Method for enhancing yield of isopentenyl pyrophosphoric acid (IPP) in chlorella through isopentenyl alcohol utilization approach (IUP) and application thereof

Abstract

The invention discloses a method for enhancing the yield of isopentenyl pyrophosphoric acid (I PP) in chlorella through an isopentenyl alcohol utilization pathway (I UP) and application of the method, the isopentenyl pyrophosphoric acid (I PP) is improved by introducing the isopentenyl alcohol utilization pathway (I UP), and a potential paclitaxel synthesis pathway is constructed. The method not only improves the yield of the IPP, but also has the advantages of sustainability and low cost, and can be widely applied to the fields of industrial production and biological medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering and metabolic engineering, and more particularly to a method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) and its application. Background Art

[0002] Terpenoids are a class of natural products with diverse structures and rich functions, possessing important applications in medicine, agriculture, and other fields. For example, paclitaxel is a highly complex diterpenoid originally extracted from the bark of the Pacific yew (Taxus brevifolia). It is a broad-spectrum anticancer drug that stabilizes microtubules and inhibits cell division, thereby preventing the proliferation and spread of cancer cells. However, due to its complex structure and low abundance in nature, paclitaxel's supply has been challenging.

[0003] Currently, the production of terpenoids relies primarily on plant extraction and chemical synthesis. However, plant extraction methods are limited by long plant growth cycles, low yields, and significant environmental impact. While chemical synthesis allows for large-scale production, it presents challenges such as complex processes, high costs, and environmental pollution. Biosynthetic pathways offer an environmentally friendly and efficient alternative, with the most common being the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.

[0004] The MVA pathway, present in archaea, eukaryotes, and some bacteria, involves six enzymatic steps, starting with acetyl-CoA (acetyl-CoA) and ultimately producing isoprenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Although the MVA pathway is common in eukaryotes, its complex enzymatic steps and strict metabolic regulation make it challenging to engineer.

[0005] The MEP pathway, primarily found in bacteria, green algae, and cyanobacteria, comprises seven enzymatic steps, starting with pyruvate and D-glyceraldehyde-3-phosphate (GAP) to produce IPP and DMAPP. Although the MEP pathway is the primary terpenoid precursor biosynthesis pathway in green algae, its poor carbon flux and multiple metabolic bottlenecks limit the efficient synthesis of target products.

[0006] Chlorella is a fast-growing green microalgae with photosynthetic capabilities, capable of synthesizing organic matter using light energy and carbon dioxide. Chlorella's rapid growth allows it to accumulate large amounts of biomass in a short period of time. It can grow in simple culture media, has a small and stable genome, and can be modified using a variety of established genetic engineering tools. It is also highly adaptable and can grow in diverse environmental conditions. These advantages make Chlorella a promising biosynthetic platform for producing high-value chemicals, including terpenoids.

[0007] In Chlorella vulgaris, the production of terpenoids is limited by the availability of the precursors IPP and DMAPP. Although genetic engineering can enhance the enzyme activity of the MEP pathway to increase precursor production, the close connection between the MEP pathway and other cellular metabolic pathways complicates and limits the regulation of metabolic flux. Furthermore, multi-gene engineering in Chlorella faces challenges such as low transgenic efficiency and unstable transgene expression. Therefore, a simpler and more efficient approach is urgently needed to increase IPP production in Chlorella to meet the production needs of high-value terpenoids. Summary of the Invention

[0008] (1) Technical problems solved

[0009] In response to the problems existing in the prior art, the present invention provides a method and application for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella through the isopentenol utilization pathway (IUP), so as to solve the problems of high cost of manual data annotation and high cost of operation and maintenance of multiple model services during medical record information extraction, and at the same time solve the problems of inaccurate medical record extraction and insufficient semantic understanding.

[0010] (2) Technical solution

[0011] To achieve the above object, the present invention provides the following technical solution: a method for increasing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) and its application, comprising the following steps:

[0012] Step 1: Select and optimize codons for the Saccharomyces cerevisiae choline kinase (ScCK) gene and the Arabidopsis thaliana isopentyl phosphate kinase (AtIPK) gene;

[0013] Step 2: Construct a fusion expression vector to fuse the ScCK and AtIPK genes with a fluorescent marker;

[0014] Step 3: introducing the fusion expression vector into Chlorella vulgaris by electroporation;

[0015] Step 4: Use antibiotics to screen transformants with high expression efficiency;

[0016] Step 5: culturing the transformant in a medium supplemented with isopentenol at different concentrations to determine the optimal IPP production conditions;

[0017] Step 6: Measure the IPP production of the transformants.

[0018] The present invention is further configured as follows: wherein the connecting peptide in the fusion expression vector is GSGSGS, which is used to connect the ScCK and AtIPK genes.

[0019] The present invention is further configured as follows: wherein the conversion step specifically comprises:

[0020] a. Mix 1.0 μg of linearized plasmid DNA with 2.5×10^7 Chlorella cells in a medium containing 40 mM sucrose;

[0021] b. Place the cell suspension in a pre-chilled electroporation cuvette and incubate at 16°C for 5 minutes.

[0022] c. Perform electroporation using an electroporator with a voltage of 500 V, a capacitance of 50 μF, and a resistance of 800 Ω;

[0023] d. The electroporated cells were transferred to a medium containing 40 mM sucrose and cultured in the dark for 16 hours.

[0024] The present invention is further configured as follows: wherein the screening step comprises:

[0025] a. The transformed Chlorella was inoculated on a plate containing 15 μg / mL hygromycin;

[0026] b. Cultivate at 25°C under continuous light for approximately 7 days to screen for monoclonal algae strains with antibiotic resistance.

[0027] The present invention is further configured as follows: wherein the concentration of prenol in the culture medium ranges from 0 mM to 25 mM.

[0028] The present invention is further configured as follows: wherein the method for measuring IPP production comprises:

[0029] a. Take 5 ml of cultured Chlorella cell suspension and collect the cells by centrifugation;

[0030] b. Resuspend the cells in cold saline and disrupt them by ultrasonication;

[0031] c. Add 100% methanol to extract IPP;

[0032] d. Purifying the extract using a solid phase extraction column;

[0033] e. The content of IPP was measured by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0034] The present invention also includes a method for enhancing the synthesis of paclitaxel using isopentenyl pyrophosphate (IPP) produced by the method according to claim 1, comprising the following steps:

[0035] a. Efficient production of IPP in Chlorella by the method;

[0036] b. Introducing additional genes required for paclitaxel biosynthesis;

[0037] c. Cultivate Chlorella vulgaris under optimized light-dark cycle conditions to maximize paclitaxel production.

[0038] The present invention is further configured as follows: wherein the light-dark cycle condition is 12 hours of light and 12 hours of darkness.

[0039] (3) Beneficial effects

[0040] Compared with the prior art, the present invention provides a method and application for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP), which has the following beneficial effects:

[0041] The present invention can simultaneously process and analyze multiple types of data, such as data combining text and images, to provide a more comprehensive understanding of medical records. The model architecture and training methods support deployments of different scales, and the capacity and services of the model can be expanded as needed; the end-to-end training method allows the model to learn directly from the raw data to the representation of the final task, reducing dependence on and adjustments in the intermediate stages. The model can continuously learn from new data and constantly adapt to emerging data problems; the model is trained on large-scale and diverse data, giving it better generalization capabilities, and can achieve good training results even in low-sample conditions, reducing dependence on professional data labelers and reducing long-term operating costs. It can adapt to different medical scenarios and tasks. DETAILED DESCRIPTION

[0042] It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0043] Example 1: Selection of target genes

[0044] Two key enzyme genes from different species were selected: the Saccharomyces cerevisiae choline kinase gene (ScCK, GenBank accession no. OP046413) and the Arabidopsis thaliana isopentyl phosphate kinase gene (AtIPK, GenBank accession no. OP046414). Codon optimization was performed simultaneously, and the linker peptide GSGSGSGS was selected to connect ScCK, AtIPK, and EGFP, maintaining normal enzyme folding and function.

[0045] Example 2: Construction of pBI-EGFP expression vector fused with ScCK and AtIPK

[0046] The gene identified and modified in Example 1 was artificially synthesized and cloned into the pBI-EGFP plasmid to construct the pBI-ScCK-GSGSGS-AtIPK-GSGSGS-EGFP expression vector.

[0047] Example 3: Transformation of Chlorella

[0048] The constructed pBI-ScCK-GSGSGS-AtIPK-GSGSGS-EGFP expression vector was introduced into the wild-type Chlorella strain by electroporation. The electroporation method was used in each transformation experiment. The specific steps were as follows: about 1.0 μg of restriction enzyme-treated linearized plasmid DNA was used in each transformation experiment. Chlorella cells were grown in SE medium to about 1.5×10 6 cells / mL. Exponentially growing cells (2.5×10 7 Cells were collected and suspended in 250 μL of culture medium supplemented with 40 mM sucrose. The cell suspension was placed in a pre-cooled disposable electroporation cuvette (4 mm spacing, Bio-Rad) and incubated at 16°C for 5 minutes. Electroporation was then performed using a BioRad Gene Pulser Xcell with the following settings (voltage 500 V, capacity 50 μF and resistance 800 Ω). The electroporated cells were transferred to 5.0 mL of culture medium containing 40 mM sucrose and kept in the dark (10 μmol m -2 s -1 ) for 16 hours.

[0049] The transformed Chlorella vulgaris was inoculated on a plate containing antibiotics (15 μg / mL hygromycin) and the plate was placed under continuous light (50 μmol m -2 s -1 ) and cultured at 25°C. After about 7 days, monoclonal algae strains with antibiotic resistance were observed.

[0050] Example 4: Screening and identification of transformants

[0051] The positive expression strains were identified by PCR. The transcription level of the target gene in the transformants was detected by RT-qPCR to verify the stability of gene expression. The specific steps of PCR were as follows: take an appropriate amount of Chlorella cells (5×10 6 Cells were added to a PCR tube containing 50 μL of 10 mM EDTA (pH 8.0) solution. After vortexing for 5-10 seconds, the tube was incubated at 100°C for 8-10 minutes. Immediately after incubation, the tube was removed and cooled on ice. After cooling, the tube was vigorously vortexed again for 5-10 seconds, and then centrifuged at 14,000 rpm for 1 minute. 1 μL of the supernatant was used as the PCR template.

[0052] Specific steps of RT-qPCR: After RNA is extracted by the Trizol method, its concentration and purity are detected by Nanodrop. Take 1.0 μg RNA and use the RNA reverse transcription kit (Takara) to prepare the reverse transcription reaction system according to the instructions. Incubate the reverse transcription reaction mixture at 45°C for 60 minutes, then incubate at 95°C for 5 minutes, immediately cool on ice and store at -20°C for later use. Take 1.0 μg RNA and prepare the reaction system according to the requirements of the qPCR kit, including primers, probes, DNA templates, polymerases, buffers, etc. Load the reaction system into the thermal cycler and perform PCR cycles according to the set program. Including steps such as DNA denaturation, primer binding, amplification and fluorescence signal detection.

[0053] According to the increase of the fluorescence signal, the threshold cycle number (Ct value) of the PCR cycle stage is analyzed by computer software, and the relative quantity of the target sequence is calculated.

[0054] Example 5: Cultivation of transformants in prenol-supplemented medium

[0055] Prepare SE medium by distributing it into multiple bottles (typically 250 mL or 500 mL Erlenmeyer flasks), with each bottle containing 150 mL of medium. Prepare a series of prenol solutions at different concentrations, for example, 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, and 25 mM. Add these prenol solutions to the pre-prepared SE medium, ensuring that the solutions are evenly mixed. Take 10 mL of the transformed Chlorella cell suspension and collect the cells by centrifugation (3000 rpm for 10 minutes). Resuspend the cell pellet in SE medium without prenol to ensure even distribution among the different concentrations of medium. Inoculate each bottle of prenol-supplemented SE medium with 5 mL of the evenly distributed cell suspension (5 mL per bottle) to a final volume of 150 mL per bottle. Place the inoculated culture bottles in a constant temperature incubator set to 25°C. The illumination conditions were a 12-hour light / 12-hour dark cycle, and the culture was continuously shaken at 120 rpm to ensure adequate mixing and gas exchange. Samples were taken at different time points in the culture (e.g., day 0, day 3, day 5, and day 7). At each sampling, 10 mL of culture medium was taken and centrifuged (3000 rpm, 10 minutes) to collect the cells. The cells were resuspended in 1 mL of cold saline and immediately subjected to cell disruption and IPP extraction.

[0056] Example 5: IPP content determination

[0057] Take 5mL of cultured Chlorella cell suspension, centrifuge (3000rpm, 10 minutes), and discard the supernatant. Resuspend the cell pellet in 1mL of cold saline and repeat washing twice. Place the washed cells in an ice bath and treat with an ultrasonic disruptor (30 seconds of ultrasound, 30 seconds of rest, repeat 5 times). Add 2mL of 100% methanol to the disrupted cell suspension and shake vigorously to mix. Centrifuge at 4°C (12000rpm, 15 minutes) and collect the supernatant. Use a C18 solid phase extraction column, pass the supernatant through the column, wash with 5mL of water first, and then elute with 5mL of 20% acetonitrile aqueous solution. Collect the eluate and concentrate it to 1mL. Configure the HPLC system with mobile phase A as 0.1% formic acid aqueous solution and mobile phase B as acetonitrile. Inject 20uL of sample and use a gradient elution program to separate IPP. Direct the effluent into the mass spectrometer for MRM detection and record the characteristic fragment ions of IPP. Peak areas were calculated by mass spectrometer software, and quantification was performed using an IPP standard curve.

[0058] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP), characterized in that: The following steps are involved: Step 1: Select and optimize codons of the Saccharomyces cerevisiae choline kinase (ScCK) gene and the Arabidopsis thaliana isopentyl phosphate kinase (AtIPK) gene; Step 2: construct a fusion expression vector to fuse the ScCK and AtIPK genes with a fluorescent marker; Step 3: introducing the fusion expression vector into Chlorella by electroporation transformation; Step 4: Use antibiotics to screen transformants with high expression efficiency; Step 5: culturing the transformant in a medium supplemented with isopentenol at different concentrations to determine the optimal IPP production conditions; Step six: measuring the IPP production of the transformants.

2. A method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) according to claim 1, characterized in that: It is stated The connecting peptide in the fusion expression vector is GSGSGS, which is used to connect the ScCK and AtIPK genes.

3. A method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) according to claim 1, characterized in that: The transformation steps specifically include: a. Mix 1.0 μg of linearized plasmid DNA with 2.5×10^7 Chlorella cells in a medium containing 40 mM sucrose; b. Place the cell suspension in a pre-cooled electroporation cuvette and incubate at 16°C for 5 minutes; c. Use an electroporator for electroporation with a voltage of 500 V, a capacity of 50 μF, and a resistance of 800 Ω; d. The electroporated cells were transferred to a medium containing 40 mM sucrose and cultured in dim light for 16 h.

4. A method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) according to claim 1, characterized in that: The screening steps include: a. The transformed Chlorella was inoculated on a plate containing 15 μg / mL hygromycin; b. Cultivate at 25°C under continuous light for about 7 days to screen out monoclonal algae strains with antibiotic resistance.

5. A method for enhancing the production of isopentenyl pyrophosphate (IPP) in Chlorella via the isopentenol utilization pathway (IUP) according to claim 1, characterized in that: It is stated The concentration of prenol in the culture medium ranged from 0 mM to 25 mM.

6. The method according to claim 1, wherein the method for measuring IPP production comprises: a. Take 5 ml of cultured Chlorella cell suspension and collect the cells by centrifugation; b. Resuspend the cells in cold saline and disrupt them with ultrasound; c. Add 100% methanol to extract IPP; d. Purifying the extract using a solid phase extraction column; e. The content of IPP was measured by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

7. A method for enhancing the synthesis of paclitaxel using isopentenyl pyrophosphate (IPP) produced according to the method of claim 1, comprising the following steps: a. Efficiently producing IPP in Chlorella by the method; b. Introduction of additional genes required for paclitaxel biosynthesis; c. Cultivate Chlorella under optimized light-dark cycle conditions to maximize the production of paclitaxel.

8. A method according to claim 7 for enhancing the synthesis of paclitaxel using isopentenyl pyrophosphate (IPP) produced according to the method of claim 1, characterized in that: It is stated The light-dark cycle conditions were 12 h light and 12 h dark.