Eukaryotic polycistron expression element obtained through machine learning guidance

Abstract

The invention discloses a eukaryotic polycistron expression element obtained through machine learning guidance. The expression element can perform tandem expression on a plurality of genes in eukaryotes such as fungi, plants and the like, and realizes control of cooperative expression or inhibition of the plurality of genes through one promoter and one transcription terminator, so that the length of an exogenous multi-gene target DNA fragment is shortened, and the working efficiency is improved. The method has relatively high practical application value in the aspects of natural product mining, yield improvement, creation of non-natural compounds, improvement of disease and pest resistance and the like by utilizing synthetic biology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a eukaryotic polycistronic expression element obtained under the guidance of machine learning. Background Art

[0002] With the continuous advancement of genetic engineering technology, its chassis cells have transitioned from simple prokaryotes such as Escherichia coli to complex eukaryotic organisms such as Saccharomyces cerevisiae and even animals and plants, such as the Saccharomyces cerevisiae cell factory for artemisinic acid and opioids, and "golden rice" containing β-carotene (Beyer, P. (2010) Golden Rice and 'Golden' cropsfor human nutrition. New Biotechnol 27, 478-481; Galanie et al. (2015) Complete biosynthesis of opioids in yeast. Science 349, 1095-1100; Paddon et al. (2013) High-level semi-synthetic production of the potent antimalarialartemisinin. Nature 496, 528-532.). In eukaryotic organisms, gene expression is usually carried out in the form of promoter-gene-terminator. However, whether it is the reconstruction of the natural product biosynthetic pathway or the optimization of traits, it usually requires the coordinated expression of multiple genes. For example, the biosynthesis of hydrocodone requires 23 genes. Given the limited number of promoters currently available, heterologous synthesis using Saccharomyces cerevisiae requires repeated use of the same promoter and terminator, increasing the probability of genetic recombination in engineered cells and genetic instability.

[0003] In 2021, Xu Yuquan and others identified two intergenic gap expression elements that can be used to construct polycistronic IGG ), and based on this, a highly efficient and controllable expression system of endogenous exogenous genes was constructed (Xu Yuquan, Yue Qun, Zhang Liwen, A highly efficient and controllable expression system of endogenous exogenous genes, ZL201911072118.4, authorization announcement date: November 23, 2021). Although this method greatly reduces the length of the target DNA fragment, reduces the experimental operation steps, and improves work efficiency. However, the number of existing expression elements is small, and a large number of repeated use will have the risk of homologous recombination. In addition, the most efficient ELISA test found IGGThe expression of downstream genes mediated by the element (5'-CAATCAAAC-3') is 55% of its endogenous "driver" gene, which needs to be further improved (Yue et al. (2023) A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nature communications 14, 4267.). IGG components to provide more stable and efficient IGG Elements are crucial for using synthetic biology to produce and increase the yield of active natural products and enhance resistance to pests and diseases in eukaryotic organisms such as Saccharomyces cerevisiae and corn. Summary of the invention

[0004] The purpose of the present invention is to provide a eukaryotic multicistronic expression element to provide a guarantee for the expression of multiple genes in eukaryotic organisms.

[0005] The present invention directs evolution of the patent sequence 5'-CAATCAAAC-3', places it between two genes, connects the stop codon of the upstream gene and the start codon of the downstream reporter gene, constructs a bicistronic, and selects clones with high fluorescence intensity by blue light to obtain efficient IGG element.

[0006] A eukaryotic polycistronic expression element obtained under the guidance of machine learning, wherein the expression element is IGG13 , IGG34 , IGG133N One or more of the above; the nucleotide sequence of IGG13 is 5'-CAATCAAAG-3'; the nucleotide sequence of IGG34 is 5'-CAACTAACA-3'; the nucleotide sequence of IGG133N is 5'-CAACCAACT-3'.

[0007] The number of open reading frames in the polycistronic is 2 or more.

[0008] The expression elements are suitable for use in fungi or plants.

[0009] The fungus is a yeast or a filamentous fungus.

[0010] The plant is a monocotyledonous plant, and the monocotyledonous plant is corn or rice.

[0011] A recombinant bacterium or a recombinant plant comprising the eukaryotic multicistronic expression element.

[0012] The application of the eukaryotic multicistronic expression element in heterologous expression.

[0013] Beneficial effects of the present invention: The expression element of the present invention IGG It can be used for heterologous expression of genes in eukaryotic organisms such as fungi and plants, and has high practical application value in the use of synthetic biology to explore natural products, increase yields, create non-natural compounds, and enhance resistance to pests and diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For different IGG Expression of the downstream gene (GFP) linked to the element in Saccharomyces cerevisiae.

[0015] Figure 2 For different IGG The upstream gene to which the element is connected ( GFP ) and downstream genes ( eYFP ) Expression in maize protoplasts; a nuclear localization signal NLS is fused to the N-terminus of GFP, and a chloroplast localization signal peptide CTP is fused to the N-terminus of YFP. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] The vector pJET1.2 / blunt Cloning Vector used in the following examples: purchased from ThermoScientific; pCas, pScURA3, and psgtRNA, see Zhang et al. (2019) (A gRNA-tRNA arrayCRISPR-Cas9 based rapid multiplexed genome editing in Saccharomycescerevisiae. Nat. Commun. 10, 1053.); Saccharomyces cerevisiae CEN.PK 2-1D: MATα; ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8C; SUC2.

[0018] Enzymes and kits: High-fidelity DNA amplification MIX and seamless cloning kit were purchased from Novozymes; DNA loading buffer and DNA marker were purchased from Cosmed; DNA purification gel recovery kit was purchased from Thermo; frozen yeast transformation kit was purchased from YMO RESEARCH; Escherichia coli DH5α competent cells were purchased from Cosmed; other reagents were domestic analytical grade products.

[0019] Culture medium: E. coli culture medium is LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). SC-Ura defective medium (2% glucose, 6.7% DifcoTM Yeast Nitrogen Base w / o AminoAcids, -Ura DO Supplement). 2% YPD medium (1% yeast extract, 2% Peptone peptone, 2% glucose). If making solid culture medium, add 2% agar powder.

[0020] Example 1 IGG Construction and screening of component library 1. Purpose of the experiment Saccharomyces cerevisiae CEN.PK 2-1D For chassis cells, with constitutive genes TDH3 As the endogenous target gene, GFP The patent sequence 5'-CAATCAAAC-3' was subjected to full-site saturation mutation as the reporter gene. Cells were sorted according to the fluorescence intensity of GFP in the transformants using a blue light lamp and flow cytometer to obtain cells with different efficiencies. IGG element.

[0021] 2. Experimental Methods (1) GFP Construction of heterologous expression cassette Replace the patent sequence 5'-CAATCAAAC-3' with 5'-NNNNNNNNN-3', design specific primers, and obtain donor DNA by one-step PCR: TDH3 Stop codon upstream fragment-FLAG-stop codon- IGG-GFP-TDH3 The fragment downstream of the stop codon.

[0022] (2) Yeast transformation According to the method described by Yue et al. (2023), pCAS-sgRNA TDH3and its corresponding donors were electroporated into CEN.PK2-1D (Yue et al. (2023) A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nature communications 14, 4267.) and cultured in a 30°C incubator for 7 days.

[0023] (3) GFP fluorescence intensity test Observe the transformant plate under blue light, pick a single colony with green fluorescence and plate it on a new SC- - Streak on Ura-deficient medium and culture in a 30°C incubator for about 2 days. IGG Primers were designed upstream and downstream of the element, and colony PCR was performed on the streaked colonies. IGG Sequence information. Pick the yeast transformants and inoculate them into 4 mL SC- - Ura liquid deficiency medium was cultured at 30℃ and 220 rpm for 24 h as seed medium. 2% was inoculated into 4 mL 2% YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h. After washing twice, the GFP fluorescence intensity was detected by an ELISA instrument.

[0024] After blue light screening, all colonies were collected and plated in SC- - After culturing in Ura medium for 24 hours, the fluorescence intensity of GFP was detected by CytoFLEX flow cytometer (Backman Coulter, USA), and the cells were sorted according to the fluorescence intensity of GFP. The starting strain was used as a reference for the non-fluorescent group, and the cells were divided into a non-fluorescent group and a fluorescent group. IGG Primers were designed upstream and downstream of the element to control the PCR product within 250 bp, and colony PCR was performed on the collected cells. After sequencing by Illuminanovaseq PE150, sequence data with different efficiencies were obtained.

[0025] 3. Experimental results and analysis Through observation under blue light, a total of 201 single colonies with green fluorescence were selected. After sequencing, it was found that most of the colonies had point mutations or fragment deletions, forming a fusion protein of TDH3 and GFP. Only 2 were constructed with the normal TDH3 stop codon upstream fragment-FLAG-stop codon-IGG-GFP-TDH3 stop codon downstream fragment, and their GFP fluorescence intensity was higher than that of the transferred patent sequence (5'-CAATCAAAC-3', IGG2 ) of yeast transformants (Figure 1 ). When base 9 mutates to G, i.e. 5'-CAATCAAAG -3' ( IGG13 ) when the GFP fluorescence intensity increased significantly. IGG2 1.28 times of the original; bases 4 and 5 mutated to C and T, and bases 8 and 9 mutated to C and A, i.e. 5'-CAACTAACA-3' ( IGG34 ), the GFP fluorescence intensity is IGG2 1.17 times of that.

[0026] After sequencing by Illumina Novaseq PE150, a total of 58,061 valid IGG Sequence, 107378 invalid IGG sequence.

[0027] Example 2 IGG Data analysis 1. Purpose of the experiment calculate IGG The minimum folding energy (MFE) of the upstream and downstream sequences of the element was combined with the fluorescence intensity of the corresponding yeast transformants for machine learning to identify the efficient IGG Components for prediction.

[0028] 2. Experimental Methods (1) IGG Calculation of the minimum folding energy MFE of the upstream and downstream sequences of the element All calculations were performed using the Vienna software package (ViennaRNA version 2.7.0). The extracted mRNA sequence window (based on which the ΔGfold calculation of the window sequence was performed) followed the following two constraints: first, in order to simulate the mRNA secondary structure and ensure complete coverage of the sample sequence, the starting position of the window was extended downward from the first nucleotide of the sample sequence; second, the window size used was 40 nucleotides. A 93 nt sample was used: including the 42 nt sequence at the end of the upstream gene (including the localization signal sequence, etc.), a 9 nt long IGG sequence, and the starting 42 nt sequence of the downstream gene (including the localization signal sequence, etc.).

[0029] The dataset was constructed with the calculated MFE values ​​at different positions as factors and the downstream GFP fluorescence expression levels (no fluorescence, low fluorescence, and high fluorescence) as output labels. The classifier model of factors and output labels was constructed using the multi-classification random forest (RF) algorithm in the Scikit-learn Python library. To obtain the best model parameters, the average accuracy of the classifier was measured by 10-fold cross validation. In each trial, a small part of the data was removed from the training set. The parameters and ranges tested were: n_estimators = 200, 500, 100, max_depth = 5, 10, 100, min_samples_split = 2,5, 10, min_samples_leaf = 1, 2, 4 and max_leaf_nodes = 10, 50, 100. The optimal parameter combination was used for the construction of the formal model. In order to evaluate the accuracy of the obtained classifier and avoid overfitting, 20% of the dataset was randomly selected as the validation set, and the remaining 80% was used as the test set. The accuracy of the classifier is evaluated using the roc_curve and roc_auc_score methods from Scikit-learn’s metrics module.

[0030] (2) Efficiency IGG Component prediction Calculate 102368 randomly generated 9 nt long IGG The MFE value of the sequence is used to predict these IGG The sequence mediates the level of expression of downstream genes.

[0031] 3. Experimental results and analysis Through IGG The calculation of the minimum folding energy MFE of the upstream and downstream sequences of the element found that the sample length was insensitive to this parameter after it was greater than 90 nt, and the window calculation results were stable over the entire reasonable range. Therefore, this window size was used to generate the presented results.

[0032] The 10-fold cross validation accuracy of the random forest classifier was 0.73±0.23, and the AUC value of ROC was 0.93. Based on the constructed random forest classifier, 102,368 items were predicted according to the MFE value. IGG The sequence mediates the level of downstream gene expression and the possibility of each level, including 86443 IGG The sequence is non-fluorescent, 4533 sequences are low fluorescent, and 11445 are high fluorescent. The probability of fluorescence level satisfies none < low < high IGG In the sequence, the probability of no fluorescence is as low as possible and the probability of high fluorescence is as high as possible, and they are sorted and one of them is selectedIGG sequence IGG133N Conduct follow-up experiments.

[0033] Table 1 IGG133N The prediction results of the random forest model

[0034] Example 3 Using IGG34 , IGG13 , IGG133N Transient expression of linked genes in maize Purpose The protoplasts isolated from maize B73 leaves were used as the chassis cells. GFP (with nuclear localization signal peptide) and eYFP (with chloroplast localization signal peptide) as the reporter gene, the IGG13 , IGG34 and Example 2 IGG133N And control IGG2 Transient expression analysis of maize protoplasts was performed to measure the fluorescence intensity and subcellular localization of GFP and eYFP in successfully transformed maize protoplasts to obtain different IGG Component efficiency in corn.

[0035] Experimental methods (1) Construction of pCG-GFP-IGG-eYFP plant expression vector by IGG2 (5'-CAATCAAAC-3') as an example, specific primers were designed and obtained by overlapping PCR. GFP-IGG2- eYFP Fragments, seamlessly clone GFP-IGG2-eYFP The fragment was constructed into the pCG vector to form the pCG-GFP-IGG2-eYFP vector. The promoter used for the reporter gene was the 35S promoter, and the terminator was the NOS terminator. The pCG-GFP-IGG13-eYFP vector, pCG-GFP-IGG34-eYFP vector, and pCG-GFP-IGG133N-eYFP vector were constructed in the same way.

[0036] (2) Maize protoplast transformation The pCG-GFP-IGG-eYFP series vectors were transformed into maize protoplasts according to the method described by Li et al. (2015) (Li et al. (2015) The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles. The Plant Journal 84(2): 296-308.) and cultured in a 23°C incubator overnight.

[0037] (3) Subcellular localization observation and GFP and eYFP fluorescence intensity test The corn protoplasts transformed in the previous step were observed under an LSM980 laser confocal microscope, and protoplast cells with green fluorescence and yellow fluorescence were selected and photographed to determine their subcellular localization and measure the fluorescence intensity of GFP and eYFP.

[0038] Experimental results and analysis Using LSM980 laser confocal microscope, three successfully transformed protoplasts were selected for each transformation vector. The subcellular localization results showed IGG2 , IGG13 , IGG34 and IGG133N The linked GFP and eYFP can be accurately localized to the nucleus and chloroplasts, respectively ( Figure 2 ). The fluorescence intensity of GFP and eYFP was measured respectively, and the fluorescence ratio of eYFP to GFP was used to compare the different IGG Efficiency. The results show IGG13 , IGG34 and IGG133N The ratio of eYFP / GFP was significantly higher than IGG2 Of which IGG13 The ratio of eYFP / GFP is IGG2 4.04 times, IGG34 The ratio of eYFP / GFP is IGG2 2.14 times, IGG133N The ratio of eYFP / GFP is IGG2 3.36 times of.

[0039] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A eukaryotic polycistronic expression element obtained by machine learning, characterized in that: The expression element is IGG13 , IGG34 , IGG133N One or more of the IGG13 The nucleotide sequence is 5'-CAATCAAAG-3'; IGG34 The nucleotide sequence is 5'-CAACTAACA-3'; IGG133N The nucleotide sequence is 5'-CAACCAACT-3'.

2. The eukaryotic multicistronic expression element obtained by machine learning guidance according to claim 1, characterized in that: The number of open reading frames in the polycistronic is 2 or more.

3. The eukaryotic multicistronic expression element obtained by machine learning guidance according to claim 1, characterized in that: The expression elements are suitable for use in fungi or plants.

4. The eukaryotic multicistronic expression element obtained by machine learning guidance according to claim 3, characterized in that: The fungus is a yeast or a filamentous fungus.

5. The eukaryotic multicistronic expression element obtained by machine learning guidance according to claim 3, characterized in that: The plant is a monocotyledonous plant, and the monocotyledonous plant is corn or rice.

6. A recombinant bacterium or recombinant plant comprising the eukaryotic multicistronic expression element of claim 1.

7. Use of the eukaryotic multicistronic expression element of claim 1 in heterologous expression.

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