Method for biosynthesizing erythritol directly using carbon dioxide and application thereof

Abstract

The application provides a method for biosynthesizing erythritol directly by using carbon dioxide and application thereof, and belongs to the field of bioengineering and synthetic biology. The application utilizes homologous recombination system for transformation, integrates phosphatase and erythrose reductase genes related to erythritol synthesis into the chromosome of wild type cyanobacteria, so as to obtain genetically engineered cyanobacteria capable of efficiently producing erythritol directly by using CO2. The application not only can directly use carbon dioxide as raw material, effectively solves the problem of raw material, but also is safe in production, so that environment-friendly, low-cost and sustainable synthesis of erythritol becomes a reality.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and synthetic biology, specifically relating to a method for the direct biosynthesis of erythritol using carbon dioxide and its application. Background Technology

[0002] Currently, the main carbon sources for microbial erythritol production are glucose and glycerol. Compared to glucose, glucose yields a higher erythritol production rate but produces more byproducts; while glycerol, although yielding a slightly lower rate, produces fewer byproducts, resulting in a higher ratio of erythritol to byproducts. Jeya et al. utilized different carbon sources, such as glucose and glycerol, to produce erythritol from strains... P. Tsukuba Comparative studies on erythritol production using KN75 revealed that the highest concentration of erythritol was produced when glucose was used as the carbon source. Mironczuk et al. used glycerol as a substrate for fermentation... Y. lipolytica Wratislavia K1 can produce erythritol with byproduct content below 10%. However, the production of erythritol using glucose and glycerol as substrates is costly, thus the discovery and utilization of novel carbon sources to replace these two have attracted much attention from researchers. With the development of synthetic biology and metabolic engineering, cyanobacteria are considered a highly promising microbial photosynthetic platform for developing photosynthetic cell factories and achieving one-stop targeted conversion of CO2 into biofuels and bio-based chemicals. Sugars are a representative class of cyanobacterial photo-driven carbon fixation synthesis products. Through the remodeling of photosynthetic metabolic networks, conversion routes for CO2 into various monosaccharides, oligosaccharides, polysaccharides, and sugar alcohols have been established. For example, Wu et al. introduced different sucrose isomerases into cyanobacteria. Synechococcus elongatus PCC 7942 achieved the synthesis and accumulation of isomaltulose in recombinant strains. Li Wei et al. encoded the gene for xylitol phosphate dehydrogenase. xpdh Import into cyanobacteria Synechococcus elongatus In PCC 7942, a pentose phosphate extension pathway was constructed in cyanobacteria, successfully creating an engineered cyanobacterial strain capable of producing xylitol using CO2. Madsen et al. derived from... Micromonas pusilla The fusion enzymes (containing mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase) were found in Escherichia coli and cyanobacteria, respectively. Synechococcus Heterologous expression in sp. PCC7002 enabled the synthesis of mannitol. However, research on the fermentation production of erythritol using cyanobacteria as a photosynthetic platform and CO2 as the sole carbon source is still scarce.

[0003] This invention utilizes a genetic engineering strategy to introduce genes related to erythritol synthesis into cyanobacteria, enabling the research on erythritol synthesis and alleviating the problem of expensive precursors or raw materials currently required. This strategy of constructing a photosynthetic platform utilizing CO2 and solar energy makes environmentally friendly, low-cost, and sustainable erythritol synthesis a reality, broadening the pathways for compound production using photosynthetic platforms. Summary of the Invention

[0004] This invention provides a method for biosynthesizing erythritol using carbon dioxide as a raw material and its application. This method not only effectively solves the raw material problem, but also ensures safe, environmentally friendly, low-cost, and sustainable production, providing a new approach to effectively improve the biosynthesis of erythritol.

[0005] The first objective of this invention is to provide a method for the direct biosynthesis of erythritol using carbon dioxide, the specific steps of which are as follows: First, the gene sequences yidA and SD, which encode phosphatase, are fused with the gene ER, which encodes erythritol reductase. Then, the fused fragment yidA-SD-ER is inserted into plasmid pAM2991 to construct the recombinant plasmid pAM-yidA-SD-ER. Finally, the recombinant plasmid pAM-yidA-SD-ER is integrated into the chromosome of wild-type cyanobacteria to obtain genetically engineered cyanobacteria capable of producing erythritol.

[0006] Furthermore, the gene yidA encoding the phosphatase is derived from... Escherichia coli K-12, the SD sequence is derived from plasmid pETDuet-1, and the gene ER encoding erythrose reductase is derived from... White magnolias DSM 70638.

[0007] Furthermore, the nucleotide sequence of the fusion fragment yidA-SD-ER is shown in SEQ ID NO.1.

[0008] Furthermore, the fusion fragment yidA-SD-ER was obtained using overlap PCR.

[0009] Furthermore, the genetically engineered cyanobacteria uses plasmid pAM2991 as an expression vector.

[0010] Furthermore, the plasmid pAM2991 contains a spectinomycin resistance selection gene and an IPTG-induced P gene. trc Promoter.

[0011] Furthermore, the recombinant plasmid is transformed using natural transformation, where the exogenous gene... Synechococcus elongatedHomologous recombination occurs at the neutral locus (NS) on chromosome PCC 7942.

[0012] Furthermore, the aforementioned Synechococcus elongatus The NS site on PCC 7942 chromosome is a neutral site I (NSI), which is also present in plasmid pAM2991.

[0013] The second objective of this invention is to provide a method for the direct biosynthesis of erythritol using carbon dioxide, and its application in the production of erythritol. The specific steps are as follows: Transformants PM were selected and placed in a suitable culture environment with appropriate temperature and light intensity for continuous aeration experiments, utilizing S... Elongated ynechococcus PCC 7942 was used as a blank control. Genetically engineered cyanobacteria PM and S were cultured. ynechococcus elongated PCC 7942's OD 730 Adjust the pH to 0.4-0.6, add 50-100 μL of 100 mmol / L IPTG for induction, and take samples every 5 days for several consecutive days. Before each sampling, add BG-11 liquid medium to 50-100 mL. Take two tubes each time; retain one tube for measuring erythritol production, and measure the other tube for OD. 730 value.

[0014] Furthermore, the culture temperature is 25-30℃.

[0015] Furthermore, the light intensity is 1500-2500 lux.

[0016] Furthermore, the gas used during continuous ventilation is a mixture of 1-5% CO2 and 95-99% air.

[0017] Further, the formulation of the BG-11 culture medium is as follows: NaNO3 (1.5-7.5 g), K2HPO4 (0.04-0.35 g), MgSO4·7H2O (0.075-0.5 g), CaCl2·2H2O (0.036-0.18 g), Citric acid (6×10⁻⁶ g), NaNO3 (1.5-7.5 g), K2HPO4 (0.04-0.35 g), MgSO4·7H2O (0.075-0.5 g), CaCl2·2H2O (0.036-0.18 g), CaCl2·2H2O (6×10⁻⁶ ... -3 -6×10 -2 g), Ferric ammonium citrate (6×10 -3 -6×10 -2 g), EDTANa2 (5×10 g) -4 -5×10 -3 g), Na2CO3 (0.02-1 g), A5 (Trace mental solution): H3PO4 (2.9×10 -3 -2.9×10-2 g), MnCl2·4H2O (1.9×10 g) -3 -1.9×10 -2 g), ZnSO4·7H2O (2.2×10 g) -4 -2.2×10 -3 g), Na2MoO4·2H2O (3.9×10 -4 -3.9×10 -3 g), CuSO4·5H2O (8×10 g) -5 -8×10 -4 g), Co(NO3)2·6H2O (5×10 -5 -5×10 -4 For solid culture medium, add 2% agar powder, then bring the volume to 1 L with distilled water, adjust the pH to 7.1 with 1 mol / L NaOH or 1 mol / L HCl, and sterilize at 121℃ for 20 min.

[0018] Furthermore, the method for detecting erythritol yield adopts GB 5009.279—2016 and high performance liquid chromatography.

[0019] Furthermore, the high performance liquid chromatography (HPLC) conditions are as follows: Shimadzu LC-2030C HPLC system, differential display: RID-20A, column: Waters Sugar-Pakl, mobile phase: ultrapure water, flow rate: 0.5 g / L, column temperature: 80℃.

[0020] Unless otherwise specified, all reagents and materials used above are commercially available. Beneficial effects

[0021] 1. This invention involves genetically modifying cyanobacteria to reconstruct the erythritol synthesis pathway within them. The resulting genetically engineered cyanobacteria can directly convert solar energy and CO2 into erythritol. This invention not only solves the problem of raw material sourcing but also ensures safe production, making environmentally friendly, low-cost, and sustainable erythritol synthesis a reality.

[0022] 2. In a specific embodiment of the present invention, after several tens of days of cultivation, the engineered cyanobacteria can produce erythritol at a concentration of more than 1.5 g / L, which opens up a new technology for the production of erythritol and has good prospects for industrial application. Attached Figure Description

[0023] Figure 1 The image shows the recombinant plasmid pAM-yidA-SD-ER of Example 1 of this invention.

[0024] Figure 2The enzyme digestion verification results are for the recombinant plasmid pAM-yidA-SD-ER in Example 1 of this invention. M represents DNA Marker, L1 represents single enzyme digestion verification of plasmid pAM2991, L2 represents single enzyme digestion verification of recombinant plasmid pAM-yidA-SD-ER, L3 represents double enzyme digestion verification of plasmid pAM2991, and L4 represents double enzyme digestion verification of recombinant plasmid pAM-yidA-SD-ER.

[0025] Figure 3 Example 2 of the present invention: Genetically engineered bacteria PM and blank control. Synechococcus elongatus The graph shows the change in erythritol production of PCC7942 over time. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described in detail below with reference to examples. These examples are used to further illustrate the content of the present invention, but do not limit the scope of protection of the present invention. All related content and modifications involved in the present invention fall within the scope of protection of the present invention.

[0027] Blue algae Synechococcus elongatus PCC 7942 is frequently used as a model strain in related experimental studies. The plasmid pAM2991 is used for integration into [the target strain] via homologous recombination. Synechococcus elongatus The target gene is expressed in the chromosome of PCC 7942.

[0028] The PCR amplification systems involved in the examples were all 25-50 μL: ①5×PrimeSTAR Buffer (Mg 2+ ① dNTP Mixture: 5-10 μL; ② upstream primer F (10 µmol / L): 2-4 μL; ③ downstream primer R (10 µmol / L): 0.5-1 μL; ④ genomic template (100-200 ng / µL): 0.5-1 μL; ⑤ Primer F (10 µmol / L): 0.25-0.5 μL; ⑥ Sterilized ddH2O to bring the total volume to 25-50 μL.

[0029] PCR amplification conditions: ① 98℃: 3 min; ② 98℃: 15 s; ③ Annealing temperature (depending on the specific primer pair): 15 s; ④ 72℃: 1 kb / min (return to ②, for a total of 10-30 cycles); ⑤ 72℃: 3 min; ⑥ 4℃: ∞.

[0030] Example 1: Construction of erythritol-producing genetically engineered cyanobacteria Construction of recombinant plasmid pAM-yidA-SD-ER extract Escherichia coli K-12 genomic DNA; The sequence of the phosphatase-corresponding gene yidA is shown in SEQ ID NO.2. Primer pairs were designed and synthesized. The sequence of the upstream primer yidA-F1 is shown in SEQ ID NO.4, and the sequence of the downstream primer yidA-R1 is shown in SEQ ID NO.5. Using the genomic DNA from step (1) as a template, PCR amplification was performed using the primers synthesized in step (2) to obtain the DNA fragment of the phosphatase corresponding gene yidA. The reaction system and conditions are as described above. extract White magnolia Genomic DNA of DSM 70638; The ER sequence of the gene corresponding to erythrose reductase is shown in SEQ ID NO.3. Primer pairs were designed and synthesized. The upstream primer ER-F1 sequence is shown in SEQ ID NO.6, and the downstream primer ER-R1 sequence is shown in SEQ ID NO.7. Using the genomic DNA from step (4) as a template, PCR amplification was performed using the primers synthesized in step (5) to obtain the DNA fragment of the erythrose reductase corresponding gene ER. The reaction system and conditions are as described above. Overlap PCR primer pairs were designed and synthesized. The upstream primer yidA-F2 for the yidA gene is shown in SEQ ID NO. 8, and the downstream primer yidA-R2 is shown in SEQ ID NO. 9. The upstream primer ER-F2 for the ER gene is shown in SEQ ID NO. 10, and the downstream primer ER-R2 is shown in SEQ ID NO. 11. Using the yidA gene obtained in step (3) as a template, PCR amplification was performed using the primers yidA-F2 and yidA-R2 synthesized in step (7) to obtain a gene with restriction enzyme sites. Echo The RI and SD sequences, as well as the fragment yidA that overlaps with the 5' end of the ER gene. + The reaction system and conditions are as shown in the instruction manual. Using the ER gene obtained in step (6) as a template, PCR amplification was performed using the primers ER-F2 and ER-R2 synthesized in step (7) to obtain a sequence with an overlap with the 3' end of the yidA gene, an SD sequence, and restriction enzyme sites. Bam HI fragment ER + The reaction system and conditions are as described above; The fragment yidA obtained in step (8) + and fragment ER +Using this as a template, PCR amplification was performed without primers to obtain the fusion fragment yidA-SD-ER. The reaction system and conditions were as described above. The fusion fragment yidA-SD-ER obtained in step (9) was diluted 5-10 times. Using the diluted fusion fragment yidA-SD-ER as a template, PCR amplification was performed using the primers yidA-F2 and ER-R2 synthesized in step (7) to obtain a large number of fusion fragments yidA-SD-ER. The reaction system and conditions are as described above. The plasmid pAM2991 and the fusion fragment yidA-SD-ER obtained in step (10) were respectively processed with restriction endonucleases. Echo RI and Bam Double digestion with HI was performed, and the digested plasmid and fusion fragment were recovered using the AxyPrep DNA Gel Extraction Kit. The required amounts of vector fragment and PCR product fragment were calculated based on a molar ratio of 1:3-6 between the recovered vector fragment and the recovered PCR product fragment. Ligation was then performed overnight at 4°C using Novizan's T4 ligase to obtain the recombinant plasmid pAM-yidA-SD-ER.

[0031] The recombinant plasmid pAM-yidA-SD-ER obtained in step (11) was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with those in GeneBank.

[0032] Primer sequences (SEQ ID NO.4~11) yidA-F1: 5'-ATGGCTATTAAACTCATTGCTATCGA-3' yidA-R1:5'-TTAATTCAGCACATACTTCTCAATAGCA-3' ER-F1: 5'-ATGTCTTCGACCTACACCCTTACTC-3' ER-R1: 5'-TCACCGTCTTGCTAGCGCG-3' yidA-F2:5'-ccg gaattc ATGGCTATTAAACTCATTGC-3' yidA-R2: 5'-CGAAGACATggtatatctccttcttTTAATTCAGCACATACTT-3' The lowercase letters marked with underscores represent the enzyme cleavage sites. Echo RI, the lowercase letters without underscores indicate the SD sequence.

[0033] ER-F2: 5'-CTGAATTAAaagaaggagatataccATGTCTTCGACCTACACC-3' ER-R2: 5'-gcg ggatcc TCACCGTCTTGCTA-3' The lowercase letters without underscores are SD sequences, while the lowercase letters with underscores are restriction enzyme sites. Bam HI.

[0034] Constructing genetically engineered cyanobacteria Transformation of cyanobacterial strains: Take 5-10 mL of cyanobacterial cells in the logarithmic growth phase, centrifuge at 4000-5000 rpm / min for 5-10 minutes to collect the cells, wash once with sterile 10 mM NaCl, and then resuspend the cells in 5-10 mL of BG-11 liquid medium. Transfer 500-750 μL of the bacterial suspension to a 1.5 mL centrifuge tube, add recombinant plasmid to a final concentration of 100-200 ng / mL, mix well, and incubate at 28-30℃ in the dark for 12-16 hours. Then, spread the mixture of cyanobacterial cells and recombinant plasmid onto BG-11 solid medium containing 20-30 µg / mL spectinomycin, and incubate under continuous illumination at 28-30℃ and 2000-2200 lux until single colonies appear on the solid medium.

[0035] (2) Obtaining genetically engineered cyanobacteria: Transformants were picked and placed in 5-10 mL of BG-11 liquid medium containing 20 µg / mL spectinomycin, and cultured continuously under light intensity of 2000-2200 lux at 28-30℃ for 10-12 days. Genomic DNA of the genetically engineered cyanobacteria was then extracted using the Ezup column-based bacterial genomic DNA extraction kit. PCR verification and sequencing were then performed using primers yidA-F2 and ER-R2 for amplification of the exogenous gene on the recombinant plasmid to obtain the genetically engineered cyanobacteria PM.

[0036] Example 2: Production of erythritol using genetically engineered cyanobacteria PM (1) Culture of recombinant strains: Transformants PM were selected and placed at 28-30℃ under a light intensity of 2000-2200 lux for continuous ventilation experiments. Elongated ynechococcus PCC 7942 was used as a blank control. Genetically engineered cyanobacteria PM and S were cultured. Elongated ynechococcus PCC 7942's OD 730Adjust the pH to 0.4-0.6, add 50-100 μL of 100 mmol / L IPTG for induction, and take samples every 5 days for a total of 30 days. Before each sampling, add BG-11 liquid medium to a final volume of 50-100 mL. Take two samples each time; retain one sample for erythritol production measurement and measure the OD in the other. 730 value.

[0037] (2) Sample preparation: After heating the sample taken in step (1) at 95-100℃ for 10-15 min, centrifuge at 4℃ and 10000-12000 rpm / min for 10-15 min, filter with a special filter membrane for water phase, and test at 4℃.

[0038] (3) Sample detection: The sample obtained in step (2) was detected by high performance liquid chromatography. The peak time of erythritol standard was 13.1 min. Genetically engineered cyanobacteria PM showed a peak at this time, confirming that the obtained sample was erythritol, and that more than 1.5 g / L of erythritol could be accumulated within several tens of days. Figure 3 Blank control Synechococcus elongatus PCC 7942 did not show a peak at this time, and no erythritol production was detected within 30 days. Figure 3 The high performance liquid chromatography (HPLC) conditions were as follows: Shimadzu LC-2030C HPLC system, differential display: RID-20A, column: Waters Sugar-Pakl, mobile phase: ultrapure water, flow rate: 0.5 g / L, column temperature: 80℃.

Claims

1. A method for directly biosynthesizing erythritol using carbon dioxide, characterized in that, First, the gene yidA and SD encoding phosphatase were fused with the gene ER encoding erythritol reductase. The nucleotide sequence of the fusion fragment yidA-SD-ER is shown in SEQ ID NO.

1. Then, the fusion fragment yidA-SD-ER was inserted into plasmid pAM2991 to construct the recombinant plasmid pAM-yidA-SD-ER. Finally, the recombinant plasmid pAM-yidA-SD-ER was integrated into the chromosome of a wild-type cyanobacterium to obtain a genetically engineered cyanobacterium capable of producing erythritol. The wild-type cyanobacterium is Synechococcus elongatus PCC7942.

2. The method for directly biosynthesizing erythritol using carbon dioxide according to claim 1, characterized in that, The SD sequence is derived from plasmid pETDuet-1.

3. The application of the method for direct biosynthesis of erythritol using carbon dioxide according to any one of claims 1-2 in the production of erythritol.

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