System and microorganism for synthesizing sesterterpene serpentin F and application thereof
阅读说明：本技术 合成二倍半萜蛇孢菌素f的系统和微生物及其应用 (System and microorganism for synthesizing sesterterpene serpentin F and application thereof ) 是由 洪葵 袁薇 邓子新 于 2019-09-09 设计创作，主要内容包括：本发明公开了一种合成二倍半萜蛇孢菌素F的系统和微生物及其应用。本发明优化了培养条件,发酵结果显示：五碳糖阿拉伯糖和木糖对比原发酵条件中甘油能显著增加蛇孢菌素F的产量；真菌来源基因倍半萜合成酶Au6298,融合标签SUMO和Trx和通过定点突变蛇孢菌素合成酶D87E,E95V,Q122D,R131K和H190F也能显著提高蛇孢菌素F的产量。本发明构建的微生物能稳定产生蛇孢菌素F的培养物,为后续进一步利用微生物产生蛇孢菌素F后修饰产物奠定了基础。(The invention discloses a system and a microorganism for synthesizing sesterterpene sporothrin F and application thereof. The invention optimizes the culture condition, and the fermentation result shows that: the five-carbon sugar arabinose and xylose can obviously increase the yield of the ophiosporin F compared with the glycerol in the original fermentation condition; the fungal source gene sesquiterpene synthetase Au6298, fusion tags SUMO and Trx and the yield of the snake sporophytin F can also be obviously improved by site-directed mutagenesis of snake sporophytin synthetase D87E, E95V, Q122D, R131K and H190F. The microorganism constructed by the invention can stably produce the culture of the snake spore bacillus F, and lays a foundation for further producing the modified product of the snake spore bacillus F by utilizing the microorganism.)
1. A system for synthesizing sesterterpene serpentin F is characterized in that: comprises the following components: synthesis related gene of snake sporophycin or its functional equivalent; the snake sporosporine synthesis related gene is an Au8003 gene optimized by a codon, and the nucleotide sequences are respectively shown in SEQ ID NO. 1; the said snake spore bacterin synthesis related gene or its function common body is set on the same carrier or different carriers;
further comprising: fungus TKYX429 derived sesquiterpene synthetase Au6298, Au13192, Au11565 or Au3446 genes; fusing labels SUMO and Trx; one or more of the mutant D87E, E95V, Q122D, R131K and H190F of the snake spore synthetic enzyme.
2. A microorganism which synthesizes sesterterpene sporothricin F, characterized in that: the microorganism comprising the system for synthesizing sesterterpene serpentin F according to claim 1; the microorganism is any one of filamentous fungi, yeast, streptomycete, bacillus or escherichia coli.
3. The microorganism for the synthesis of sesterterpene serpentin F according to claim 2, characterized in that: the microorganism is one or more of nucleic acid sequences of the synthesis system for sesterterpene serpentin F according to claim 1 integrated in genome DNA; the microorganism is one or more of escherichia coli containing plasmids pWHU4003, pWHU4004-pWHU4009, pWHU4014-pWHU4016 or pWHU4003(1) - (13): the plasmid promoters are strong T7 promoters, replicons are pBBR322 replicons with medium copy number and all contain codon-optimized snake sporosin synthetase genes; the carbon source of the culture medium for producing the ophiosporin F by the microorganism comprises pentose, arabinose, xylose or ammonium tartrate, citric acid, glycerol and glucose.
4. Use of a system for the synthesis of sesterterpene serpentin F according to claim 1 for the production of a serpentin.
5. Use of a microorganism as defined in claim 2 or 3 for the synthesis of sesterterpene serpentin F in the production of a serpentin.
Technical Field
The invention belongs to the field of synthetic biology, genetic engineering and enzyme engineering, and particularly relates to a system and a microorganism for synthesizing sesterterpene serpentin F and application thereof.
Background
The class of ophiosporins (Ophiobolins, ophs) are sesterterpenoids with a characteristic 5-8-5 tricyclic mother nucleus structure (Chiba R, Minami A, Gomi K, Oikawa H. identification of ophiobolin Fsynthsase by a genetic minor approach: a sesterpen synthase from Aspergillus clavatus. organic letters,2013,15(3): 594) 597.). By 2019, 56 natural product-derived ophs compounds were reported (TianW, Deng Z, Hong K. the biological activity of the segmented-Type ophiobolins [ J ]. Marine drugs,2017,15(7): 229; Zhu T, Lu Z, Fan J, et al. The compounds show antitumor, broad-spectrum antibacterial, nematicidal and antiviral activity (Tian W, Deng Z, Hong K. the biological activities of sesterterpinenid-Type ophiobolins [ J ]. Marine drugs,2017,15(7): 229.). The Ophs backbone synthesis is a chimeric terpene synthase synthesis and is called chimeric because it has two domains: a chain extension domain responsible for the synthesis of the five carbon precursor isopentenyl pyrophosphate and dimethylallyl pyrophosphate as nilylgeranyl pyrophosphate (GFPP); a cyclization domain capable of cyclizing GFPP to a 5-8-5 tricyclic oph mother nucleus (Chiba R, Minami A, Gomi K, Oikawa H (2013) Identification of an opiurobacterial F synthase by a genetic engineering approach: a segaterepene synthase from Aspergillus clavatus. organic letters,2013,15(3): 594-597.). In the native fungus, the yields of ophs are not only related to oph synthetases, but also to the sesquiterpenes Au6298, the diterpenes Au13192 and Au11565, the triterpene Au3446 synthetase-associated gene clusters (Chai H, YinR, Liu Y, et al.
The current production methods of terpenoids mainly comprise 3 methods: extraction method, chemical synthesis method and biological synthesis method. The isolation and chemical synthesis of Ophs from the original strain presents practical difficulties to supply this class of compounds in large quantities. First, the wild-type fungus TKYX429 is capable of producing ophs compounds, but there are several difficulties if analyzed from the perspective of industrial strains: the strain can synthesize other types of compounds besides the ophs product, such as metabolic products of sesquiterpene, isochroman, sterol, cyclodipeptide and the like, so that the strain is not beneficial to separating and obtaining oph monomeric compounds with high purity; secondly, the synthesized ophs homologues have complex components, which leads to particular difficulties in the preparation of monomeric compounds; again, the solid fermentation process results in the raw fermentation material easily remaining in the final product, thereby increasing the difficulty of later separation and purification (Chai H, Yin R, Liu Y, et. Sesterterpine thermophilic biosynthesis enclosing multiple genes Cluster in Aspergillus oryzae [ J ]. Scientific reports,2016,6: 27181.). Since the first paper 1977 published a framework for oph synthesis, the total synthesis of ophs has been limited to laboratory scale over decades (Brill Z G, Grover H K, maione TJ. organic selective synthesis of an animal tissue virus a programmed radial cassette [ J ] Science,2016,352(6289):1078 and 1082.). Other strategies are therefore needed to supply inexpensive quantities of oph compound.
The synthesis of terpenoids using synthetic biological approaches with heterologous hosts has indicated ways to drive the industrialization of this class of compounds (George KW, Alonso-Guiierrez J, Keasling JD, Lee TS Isoprenoid drivers, biofials, and chemicals-artemisinins, farnesene, andbyeyond. in: Schrader J., Bohlmann J. (eds) Biotechnology of Isoprenoid. adv. biochem. Eng./Biotechnology, vol148.Springer, Cham,2015: 355-389.). Terpenoids are heterogeneously expressed in escherichia coli chassis cells by a large group of subjects at home and abroad. Jay Keasling teaches laboratories to perform the biosynthesis of sesquiterpene artemisinin precursors via the synthetic pathway of the mevalonate pathway in Escherichia coli and Liu Tian gang teaches overexpression of the lycopene-related synthetic pathway in Escherichia coli (Martin V J J, Pitera D J, Withers S T, et al. engineering a metabolic pathway in Escherichia coli for production of terpene [ J ]. Nature biotechnology 2003,21(7): 796; Zhu F, Lu L, Fu S, et al. targeted engineering anode up of lysine production in Escherichia coli [ J ]. Process Biochemistry 2015,50(3): 346 341).
Coli expression due to the lack of post-translational processing mechanisms in the system, insoluble inclusion bodies are formed during the expression of foreign proteins due to incorrect folding of the protein. According to the specification of expression vector pET series of Novagen company, the proportion and the activity of the soluble part of the target protein can be obviously improved by correctly selecting a proper vector and host bacterium combination. For example, thioredoxin (TrxA) has a function of catalyzing reduction of protein disulfide bonds, can help correct folding of proteins, and can increase solubility of foreign proteins through co-expression; the small molecule ubiquitin-like modified protein (SUMO) is a molecular chaperone, has a structure similar to ubiquitin, wherein beta is a globular folding structure with a sheet layer wound with alpha layer spiral, has a highly hydrophobic core, and provides a site for folding of fusion protein, so that the solubility and the stability of foreign protein can be improved. According to the previous literature, the only fusion protein selected by chimeric terpene synthase is glutathione-S-transferase (GST), which is classical but has a large protein tag and easily affects the enzymatic activity (Chen M, Chou W K W, Toyomasu T, et al.Structure and function of biochemical pathway, a heterocyclic biochemical pathway [ J ]. ACS chemical biology 2016,11(4): 889-899.). This can result in: the fusion of other suitable protein labels is very beneficial to improve the heterologous expression yield of the compounds.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a system and a microorganism for synthesizing sesterterpene sporotrichostatin F and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a system for synthesizing sesterterpene serpentin F, characterized in that: comprises the following components: synthesis related gene of snake sporophycin or its functional equivalent; the snake sporosporine synthesis related gene is an Au8003 gene optimized by a codon, and the nucleotide sequences are respectively shown in SEQ ID NO. 1; the said snake spore bacterin synthesis related gene or its function common body is set on the same carrier or different carriers;
further comprising: fungus TKYX429 derived sesquiterpene synthetase Au6298, Au13192, Au11565 or Au3446 genes; fusing labels SUMO and Trx; one or more of the mutant D87E, E95V, Q122D, R131K and H190F of the snake spore synthetic enzyme.
In a second aspect, the present invention provides a microorganism which synthesizes sesterterpene sporothricin F, characterized in that: the microorganism comprising the system for synthesizing sesterterpene serpentin F according to
Further, the microorganism is a microorganism having one or more of the nucleic acid sequences of the synthesis system for sesterterpene serpentin F according to
In a third aspect, the invention provides the application of the system for synthesizing sesterterpene serpentin F in producing the serpentin.
In a fourth aspect, the present invention provides the use of a microorganism as described above for the synthesis of sesterterpene sporothricinF in the production of a sporothricin.
The fungus TKYX429 source Au8003 gene is optimized in codon in an escherichia coli system and has a base sequence in a sequence table SEQ ID NO. 1.
The system produced in the present invention comprises a gene involved in the synthesis of a snake sporocin or a functional equivalent (isofunctional gene) thereof.
The microorganism is at least one selected from the group consisting of bacteria, fungi, actinomycetes, spirochetes, mycoplasmas, chlamydia, rickettsiae, viruses and yeasts. The microorganism has a precursor supply pathway. Preferably, Escherichia coli comprising plasmid pWHU 4003; the plasmid pWHU4003 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID No. 2.
The strain for producing the snake spore mycin F is introduced into a culture medium containing a carbon source for fermentation to obtain the snake spore mycin F. The carbon source is preferably glycerol, glucose, ammonium tartrate, citric acid, arabinose or xylose.
The culture medium is preferably one of the following culture media:
2 xTYGly: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 20mL/L glycerol, pH 7.2;
2 xTYGlu: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L glucose, pH 7.2;
2 xTYAm: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L ammonium tartrate, pH 7.2;
2 xTYCit: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L citric acid, pH 7.2;
2 xTYRaA: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L arabinose, pH 7.2;
2 xTYXylo: 16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L xylose, pH 7.2;
the culture medium is 2 xTYRaA (16g/L tryptone, 10g/L yeast extract, 5g/L sodium chloride, 15.2g glycerol, 10g/L arabinose, pH 7.2).
Pyrophosphoric acid intermediates C15(Au6298), C20(Au11565 and Au13192) or C30(Au3446) of different chain extension lengths were supplied.
The genes related to the synthesis of C15(Au6298), C20(Au11565 and Au13192), C30(Au3446) and oph or the functional common bodies thereof are arranged on the same carrier or carriers different from each other.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4004; the plasmid pWHU4004 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID No. 3.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4005; the plasmid pWHU4005 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID No. 4.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4006; the plasmid pWHU4006 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID No. 5.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4007; the plasmid pWHU4007 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID NO. 6.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4008; the promoter of the plasmid pWHU4008 is a strong promoter of T7, the replicon is a replicon which is only a moderate copy replicon of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID NO. 7.
The microorganism for producing the ophiosporin F contains escherichia coli of a plasmid pWHU 4009; the plasmid pWHU4009 promoter is a strong T7 promoter, the replicon is a replicon with only moderate copies of pBBR322, and the sequence of the replicon-free vector is shown in SEQ ID No. 8.
The microorganism for producing the ophiosporin F contains Escherichia coli of a plasmid pWHU 4004.
Fusion protein modification oph synthetase: a fusion tag of glutathione mercaptotransferase (GST), small molecule ubiquitin related modified protein (SUMO) and thioredoxin (Trx) is adopted to modify oph synthetase so as to improve folding or soluble expression of the protein.
The microorganism for producing the ophiosporin F is escherichia coli of a plasmid pWHU 4014; the plasmid pWHU4014 promoter is a strong T7 promoter, the replicon is a replicon with medium copy pBBR322 only, and a sequence without a framework vector is shown in SEQ ID No. 9.
The microorganism for producing the ophiosporin F is escherichia coli of a plasmid pWHU 4015; the plasmid pWHU4015 promoter is a strong T7 promoter, the replicon is a replicon with medium copy pBBR322 only, and a sequence without a framework vector is shown in SEQ ID No. 10.
The microorganism for producing the ophiosporin F is escherichia coli of a plasmid pWHU 4016; the plasmid pWHU4016 promoter is a strong T7 promoter, the replicon is a replicon with medium copy pBBR322 only, and the sequence of the replicon-free vector is shown in SEQ ID No. 11.
The SUMO fusion modified snake sporoderm F synthesis related gene or the functional community thereof is co-expressed.
Site-directed mutagenesis of the snake spore mycin synthetase: the amino acid preference of the oph synthetase was derived based on the phylogenetic-based method using the NCBI database. Based on the comparison with Au8003 protein, 13 points to be mutated, S38A, D87E, E95V, Q122D, R131K, A134Q, L144I, H190F, M224L, Y231F, Y231W, N238A and A274L are found. Thus, strains with improved yield were found from the mutants.
The microorganism for producing the snake sporocin F has mutant sites of snake sporocin synthetase in S38A, D87E, E95V, Q122D, R131K, A134Q, L144I, H190F, M224L, Y231F, Y231W, N238A and A274L.
The mutant strains D87E, E95V, Q122D, R131K and H190F.
The invention aims to increase the expression of any gene or functional equivalent thereof in the system by improving the ability of producing the snake spore bacterin F.
In conclusion, the invention has the following advantages and beneficial effects:
(1) the invention utilizes escherichia coli engineering bacteria to highly express sesterterpene compound staurosporine F.
(2) By optimizing the carbon source of the culture medium, the synthesis amount of the snake sporophycin is increased and obvious, wherein the five-carbon sugar arabinose and xylose are most obvious.
(3) The invention utilizes terpene synthetases derived from fungus TKYX429, wherein Au6298, Au13192 and Au11565 obviously improve the yield of the snake sporophycin F.
(4) In order to increase the soluble expression of the bifunctional enzyme Au8003, three GST, SUMO and Trx fusion tags are selected, wherein the SUMO is most significant in improving the yield of the snake sporosporins.
(5) Compared with the traditional mutant library establishment, the mutant strains to be screened based on phylogeny have fewer numbers and high positive rate (38%).
Drawings
FIG. 1 is a structural diagram of a common precursor of the serpentine compounds of the present invention, i.e., serpentine F;
FIG. 2 is a schematic diagram of plasmid pWHU4003 of the present invention;
FIG. 3 is a GC-MS spectrum of a snake sporocin F of the invention;
FIG. 4 is a standard curve for the quantification of snake sporocin F according to the present invention;
FIG. 5 is a diagram of carbon source optimization according to the present invention;
FIG. 6 is a schematic diagram of plasmid pWHU4004 of the present invention;
FIG. 7 is a schematic diagram of plasmid pWHU4005 of the present invention;
FIG. 8 is a schematic diagram of plasmid pWHU4006 of the present invention;
FIG. 9 is a schematic diagram of plasmid pWHU4007 of the present invention;
FIG. 10 is a schematic diagram of plasmid pWHU4008 of the present invention;
FIG. 11 is a schematic diagram of plasmid pWHU4009 of the present invention;
FIG. 12 is a graph showing the effect of the fungal-derived synthetic pathway-related genes of the present invention on the yield of cyclosporin F;
FIG. 13 is a schematic diagram of plasmid pWHU4014 of the present invention;
FIG. 14 is a schematic diagram of plasmid pWHU4015 of the present invention;
FIG. 15 is a schematic diagram of plasmid pWHU4016 of the present invention;
FIG. 16 is a graph showing the effect of modification of a snake-sporin synthase by a fusion protein of the present invention on the production of snake-sporin F;
FIG. 17 is a graph showing the effect of the phylogenetically directed mutant cyclosporin synthetases of the present invention on the production of cyclosporin F.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, but the present invention should not be construed as being limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Unless otherwise specified, the oligonucleotide primers of the present invention were synthesized by Wuhan Pongjiu biotech GmbH, and the DNA sequencing was performed by Wuhan Pongjiu biotech GmbH. The competent cells Top10 and BL21(DE3) were purchased from Hakken Biotech Co., Ltd, the restriction enzymes were purchased from NEB Co., Ltd, the T4 ligase was purchased from Takara bioengineering Co., Ltd, and the plasmid extraction, gel recovery and enzyme digestion system recovery were carried out using the reagent kit of omega Bio-Tek Co., Ltd, according to the instructions. Ampicillin, kanamycin and chloramphenicol antibiotics were purchased from Biosharp, and media components such as glycerol, glucose, ammonium tartrate, citric acid, arabinose and xylose were purchased from national drug group chemical agents, ltd.
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