PCR verification of transformant after conjugation transfer (left: plasmid p15A-cm-1.29 enzyme digestion map obtained by direct cloning of BGC1.29; in the middle: modified plasmid p15A-apra-1.29 enzyme digestion map; right: J1074/BGC1.29 colony PCR).
Fig. 4: heterologous expression in Streptomyces albus J1074 and GC-MS analysis results after BGC1.29 promoter engineering modification;
a: it was assumed in BGC1.29 that the original promoter was replaced upstream of terpene synthase SdTS29 with constitutive promoter SP 44; b: enzyme digestion verification of plasmid modified by BGC1.29 promoter and colony PCR verification of transformant after conjugation transfer; c: GC-MS detection of the promoter modified BGC1.29 heterologous expression strain J1074/BGC1.29-P fermentation crude extract shows 2 difference peaks, and is presumed to be sesquiterpene.
Fig. 5: BGC1.29 heterologous expression of structure of compound 1.
Fig. 6: construction of an in-vivo expression system of SdTS29 and SdP450 escherichia coli and GC-MS detection of a fermentation product;
a: construction of an expression system of the assumed terpene synthase SdTS29 and the assumed P450 monooxygenase SdP450 in escherichia coli in BGC1.29; b: GC-MS analysis of the heterologous expression strain of SdTS29 E.coli and the heterologous expression strains of SdTS29, sdP450 revealed that the production of the compound isodauc-8-en-11-ol (1) could be detected for SdTS29 expression, whereas SdP450 did not function in this E.coli.
Fig. 7: sesquiterpene alcohol isodac-8-en-11-ol (1) 1 H NMR spectrum (a); isodauc-8-en-11-ol (1) 13 C NMR spectrum (B); DEPT profile (C) of isobauc-8-en-11-ol (1); HSQC profile (D) of isobauc-8-en-11-ol (1); HMQC profile (E) of isobauc-8-en-11-ol (1); 1H-1H COSY spectrum (F) of isobauc-8-en-11-ol (1); NOSEY profile (G) of isobauc-8-en-11-ol (1).
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
Specifically, the application uses the anti-smash gene cluster prediction result to show that a gene cluster (named as BGC 1.29) with lower similarity with the known terpenoid gene cluster exists in Streptomyces tanashiensis Kala DSM (DSM 731), and the functional verification of the gene cluster is helpful for exploring more terpenoid natural products with new frameworks, new activities and new mechanisms, activating the silent terpenoid gene cluster and constructing a high-yield strain of the corresponding terpenoid.
The streptomyces-derived sesquiterpene biosynthesis gene cluster is named as BGC1.29, and the gene cluster composition is shown in figure 1; the gene cluster sequence was derived from Streptomyces DSM731 (Streptomyces tanashiensis Kala DSM 731), the gene cluster encodes 19 genes (NCBI SEQ ID NO: CP084204.1, gene cluster position 7903770-7924722), and the predicted functions are shown in Table 1.
The streptomycete-derived sesquiterpene synthase coding gene is named SdTS29, and the nucleotide sequence of the gene is shown as SEQ NO. 1; the gene sequence is derived from Streptomyces DSM731 (Streptomyces tanashiensis Kala DSM 731), and the nucleotide sequence thereof consists of 1032 bases, and is predicted to encode sesquiterpene synthases.
First, applicants performed bioinformatic analyses of possible terpene gene clusters and terpene synthase genes: (1) By anti smash analysis, a total of 7 possible terpenoid BGCs were found in the streptomyces DSM731 (Streptomyces tanashiensis Kala DSM 731) genome, presumably with the function of expressing terpenoid compounds, wherein 4 gene cluster predicted products did not show similarity to known compounds (table 2). Therefore, the applicant directly clones and verifies the functions of one of the terpene biosynthesis gene clusters BGC 1.29. (2) The terpene synthases in the supposedly terpene biosynthesis gene cluster are subjected to multi-sequence comparison with known terpene synthases from different microorganisms by using CLUSTALW, and then the catalytic type and the function of the terpene synthases are preliminarily deduced through the sequence editing website ENDscript/ESPribept comparison of conserved sequences. The multiple sequence alignment (FIG. 2A) shows that SdTS29 contains highly conserved DDxxD/E and NSE/DTE aspartate rich motifs in type I terpene synthases. And selecting terpene synthases from different microorganism sources and carrying out sequence alignment with the terpene synthase SdTS29, and constructing a phylogenetic evolutionary tree, wherein yellow, orange, blue and green branches in the figure 2B respectively represent the half terpene synthases, the monoterpene synthases, the sesquiterpene synthases and the diterpene synthases. The results showed that SdTS29 was clustered in the same group as sesquiterpene synthases.
Next, the application discloses two heterologous expression engineering bacteria of a biosynthesis gene cluster BGC1.29 for producing sesquiterpenes by taking streptomycete as a host:
(1) The application discloses an engineering bacterium for producing heterologous expression of a terpenoid biosynthesis gene cluster, which is named Streptomyces albus J1074/BGC1.29 and is obtained by directly cloning the gene cluster BGC1.29 from Streptomyces tanashiensis Kala DSM731, adding a conjugation transfer and site-specific recombination element, and integrating the gene cluster into a Streptomyces albus J1074 genome through indirect transfer between species; the genotype was Streptomyces albus J1074, p15A-apra harbored BGC1.29 integrated into the s.albus J1074 genome, apramycin resistance (apramycin resistance) (fig. 3B).
(2) The application discloses a heterologous expression engineering bacterium for a terpene biosynthesis gene cluster after promoter engineering modification, which is named Streptomyces albus J1074/P-BGC1.29, and is obtained by directly cloning a gene cluster BGC1.29 derived from Streptomyces tanashiensis Kala DSM731, adding a joint transfer and site-specific recombination element, replacing a primary promoter with a strong promoter SP44 at the upstream of a terpene synthase gene SdTS29, and integrating the primary promoter into a Streptomyces albus J1074 genome through indirect transfer; BGC1.29, which has genotype Streptomyces albus J1074, p15A-apra harbors the insert promoter, was integrated into the S.albus J1074 genome, apramycin resistance (apramycin resistance), hygromycin resistance (hygromycin resistance), constitutive strong promoter SP44 (FIG. 4A).
Then, the application discloses a heterologous expression engineering bacterium for detecting the terpene synthase gene from BGC1.29 by taking escherichia coli as a host, wherein the engineering bacterium is named Escherichia coli F1 SdTS29 (abbreviated as F1) is obtained by inserting pMHI and pFZ81 plasmids (comprising genes required by MVA pathway and responsible for providing terpene synthesis precursors) and a possible terpene synthase encoding gene SdTS29 in a gene cluster BGC1.29 into pGB231 plasmid to obtain plasmid pGB231-1, and transferring Escherichia coli BL; the genotypes of the recombinant DNA are Escherichia coli BL, pMHI, pFZ81, pGB231-1 carrying SdTS29 and introducing BL21, chloramphenicol resistance (chloramphenicol resistance) and carpenterNatamycin resistance (kanamycin resistance), promoter T7, inducible promoter Lac (fig. 6A).
Meanwhile, the application discloses a construction process of engineering bacteria based on the escherichia coli as a host, which is used for detecting the cytochrome oxidase P450 function in the gene cluster BGC1.29, and the engineering bacteria is named Escherichia coli F2 SdTS29-SdP450 (abbreviated as F2), the plasmid pGB231-2 obtained by inserting pMHI and pFZ81 plasmids and a possible cytochrome oxidase P450 coding gene SdP450 in a gene cluster BGC1.29 into pGB231-1 plasmid SdTS29 downstream of the gene is transferred into Escherichia coli BL; genotypes Escherichia coli BL, pMHI, pFZ81 and pGB231-2 carrying SdTS29 and SdP were introduced into BL21, chloramphenicol resistance (chloramphenicol resistance), kanamycin resistance (kanamycin resistance), promoter T7 and inducible promoter Lac (FIG. 6A).
Experiments prove that: (1) Compared with Streptomyces albus J1074/BGC1.29 and wild strain Streptomyces tanashiensis Kala DSM731, the heterologous expression engineering bacterium modified by the BGC1.29 promoter engineering provided by the application successfully activates gene cluster expression and detects the generation of sesquiterpene products. Further separating and purifying the fermented crude extract, and identifying a sesquiterpene isodauc-8-en-11-ol. (2) Compared with the escherichia coli strain which only contains the introduced MVA pathway to synthesize the general precursor of terpenes, the heterologous expression engineering bacteria F1 and F2 of the escherichia coli of the terpene synthase SdTS29 and the escherichia coli of the cytochrome P450 enzyme SdP, and the detection of the expression of the sesquiterpene alcohol isoauc-8-en-11-ol proves that the SdTS29 is isoauc-8-en-11-ol synthase and SdP450 does not function.
In the following examples, materials, reagents and the like used, unless otherwise specified, were obtained commercially. The experimental methods are all conventional methods in the art unless specifically stated.
Wherein: coli E.coli ET12567/pUZ8002, from John Innes institute, england; streptomyces Streptomyces tanashiensis Kala DSM731 is from DSMZ collection of Germany.
Coli e.coli BL21 (DE 3) and Streptomyces albus J1074 are common bacterial heterologous expression hosts. E.coli GBdir and E.coli GBred are used for direct cloning of gene clusters (line homologous recombination) and general cloning of genes (line loop homologous recombination); plasmids pGB231, pMHI and PFZ81, which provide terpene precursor synthetic genes, were from the university of Wuhan Liu Tian subject group. Other vectors are all commonly used in laboratories. Gene sequencing in plasmid construction was done by the biological engineering (Shanghai) Co., ltd.
Formulation of SM medium (per liter): yeast extract 2g, glucose 60g, naCl 2g, K 2 HPO 4 0.5g、(NH 4 ) 2 SO 4 2g、MgSO 4 ·7H 2 O 0.1g，FeSO 4 ·7H 2 O 0.05g、ZnSO 4 ·7H 2 O 0.05g、MnSO 4 ·7H 2 O 0.05g，CaCO 3 5g, pH was adjusted to 7.2.
Formulation of TSB broth (per liter): 30g of trypticase soy peptone.
Formulation of MS solid medium (per liter): 20g of soybean powder, 20g of mannitol and 20g of agar powder, and adding 2.5M MgCl when in use 2 The final concentration was 10mM.
Example 1: constructing a BGC1.29 heterologous expression engineering bacterium, detecting heterologous host metabolites through experiments, and analyzing the expression condition of BGC1.29
The steps of constructing the BGC1.29 heterologous expression engineering bacteria are shown in FIG. 3A:
(1) Selecting cleavage sites BstXI at two sides of a BGC1.29 sequence (NCBI sequence number: CP084204.1, gene cluster position: 7903770-7924722) in the streptomyces DSM731 genome, and performing cleavage on the DSM731 genome to obtain a BGC1.29 fragment;
(2) The p15A-cm-tetR-tetO-hyg-ccdB plasmid is used as a cloning template of the linear vector fragment, and the primer sequences are as follows:
dsm29-cm-F:GTC CTC GCC TAC AAG GTC TTC CGC GCC TGG GTC TCC GAG TTC CAG TCC CTC CCC GAA CTC GAC TCC AAC GAG ATC CGA AAA CCC CAA G；
dsm29-cm-R:TTG GGG TCG GCC GCG CGG TGG TCC ACG TCC CAC CGC TGA CCG CGC TCG CTG ACC AGG ACG TAG TCG TGC TAG ATC CTT TCT CCT CTT T。
the PCR product obtained by PCR amplification (30 cycles; 98 ℃,10s;43 ℃,15s;72 ℃,2 min) contains homologous fragments (70 bp) on the upstream and downstream sides of BGC1.29 and a p15A-cm carrier fragment;
(3) Introducing the PCR product obtained in the step (2) and the BGC1.29 fragment obtained in the step (1) into escherichia coli GBdir, and performing homologous recombination and Bsu36I digestion and screening to obtain a recombinant plasmid p15A-cm-1.29 related to the BGC1.29 (left in FIG. 3C);
(4) As shown in FIG. 3B, plasmid pR6K-oriT-phiC31-Apra was digested with AseI to obtain a conjugation-transfer and site-specific recombination fragment Apra-oriT-attP-int-phiC31 with homology arms on both sides of chloramphenicol (cm) resistance gene on the direct cloning vector;
(5) And (3) jointly electroconverting the fragment Apra-oriT-attP-int-phiC31 obtained in the step (4) and the plasmid p15A-cm-1.29 into escherichia coli GBred, screening positive clones, and extracting the plasmid for enzyme digestion verification. FIG. 3C (middle) XmnI cleavage results indicate successful construction of plasmid p15A-apra-1.29;
(6) Electrically transforming the plasmid p15A-apra-1.29 obtained in the step (5) into escherichia coli ET12567/pUZ8002, and screening to obtain positive clones;
(7) ET 12567/puc 8002 cells containing plasmid p15A-apra-1.29 (containing intact BGC 1.29) were mixed with heterologous host bacteria Streptomyces albus J1074 spores by interspecific binding transfer, allowing transfer of plasmid p15A-apra-1.29 into the latter (J1074), and the colony PCR results in fig. 3C (right) indicated successful integration of the BGC1.29 gene cluster into the J1074 genome; namely, a BGC1.29 heterologous expression engineering bacterium is obtained, the bacterium is named Streptomyces albus J1074/BGC1.29, the genotype is Streptomyces albus J1074, and p15A-apra carries complete BGC1.29 gene cluster and is introduced into J1074 and apramycin resistance (apramycin resistance).
Detection of the crude fermentation extract by GC-MS showed that no difference peak was detected in heterologous expression of BGC1.29 (engineering bacterium J1074/BGC 1.29) relative to the starting strain J1074, assuming that BGC1.29 is still in a silent state in J1074.
Example 2: constructing a BGC1.29 promoter engineering modified heterologous expression engineering bacterium, detecting heterologous host metabolites by experiment, and analyzing the expression condition of BGC1.29 after promoter modification
Constructing a promoter engineering modified BGC1.29 heterologous expression engineering bacterium, wherein the steps are shown in FIG. 4A:
(1) The SP44-hyg-ermE21 fragment is used as a template, and the primer sequences are as follows:
dsm29-P-F：CAT CCA CAG CAG GGG GGC TTC GAC GGT ACG TAC TGT CAT TAC ACC AGA CTT TAC AAC ACC；
dsm29-P-R：CGG CGC CCG CCT CCT GGA CTG GCC GGA CCA GCC CCT GTA GCT ACG ATG TAT CAG GCG CCG。
AC ACC AG is the Ribosome Binding Site (RBS) downstream of promoter SP44.
Amplifying by PCR (30 cycles; 98 ℃,10s;50 ℃,15s;72 ℃,1 min), obtaining SP44-hyg fragments;
(2) Introducing the PCR product obtained in the step (1) and the P15A-apra-1.29 plasmid obtained in the step (5) into escherichia coli GBred together, carrying out homologous recombination screening to obtain a recombinant plasmid P15A-apra-P-1.29 (a constitutive strong promoter is inserted upstream of an SdTS29 gene), and carrying out MluCI enzyme digestion on the result of FIG. 4B (left) to show that the P15A-apra-P-1.29 plasmid is successfully constructed;
(3) Electrotransformation of the plasmid P15A-apra-1.29-P obtained in the step (2) into E.coli ET12567/pUZ8002, and screening positive clones;
(4) ET 12567/puc 8002 cells containing plasmid P15A-apra-1.29-P were mixed with heterologous host bacteria Streptomyces albus J1074 spores by interspecific binding transfer, allowing transfer of plasmid P15A-apra-P-1.29 into the latter (J1074), and the colony PCR results in fig. 4B (right) indicated successful integration of the P-BGC1.29 gene cluster into the J1074 genome; namely, a P-BGC1.29 heterologous expression engineering bacterium is obtained, the bacterium is named Streptomyces albus J1074/P-BGC1.29, the genotype of the bacterium is Streptomyces albus J1074, and P15A-apra carries the P-BGC1.29 gene cluster modified by the promoter to be introduced into J1074, the apramycin resistance (apramycin resistance), the hygromycin resistance (hygromycin resistance) and the constitutive promoter SP44.
The detection of the fermentation crude extract by using GC-MS shows that compared with the original strains J1074 and BGC1.29 heterologous expression engineering bacteria J1074/BGC1.29 and wild type DSM731, the modified BGC1.29 heterologous expression engineering bacteria J1074/P-BGC1.29 obtained by the application has the characteristic that the insertion of the promoter successfully activates the expression of BGC1.29, and two difference peaks are detected at 12.67min and 14.55min (figure 4C), which has the characteristic of sesquiterpene.
Example 3: large-batch fermentation promoter modified BGC1.29 heterologous expression engineering bacteria, separating and purifying fermentation products, and identifying compound structures
(1) Heterologous expression engineering bacteria J1074/P-BGC1.29 are fermented for 10L in a large batch, and the obtained fermentation crude extract is further purified by forward silica gel column separation and semi-preparative liquid chromatography, so as to be separated into 1mg of compound 1;
(2) The resulting compound was dissolved in deuterated methanol (MeOD-d 4 ) Nuclear magnetic resonance spectroscopy (NMR) detection was performed, and it was found by literature comparison 1 H， 13 C and 2D NMR data (shown in Table 3 and FIG. 7) and the known compound isodauc-8-en-11-ol (C) 15 H 26 O) data match, identifying Compound 1 as sesquiterpene alcohol isodauc-8-en-11-ol (FIG. 5).
Example 4: constructing a SdTS29 heterologous expression engineering bacterium and SdTS29 and SdP450 heterologous expression engineering bacterium, detecting heterologous host metabolites by experiment, and analyzing the influence on expression of terpenoid products
The steps of constructing the SdTS29 heterologous expression engineering bacteria and the SdTS29 and SdP450 heterologous expression engineering bacteria are shown in FIG. 6A:
(1) Construction of a heterologous expression System of SdTS29 E.coli
PCR amplification of SdTS29 using Streptomyces DSM731 total DNA as template, the primer sequences are as follows:
SdTS29-F：CTC GAG TGC GGC CGC AAG CTT GTC GAC GGA GCT CGA ATT CTC ACT CCG TGAACG GTC C；
SdTS29-RBS-R：CTAGAAATA ATT TTG TTT AAC TTT AAG AAG GAG ATATAC CAT GAC AGT ACG TAC CGT C；
SdTS29-R：AGC CAG AAAACG ATT ATC TGC ATT TAC CCA GCT TAA ATAACT AGA AAT AAT TTT GTT T。
TTAACTTTAAGAAGGAG is the RBS site.
PCR amplification was performed using SdTS29-F and SdTS29-RBS-R primer pairs (30 cycles; 98 ℃,10s;49 ℃,15s;72 ℃,1 min). And then using the obtained fragments as templates, adopting a SdTS29-F and SdTS29-R primer pair for amplification (30 cycles; 98 ℃,10s;37 ℃,15s;72 ℃,1 min), and finally obtaining PCR products comprising the SdTS29 fragments, fragments containing RBS and fragments homologous to the upstream and downstream of the vector pGB231 (40 bp).
The PCR fragment was introduced into E.coli GBdir together with the expression vector pGB231 treated by double cleavage with SacI and EcoRI, and subjected to homologous recombination to obtain recombinant plasmid pGB231-1 concerning SdTS29.
pGB231 was co-transformed with pMHI and PFZ81 (containing genes required for the MVA pathway responsible for providing terpene synthesis precursors) into the E.coli BL21 (DE 3) chassis to give mutant F0.
pGB231-1 was co-transformed with pMHI and PFZ81 into E.coli BL21 (DE 3) chassis to give mutant F1.
(2) Construction of a heterologous expression System for E.coli containing SdP450
PCR amplification SdP with Streptomyces DSM731 total DNA as template, primer sequences are as follows:
SdP450-F：CTC GAG TGC GGC CGC AAG CTT GTC GAC GGAGCT CGA ATT CCT ACC GCC TGC GCAG；
SdP450-RBS-R：CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATATAC CAT GAC CAT CCC ACC CAC C；
SdP450-R：ACC CCG CTA CGC CTC CTC CGA CGG ACC GTT CAC GGA GTG ACT AGAAAT AAT TTT GTT T。
TTAACTTTAAGAAGGAG is the RBS site.
PCR amplification was performed using SdP-F and SdP-RBS-R primer pairs (30 cycles; 98 ℃,10s;50 ℃,15s;72 ℃,1 min). And amplifying the obtained fragments by using SdP450-F and SdP450-R primer pairs (30 cycles; 98 ℃,10s;37 ℃,15s;72 ℃ for 1 min) with the obtained fragments as templates, wherein the finally obtained PCR product comprises SdP fragments, fragments containing RBS and fragments homologous to the upstream and downstream of the vector pGB231-1 (40 bp).
The PCR fragment was introduced into E.coli GBdir together with the expression vector pGB231-1 treated by double cleavage with SacI and EcoRI, and subjected to homologous recombination to obtain recombinant plasmid pGB231-2 concerning SdP 450.
pGB231-2 was co-transformed with pMHI and PFZ81 into E.coli BL21 (DE 3) chassis to give mutant F2.
(3) SdTS29 and SdP450 are expressed in E.coli BL21 (DE 3), and GC-MS detects metabolites
The strain was cultured overnight at 37℃in LB-resistant liquid medium containing 50mg/L Kan and 34mg/L Cm, inoculated in 50mL of the corresponding resistant LB medium at an inoculum size of 2% (v/v) the next day, and cultured at 37℃for 2 hours (OD 600 Reaching 0.6-0.8). 0.1mM IPTG inducer was added to induce expression at 16℃for 18-20 hours, followed by culturing at 28℃for 3d. The crude extract of the fermentation broth was tested by GC-MS and analyzed for the production of metabolites.
Experiments show that: the F1 and F2 strains produced only Compound 1 compared to control F0 (see FIG. 6B), indicating that SdTS29 is isodauc-8-en-11-ol synthase and SdP450 did not function in this cell.
Table 1 prediction of Gene function in BGC1.29 Gene clusters
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TABLE 2 putative terpenoid biosynthesis Gene Cluster in Streptomyces DSM731 genome
TABLE 3 Isodauc-8-en-11-ol (1) 13 C NMR、 1 H NMR data
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a 151MHz, meOD-d 4 . b 600MHz, meOD-d 4 .
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. A sesquiterpene synthase encoding gene, wherein said encoding gene is designated as SdTS29, said sesquiterpene synthase encoding gene having any of the nucleotide sequences (a 1) - (a 4):
(a1) A nucleotide sequence shown as SEQ ID NO. 1; the nucleotide sequence consists of 1032 bases and codes for isodacu c-8-en-11-ol synthase;
(a2) A sequence formed by substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence as shown in (a 1);
(a3) A nucleic acid molecule having 50% or more identity to the nucleotide sequence defined in (a 1) or (a 2) and encoding said fusion protein;
(a4) A nucleotide sequence capable of hybridizing to the nucleotide sequence according to any one of (a 1) to (a 3) under stringent conditions and encoding an isobauc-8-en-11-ol synthase.
2. A sesquiterpene synthesis gene cluster designated BGC1.29 comprising at least the sesquiterpene synthase encoding gene of claim 1; it is located at 7903770-7924722 bits on NCBI sequence number CP 084204.1.
3. A host bacterium comprising the sesquiterpene synthase encoding gene of claim 1, the sesquiterpene synthesis gene cluster of claim 2, or the ability to express isobauc-8-en-11-ol.
4. The host bacterium of claim 3, wherein said host bacterium is Streptomyces.
5. The host bacterium according to claim 3 or 4, wherein the method for constructing the host bacterium comprises the steps of:
directly cloning the gene cluster BGC1.29 of Streptomyces tanashiensis Kala DSM731, adding a conjugation transfer and site-specific recombination element, and integrating the gene cluster BGC1.29 onto the genome of the streptomyces strain through indirect transfer between seeds;
the Streptomyces roseoflavus strain is Streptomyces albus J1074;
at this time, the host bacterium obtained by construction is named Streptomyces albus J1074/BGC1.29; the genotype of the gene is Streptomyces albus J1074, the p15A-apra carries BGC1.29 to be integrated on the S.albus J1074 genome, the genotype of the gene is Streptomyces albus J1074, the p15A-apra carries BGC1.29 to be integrated on the S.albus J1074 genome, and the gene is apomycin resistant.
6. The host bacterium according to claim 3 or 4, wherein the host bacterium is constructed by:
directly cloning the gene cluster BGC1.29 from Streptomyces tanashiensis Kala DSM731, adding a conjugation transfer and site-specific recombination element, replacing a primary promoter with a strong promoter SP44 at the upstream of a terpene synthase gene SdTS29, and integrating the primary promoter into the genome of a streptomycete strain through indirect transfer between species;
the Streptomyces roseoflavus strain can be Streptomyces albus J1074;
at this time, the host bacterium obtained by construction is named Streptomyces albus J1074/P-BGC1.29; the genotype of which is Streptomyces albus J1074, p15A-apra carries the BGC1.29 of the insert promoter integrated into the S.albus J1074 genome, the apramycin resistance, the hygromycin resistance and the constitutive strong promoter SP44.
7. Use of a sesquiterpene synthase encoding gene according to claim 1, a sesquiterpene synthesis gene cluster according to claim 2 or a host bacterium according to any one of claims 3-6 for biosynthesis and/or promotion of biosynthesis of terpenoids;
further, the terpenoid is specifically sesquiterpene alcohol isodauc-8-en-11-ol.
8. A method of biosynthesizing sesquiterpene alcohol isodauc-8-en-11-ol, said method comprising: culturing the host bacterium according to any one of claims 3 to 6, and isolating and purifying to obtain the sesquiterpene alcohol isodauc-8-en-11-ol.
9. The engineering bacterium for detecting the function of terpene synthases in the gene cluster BGC1.29 is characterized in that the engineering bacterium is obtained by transferring pMHI, pFZ81 and a plasmid pGB231-1 obtained by inserting a terpene synthase encoding gene SdTS29 into pGB231 plasmid into an original strain escherichia coli;
the escherichia coli of the initial strain is Escherichia coli BL;
at this time, the obtained engineering bacterium was named Escherichia coli F1 SdTS29 The genotypes of the gene are Escherichia coli BL, pMHI, pFZ81 and pGB231-1 carrying SdTS29 and introduced into BL21, chloramphenicol resistance, kanamycin resistance, a promoter T7 and an inducible promoter Lac;
or the engineering bacteria are obtained by transferring plasmid pGB231-2 obtained by inserting pMHI and pFZ81 and a cytochrome oxidase P450 coding gene SdP into pGB231-1 plasmid SdTS29 downstream of the genes into an original strain escherichia coli;
the escherichia coli of the initial strain is Escherichia coli BL;
at this time, the obtained engineering bacterium was named Escherichia coli F2 SdTS29-SdP450 Genotypes Escherichia coli BL, pMHI, pFZ81 and pGB231-2 carrying SdTS29 and SdP were introduced into BL21, chloramphenicol resistance, kanamycin resistance, promoter T7 and inducible promoter Lac.
10. The application of the engineering bacteria in detecting related coding genes and functions of the gene cluster BGC 1.29.
CN202311109983.8A 2023-08-30 2023-08-30 Streptomyces-derived sesquiterpene synthase encoding gene, genetically engineered bacterium and application thereof Pending CN117106807A (en)

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