CN111088254B - Endogenous carried exogenous gene efficient controllable expression system - Google Patents
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Abstract
The invention relates to the technical field of biology, and discloses an endogenously carried exogenous gene high-efficiency controllable expression system, which comprises sequences connected in sequence: the DNA fragments of the host target gene termination codon upstream segment containing the termination codon, the fungal operon gene spacer region, the target gene and the host target gene termination codon downstream segment are connected with the target gene by using the fungal operon gene spacer region sequence, and are inserted into the downstream of the host cell expression gene termination codon by using the fixed-point gene insertion mode, and the transcription expression of the target gene is completed by using the promoter of the target gene. The expression system simplifies experimental operation steps, improves working efficiency, increases controllability and stability of target gene expression, reduces requirements of multi-gene heterologous expression on diversity of known promoters, and has high practical application value in aspects of natural product excavation, yield improvement, non-natural compound creation and the like by utilizing synthetic biology.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a foreign gene fixed-point quantitative timing expression system based on a fungal operon, which comprises the following steps: an endogenously carried exogenous gene high-efficiency controllable expression system.
Background
Natural products are an important source for the creation of modern drugs. However, the difficulty of obtaining natural products with new structures and practical values by using the traditional screening technology is increasing, and in addition, a part of known active natural products have scarce sources, low natural content, complex components and difficult complete separation. The development of genome sequencing technologies and the elucidation of the synthetic pathways for natural products have made it possible to produce these valuable natural products by heterologous biosynthetic techniques. Synthetic biology techniques, with their ability to transfer functional elements and modules "across species", are increasingly becoming the primary means of addressing these problems. At present, based on genome sequencing, the production of important active natural products such as artemisinin precursor arteannuic acid, opioid and the like in a cell factory is completed by utilizing a synthetic biological method.
The genetic background of the fungal underpan cells such as saccharomyces cerevisiae, aspergillus nidulans and the like is clear, and the genetic operation is simpler compared with other eukaryotes. Compared with prokaryotic underpan cells such as escherichia coli and actinomycetes, the biosynthesis enzymes derived from eukaryotes are more easily and successfully expressed in fungi and play the catalytic role. In fungi, genes are independently regulated and transcribed into monocistronic mRNA, and thus the expression of foreign genes therein also needs to be performed in the form of a promoter-gene-terminator module. However, synthesis of natural products usually involves expression of multiple genes, which requires separate promoters and transcription terminators for each gene, resulting in complicated experimental procedures and lengthy target DNA fragments. Especially, under the condition of limited number of the promoters selected at present, the repeated use of the same promoter and terminator is often required for the expression of multiple genes, which greatly increases the probability of homologous recombination of genes in transformants.
In 2015, operon structures were first found in fungi. Yue (Yue Q, Chen L, Li Y, Bills G, Zhang X, Xiaong M, Li S, Chen Y, Wang C, Niu X, An Z, Liu X. 2015. Functional optics in secondary metabolism gene clusters inGlarea lozoyensis(Fungi, Ascomycota, Leotomyces.) mBio6: e 00703-15.) in the production of echinocandin, an important antifungal drugGlarea lozoyensisIn the identification and identification of fungal operonglpks3-glnrps7The intergenic region is 26 bp, and the two are directly translated into 2 proteins after being transcribed into 1 mature bicistronic mRNA. If it is to be operatedThe substructure is applied to the heterologous expression of multiple genes in fungi, so that the length of a target DNA fragment is greatly reduced, experimental operation steps are reduced, and the working efficiency is improved.
Disclosure of Invention
The term "exogenous gene site-directed quantitative timed expression system based on fungal operon" mainly refers to the use of the special intergenic region of the operon to link the host target gene and the exogenous gene, and the term "endogenously carried exogenous gene" in the "highly controllable expression system of endogenously carried exogenous gene" also refers to the use of the special intergenic region of the operon to link the host target gene (endogenous gene) and the exogenous gene ". Therefore, the object of the present invention is to provide a fungal operon-based exogenous gene site-directed quantitative timed expression system, which comprises: an endogenously carried exogenous gene high-efficiency controllable expression system.
The technical scheme of the invention is summarized as follows:
an endogenously carried exogenous gene high-efficiency controllable expression system is characterized by that it utilizes fungal operon gene spacer sequence to connect target gene, and utilizes the gene insertion mode to insert it into the downstream of host cell expression gene stop codon, and under the condition of not destroying expression of original gene (i.e. target gene), utilizes the promoter of target gene to implement transcription expression of target gene.
The technical scheme provided by the invention is as follows:
an operon gene spacer having the sequence 5'-GAGTCAATCAAACACTC-3'.
A fungal operon-based exogenous gene site-directed quantitative timed expression system comprising sequentially linked sequences:
a DNA segment containing the upstream segment of the stop codon of the host target gene, the intergenic region of the fungal operon, the target gene and the downstream segment of the stop codon of the host target gene,
wherein the fungal operon intergenic region has the following sequence:
5 '-CAATCAAAC-3', or 1 to 8 nucleotides added to its 5 'end and 1 to 9 nucleotides added to its 3' end, wherein the addition of nucleotides corresponds to 5'-TGTTGAGTCAATCAAACACTCAACAG-3', preferably 5'-TGTTGAGTCAATCAAACACTCAACAG-3', 5'-GAGTCAATCAAACACTC-3'.
The expression system: a screening marker gene is also connected between the target gene and the DNA fragment of the downstream fragment of the stop codon of the host target gene; the target gene is one or more, preferably 2, 3 or 4.
The expression system is suitable for fungi or plants, wherein the fungi are especially yeasts and filamentous fungi, such as Saccharomyces cerevisiae.
The invention also provides a recombinant bacterium or a recombinant plant containing the operon or the expression system.
The invention also provides the use of a fungal operon intergenic spacer having the following sequence:
5 '-CAATCAAAC-3', or 1 to 8 nucleotides added to its 5 'end and 1 to 9 nucleotides added to its 3' end, wherein the addition of nucleotides corresponds to 5'-TGTTGAGTCAATCAAACACTCAACAG-3', preferably 5'-TGTTGAGTCAATCAAACACTCAACAG-3', 5'-GAGTCAATCAAACACTC-3'.
In the above application, the heterologous expression is a fungus or a plant, wherein the fungus is specifically a yeast, a filamentous fungus, such as Saccharomyces cerevisiae.
Meanwhile, the invention also provides application of the expression system in expressing heterologous genes, which is to transform the expression system into a host to realize expression of the heterologous genes.
Specifically, the embodiments of the present invention are as follows.
1) Determination of fungal operon intergenic region sequence. The original sequence was 5'-TGTTGAGTCAATCAAACACTCAACAG-3' and the basic sequence was 5 '-CAATCAAAC-3'. The preferred sequence is 5'-GAGTCAATCAAACACTC-3'.
2) Designing a primer according to a fungal operon gene spacer region sequence, a target gene sequence, a screening marker gene sequence and a host target gene sequence, amplifying the target gene, the upstream and downstream fragments of a host target gene stop codon and the screening marker gene through PCR, further performing connection and cloning (or amplification) through seamless cloning (or fusion PCR), and finally obtaining a DNA fragment containing a host target gene stop codon upstream fragment (containing a stop codon) -fungal operon gene spacer region-target gene-screening marker gene-host target gene stop codon downstream fragment through PCR.
3) The obtained DNA fragment is transferred into a host cell by transformation, and the targeted insertion of the target gene is performed.
4) Screening the obtained transformant by PCR to obtain a positive transformant expressing the target gene.
The exogenous gene expression system can be used for heterologous expression of eukaryotic genes such as fungi and plants, and has higher practical application value in aspects of natural product excavation, yield improvement, non-natural compound creation and the like by utilizing synthetic biology.
The exogenous gene expression system of the present invention does not need to provide a promoter and a transcription terminator for the target gene alone, and can insert the target gene into the downstream of any gene of a host under the condition of not destroying the original gene expression of the host cell, and the transcription expression of the target gene can be controlled by using the promoter and the terminator of the target gene. The method not only simplifies the experimental operation steps and improves the working efficiency, but also greatly increases the controllability and stability of the target gene expression, reduces the requirement of the multi-gene heterologous expression on the diversity of the known promoter, and greatly reduces the length of the target DNA fragment.
Drawings
FIG. 1 is a schematic diagram of a construction strategy of a foreign gene site-directed quantitative timing expression system (endogenous carried foreign gene high-efficiency controllable expression system) based on a fungal operon.
FIG. 2 comparison of fungal operon spacer regions, wherein (A) the fungal operon spacer sequence and secondary structure; (B) effect of intergenic region sequences on downstream gene expression.
FIG. 3 production of DLD and its methylation products in LtLasS1 and LtLasS2 and HsOMT heterologous expression transformants.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Vector pJET1.2/blunt Cloning Vector: purchased from Thermo Scientific, inc.
Saccharomyces cerevisiae BJ 5464: MAT alpha ura3-52 HIS 3-delta 200 leu 2-delta 1 trp1 pep4 HIS3 prb1 delta 1.6R can1 GAL. American model culture Collection Bank (ATCC, accession number www.atcc.org /), ATCC No. 208288.
Enzyme and kit:
high fidelity DNA amplification MIX, seamless cloning kit purchased from nuozokenza;
DNA loading buffer and DNA marker were purchased from Kangsu century Co;
the DNA purification gel recovery kit is purchased from Corning;
the frozen yeast transformation kit was purchased from YMO RESEARCH Biometrics;
coli DH5 α was obtained from Cisco as a century company;
the reagent for liquid phase detection is chromatographically pure, and other reagents are domestic analytical pure products.
Culture medium:
the E.coli medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). SC--Trp or SC--Trp/-Leu or SC--Trp/-Leu/-ura deficient Medium (1% glucose, 6.7% Difco)TM Yeast Nitrogen Base w/o Amino Acids、-Trp or-Trp/-Leu or-Trp/-Leu/-ura DO Supplement). YPD low-sugar medium (1% yeast extract, 2% Peptone, 1% glucose). If solid culture medium is prepared, 2% agar powder is added.
Example 1 comparison of fungal operon sequence spacers
1. Purpose of experiment
Auxotrophic saccharomyces cerevisiae BJ5464-NpgA is taken as a chassis cell, glycosyltransferase-methyltransferase module coding genes BbGT and BbMT reported earlier by the research team are taken as reporter genes (Xie L, Zhang L, Wang C, Wang X, Xu YM, Yu H, Wu P, Li S, Han L, Gunatilaka AAL, Wei X, Lin M, Molnar I, Xu Y.2018. Methylglycosylation of aromatic amino and phenoolic moieties of drug-lipolysis biorganism, Proc Natl Acad Sci USA 115: E4980-E4989.), base substitution or deletion is designed aiming at a 26 bp gene spacer original sequence, the liquid chromatography is used for detecting the sugar methylation efficiency of the BbGT and the BbMT to the substrate in the positive transformant, and key elements such as motif or secondary structure influencing the translation of downstream genes in a gene spacer region sequence are clarified.
2. The experimental method comprises the following steps:
（1）BbGTandBbMTconstruction of heterologous expression cassettes
Based on the information of the original sequence (5'-TGTTGAGTCAATCAAACACTCAACAG-3') of the 26 bp intergenic region, a series of specific primers containing point mutation or fragment deletion are designed, and the primers are fused with the gene by PCRBbGT、BbMTIs connected with thenADH2The transcription terminator of (1),trp1The promoter and the gene sequence of (a) are connected and in the first geneBbGTThe upstream of the strain is connected with a saccharomyces cerevisiae glucosidase geneEXG1Part of the upstream fragment of (1) intrp1Downstream addition of genesEXG1Finally obtaining a series of fragments of about 4.2 kb with different intergenic sequencesBbGTAndBbMTa heterologous expression cassette.
(2) Yeast transformation
The heterologous expression cassette was transformed into Saccharomyces cerevisiae according to the instructions of the frozen yeast transformation kit. The obtained yeast transformants were verified by PCR. The obtained positive yeast transformant is streaked and cultured in a new SC--Cultured on Trp-deficient medium in an incubator at 30 ℃ for about 2 days.
(3) Fermentation culture
Adopts two-step fermentation technologyFirstly, inoculating a proper amount of yeast transformant thallus to 10 mL of SC--Culturing in Trp liquid defect culture medium at 30 deg.C and 200 rpm for about 16 hr, adding equal volume YPD low sugar culture medium, and adding 0.2 mg kaempferol pure product dissolved in methanol, and culturing for 48 hr; extracting the fermentation product by using ethyl acetate, wherein the ratio of the ethyl acetate to the fermentation liquid is 1: 1; after rotary evaporation, the dry extract was obtained and 1 mL of methanol was used to reconstitute the extract.
(4) Liquid Chromatography (LC) detection:
1 mL of the obtained fermentation product is subjected to high-speed centrifugation and filtration by a 0.22 mu m filter membrane, and then is detected by ultra-high performance liquid chromatography.
The liquid chromatography analyzer is an Agilent 1290 ultra performance liquid chromatography, and the chromatographic column is an Agilent ZORBAX Eclipse Plus C18 RRHD column (1.8 mu m, 50 mm x 2.1 mm); the total flow rate of the mobile phase is 0.35 mL min-1(ii) a The mobile phase is a mixture of a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.1 percent (volume ratio) formic acid aqueous solution, and the mobile phase B is 0.1 percent (volume ratio) formic acid acetonitrile; total elution time was 10 minutes; the elution process is as follows: the volume ratio of the mobile phase B to the mobile phase is linearly increased from 10% to 50% in 0-4 min, the volume ratio of the mobile phase B to the mobile phase is linearly increased from 50% to 95% in 4-8 min, the volume ratio of the mobile phase B to the mobile phase is 95% in 8-9.3 min, and the volume ratio of the mobile phase B to the mobile phase is linearly decreased from 95% to 10% in 9.3-10 min; the column temperature is 40 ℃, the sample injection amount is 2 mu L, and the post-column effluent directly enters mass spectrometry detection without shunting.
3. Experimental results and analysis:
it was found by LC detection that different intergenic sequences influence the expression of the downstream genes, i.e.the efficiency of the sugar methylation of the substrate kaempferol by the corresponding yeast transformants (FIG. 2). Compared with the yeast transformant transformed with the original sequence, the kaempferol sugar methylation efficiency of the gene spacer region only remains a ring structure, namely 5 '-CAATCAAAC-3' (the sequence (2) in figure 2), has no significant change, and when the sequence is further shortened, kaempferol cannot carry out sugar methylation, which indicates that the sequence (2) is the basic sequence of downstream gene expression, namely 5 '-CAATCAAAC-3'. Except that T at position 4 in the sequence No. 2 is changed into A, point mutation in the sequence mostly causes rapid reduction or disappearance of methylation efficiency of kaempferol sugar, and the base in the sequence No. 2 is a key base for downstream gene expression. When the intergenic region is 5'-GAGTCAATCAAACACTC-3' (sequence No. (4) in fig. 2), kaempferol sugar methylation efficiency is significantly improved, so we chose it as the preferred sequence, i.e. 5'-GAGTCAATCAAACACTC-3'.
Example 2, exogenous gene site-directed quantitative timed expression system based on fungal operon endogenously carried exogenous gene highly efficient controllable expression system) in desmethyl-lasiodiplodin (DLD) and its methylation product biosynthetic pathway reconstitution.
1. Purpose of experiment
An auxotrophic saccharomyces cerevisiae BJ5464-NpgA is taken as a chassis cell, an optimized sequence is taken as a gene spacer region, a foreign gene site-directed quantitative and timed expression system (an endogenously carried foreign gene high-efficiency controllable expression system) based on a fungal operon is utilized, and the biosynthesis pathways (Xu Y, Zhou T, Epinosa-arthritis P, Tang Y, Zhan J, Molnar I.2014. insight into the biosynthesis pathway of DLD and methylation products thereof, which are discovered in the early stage of research team of the inventor, are reconstructedSaccharomyces cerevisiaeACS Chem Biol 9:1119-1127 Wang X, Wang C, Duan L, Zhang L, Liu H, Xu YM, Liu Q, Mao T, Zhang W, Chen M, Lin M, Gunatilaka AAL, Xu Y, Molnar I.2019 radial characterization of O-methyl selectivity for combinatorial biological activity of benzene two lactic acid scans, J Am Chem Soc 141:4355-4364 transformants) detecting DLD produced in the positive and its methylated products by LC.
2. The experimental method comprises the following steps:
(1) construction of DLD biosynthesis key enzymes LtLasS1 and LtLasS2 and an oxygen methyltransferase HsOMT heterologous expression cassette.
Selecting expression genes FBA1, TDH3 and EXG1 in saccharomyces cerevisiae as target genes, designing specific primers according to the information of preferred sequences of LtLasS1, LtLasS2, FBA1, TDH3 and EXG1 and gene spacers, obtaining corresponding fragments through PCR, connecting the fragments to pJET1.2/blunt Cloning Vector through multi-fragment seamless Cloning, obtaining DNA fragments of upstream fragments of stop codons of host target genes (containing stop codons) -fungal operon spacers-target genes-screening marker genes-downstream fragments of stop codons of host target genes through colony PCR of positive clones, namely: uFBA1- (4) sequence-LtLasS 1-Trp1-dFBA1, uTDH3- (4) sequence-LtLasS 2-Leu 2-dTDDH 3 and uEXG1- (4) sequence-LtLasS 2-Ura3-dEXG 1.
(2) Yeast transformation
Heterologous expression cassettes for LtLasS1 and LtLasS2 or LtLasS1, LtLasS2 and HsOMT were co-transferred into Saccharomyces cerevisiae according to the instructions of the frozen yeast transformation kit. The obtained yeast transformants were verified by PCR. The obtained positive yeast transformant is streaked and cultured in a new SC--Trp/-Leu or SC--Trp/-Leu/-Cultured on ura-deficient medium in an incubator at 30 ℃ for about 2 days.
(3) Fermentation culture
Adopts a two-step fermentation technology, firstly inoculates a proper amount of yeast transformant thalli to 10 mL of SC--Trp/-Leu or SC--Trp/-Leu/-Culturing in ura liquid defect culture medium at 30 deg.C and 200 rpm for about 16 h, adding equal volume YPD low sugar culture medium, and culturing for 48 h; extracting the fermentation product by using ethyl acetate, wherein the ratio of the ethyl acetate to the fermentation liquid is 1: 1; after rotary evaporation, the dry extract was obtained and 1 mL of methanol was used to reconstitute the extract.
(4) Liquid phase mass spectrum detection:
the same procedure as in example 1 was followed for the detection by liquid chromatography.
3. Experimental results and analysis:
the present inventors successfully detected DLD in yeast-positive transformants expressing ltlas 1 and ltlas 2 by LC and successfully detected 5-oxymethyl-DLD in heterologously expressing positive transformants expressing ltlas 1, ltlas 2 and HsOMT (fig. 3). The application of the exogenous gene site-directed quantitative timing expression system (endogenously carried exogenous gene high-efficiency controllable expression system) based on fungal operon to the multi-gene heterologous expression is also the successful application of the system to the combined biosynthesis.
Claims (11)
1. An operon gene spacer having the sequence 5'-GAGTCAATCAAACACTC-3'.
2. An endogenously-carried exogenous gene high-efficiency controllable expression system, which comprises sequences connected in sequence:
a DNA segment containing the upstream segment of the stop codon of the host target gene, the intergenic region of the fungal operon, the target gene and the downstream segment of the stop codon of the host target gene,
wherein the fungal operon intergenic region has a sequence:
5 '-CAATCAAAC-3', or 5'-GAGTCAATCAAACACTC-3'.
3. The expression system according to claim 2, wherein a selection marker gene is further ligated between the DNA fragments of the target gene and the downstream region of the stop codon of the host target gene.
4. The expression system of claim 2, wherein the target gene is one or more.
5. The expression system of claim 2, wherein the expression system is suitable for fungi.
6. The expression system of claim 5, wherein the expression system is suitable for yeast, filamentous fungi.
7. A recombinant bacterium comprising the operon spacer of claim 1 or the expression system of any one of claims 2 to 5.
8. Use of a fungal operon intergenic spacer for heterologous expression, wherein the fungal operon intergenic spacer has the sequence: 5 '-CAATCAAAC-3', or 5'-GAGTCAATCAAACACTC-3'.
9. Use according to claim 8, wherein the heterologous expression is in a fungus.
10. Use according to claim 9, wherein the fungus is a yeast, filamentous fungus.
11. Use of an expression system according to any one of claims 2 to 6 for the expression of a heterologous gene by transforming the expression system into a host to effect expression of the heterologous gene.
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| WO2021088370A1 (en) | 2021-05-14 |
| CN111088254A (en) | 2020-05-01 |
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