A method for producing succinic acid by aerobic fermentation of escherichia coli

Abstract

The application discloses a method for producing succinic acid by Escherichia coli aerobic fermentation, and belongs to the field of bioengineering. In the application, sdhA, iclR, icd, poxB and ptsG are continuously knocked out in Escherichia coli BL21 (DE3), and gltA-aceA is constitutively overexpressed in the engineering bacteria, so that the yield of succinic acid reaches 4.49 g / L in shake flask fermentation of the engineering bacteria B6-gltA-aceA, reaches 85.6% of the theoretical conversion rate, and the yield is increased by 855.3% compared with that of the genetic engineering bacteria B1, and the accumulation amount of acetic acid is 0.24 g / L, which is beneficial to the industrialized production of succinic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing succinic acid by aerobic fermentation of Escherichia coli, belonging to the field of bioengineering technology. BACKGROUND

[0002] Succinic acid, also known as succinic acid, has a molecular weight of 118.088 and is a colorless crystal. It is an important metabolic intermediate in the tricarboxylic acid cycle of living organisms and is also an important C4 platform compound. It is often used as a starting material for the synthesis of general chemicals, such as 1,4-butanediol, tetrahydrofuran, succinonitrile, succinic anhydride, and 2-pyrrolidone, etc. Therefore, it is widely used in the fields of food, medicine, and agriculture. In addition, as an important organic synthesis intermediate, succinic acid is widely used in the synthesis of a variety of degradable polyesters, such as polybutylene succinate (PBS) and polyhexylene succinate (PHS), etc. Compared with other biodegradable plastics, PBS not only has excellent mechanical properties, but also has a very reasonable price and a large market demand. Many countries and regions around the world have begun to implement "ban on plastic, limit on plastic" policies, and biodegradable materials have ushered in a good development opportunity. Experts estimate that the annual demand for PBS in China will exceed 3 million tons. Based on the calculation that 0.62 tons of succinic acid are needed to produce 1 ton of PBS, 1.8 million tons of succinic acid are needed annually. According to global succinic acid production capacity data, China's succinic acid production capacity was about 55,000 tons in 2021, accounting for 48% of the global market. Due to technical and cost constraints, the current market size of succinic acid is still relatively low, and the production capacity is far from meeting market demand.

[0003] With the rapid development of metabolic engineering and synthetic biology, the use of small-pollution, low-cost, and sustainable biological methods to produce succinic acid has attracted widespread attention from researchers. Escherichia coli is selected as the most potential succinic acid-producing strain due to its clear genetic background, ease of modification, ease of cultivation, and utilization of carbon sources. Under aerobic conditions, cells can grow rapidly and accumulate a large amount of bacterial cells, but succinic acid cannot be accumulated under aerobic conditions, and the addition of a large amount of glucose during fermentation can easily lead to acetic acid overflow. Therefore, under the condition of adding substrate glucose, it is a technical problem that needs to be solved to make Escherichia coli accumulate succinic acid while growing rapidly and reduce the accumulation of by-products such as acetic acid. SUMMARY

[0004] The present application is to knock out one or more of the sdhA, sdhB, iclR, icd, poxB, and ptsG genes in Escherichia coli BL21 (DE3) through metabolic engineering, and to use pCDFDuet-1 as an expression vector to constitutively overexpress the citrate synthase gene (gltA) and isocitrate lyase gene (aceA) derived from Escherichia coli. This allows Escherichia coli to accumulate a large amount of succinic acid under aerobic conditions and reduce the content of by-products such as acetic acid.

[0005] The first object of the present application is to construct an engineered Escherichia coli for aerobic production of succinic acid, wherein the engineered Escherichia coli is constructed by knocking out the genes sdhA (succinate dehydrogenase), iclR (transcription factor), icd (isocitrate dehydrogenase), poxB (pyruvate dehydrogenase), and ptsG (pyruvate dehydrogenase) in the Escherichia coli and overexpressing the genes gltA (citrate synthase) and aceA (isocitrate lyase) in the Escherichia coli.

[0006] The present application also provides a method for constructing the engineered Escherichia coli, comprising at least one of the following steps:

[0007] (1) knocking out the gene sdhA (succinate dehydrogenase) shown in SEQ ID NO. 1 in Escherichia coli BL21 (DE3) by the method of CRISPR / CAS9, and the obtained engineered Escherichia coli BL21 (DE3) ΔsdhA with the deleted sdhA gene is named B1.

[0008] (2) knocking out the gene sdhB (succinate dehydrogenase) shown in SEQ ID NO. 2 in Escherichia coli BL21 (DE3) ΔsdhA by the method of CRISPR / CAS9, and the obtained engineered Escherichia coli BL21 (DE3) ΔsdhA ΔsdhB with the deleted sdhB gene is named B2.

[0009] (3) knocking out the gene iclR (transcription factor) shown in SEQ ID NO. 3 in Escherichia coli BL21 (DE3) ΔsdhA by the method of CRISPR / CAS9, and the obtained engineered Escherichia coli BL21 (DE3) ΔsdhA ΔiclR with the deleted iclR gene is named B3.

[0010] (4) knocking out the gene icd (isocitrate dehydrogenase) shown in SEQ ID NO. 4 in Escherichia coli BL21 (DE3) ΔsdhA ΔiclR by the method of CRISPR / CAS9, and the obtained engineered Escherichia coli BL21 (DE3) ΔsdhA ΔiclR Δicd with the deleted icd gene is named B4.

[0011] (5) knocking out the gene poxB (pyruvate dehydrogenase) shown in SEQ ID NO. 5 in Escherichia coli BL21 (DE3) ΔsdhA ΔiclR Δicd by the method of CRISPR / CAS9, and the obtained engineered Escherichia coli BL21 (DE3) ΔsdhA ΔiclR Δicd ΔpoxB with the deleted poxB gene is named B5.

[0012] (6) Knock out the gene ptsG (phosphotransferase system II) as shown in SEQ ID NO. 6 in E. coli BL21 (DE3) ΔsdhA ΔiclR Δicd ΔpoxB by CRISPR / CAS9 method, and the obtained E. coli engineering strain BL21 (DE3) ΔsdhA ΔiclR Δicd ΔpoxB ΔptsG with the deleted poxB gene is named as B6.

[0013] (7) Construct the plasmids pJUN-gltA, pJUN-aceA and pJUN-gltA-aceA for strengthening the glyoxylate pathway: take pCDFDuet-1 as the plasmid backbone, and take the constitutive promoter PUTRinfc-rplt as shown in SEQ ID NO. 9 to express gltA as shown in SEQ ID NO. 7, aceA as shown in SEQ ID NO. 8 and gltA-aceA, respectively, and the plasmid map is as shown in SEQ ID NO. 10. Figure 1 Transform the plasmids pJUN-gltA, pJUN-aceA and pJUN-gltA-aceA into the E. coli engineering strain B6, respectively, and the obtained strains are named as B6-gltA, B6-aceA and B6-gltA-aceA, respectively.

[0014] A second object of the present application is to provide a method for producing succinic acid by aerobic fermentation using the constructed E. coli engineering strain.

[0015] Cultivate the E. coli engineering strain at 30-40°C for 10-24h, inoculate into a 250mL triangular flask containing 50mL of M9 modified medium at an inoculation amount of 1-3%, and place the flask at 30-40°C with a rotation speed of 200-250r / min to perform shake flask fermentation for producing succinic acid.

[0016] In an embodiment of the present application, the M9 modified medium contains, in terms of g / L, Na2HPO46-8, KH2PO42-4, NH4Cl 0.5-1.5, NaCl 0.5-1, MgSO40.15-0.3, CaCl20.1-0.2, tryptone 7-8, yeast powder 2-3 and glucose 8-9.

[0017] The present application also provides the use of the E. coli engineering strain or the method in aerobic fermentation for preparing succinic acid or a product containing succinic acid.

[0018] Advantages

[0019] The present application can accumulate succinic acid in large quantities and reduce the content of by-product acetic acid under aerobic conditions by continuously knocking out sdhA, iclR, icd, poxB and ptsG genes in E. coli BL21 (DE3) through metabolic engineering, and using pCDFDuet-1 as an expression vector to constitutively overexpress the citrate synthase gene (gltA) and isocitrate lyase gene (aceA) derived from E. coli. The yield of succinic acid of the recombinant bacteria B6-gltA-aceA constructed by the present application reaches 4.49 g / L after 48 h fermentation, which is increased by 855.3% compared with B1, and reaches 85.6% of the theoretical conversion rate; the accumulation of acetic acid is 0.24 g / L, which is beneficial to the industrialized production of succinic acid. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the aerobic synthesis pathway of succinic acid and the related plasmid map of the present application.

[0021] Figure 2 The figure is the shake flask fermentation results of all strains of the present application. DETAILED DESCRIPTION

[0022] The culture medium used in the present application is as follows:

[0023] The seed culture medium is LB medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0024] The LB solid culture medium is LB medium with the addition of 1.8% agar powder.

[0025] The LB solid culture medium with different resistances is LB solid culture medium with the addition of corresponding antibiotics with a final concentration of 50 μg / mL.

[0026] The fermentation medium is M9 modified medium (g / L): Na2HPO4 6.78, KH2PO4 3, NH4Cl 1, NaCl 0.5, MgSO4 0.24 (sterilized separately), CaCl2 0.115 (sterilized separately), tryptone 8, yeast powder 2, glucose 8 (sterilized separately).

[0027] The strains and plasmids used in the present application are shown in Table 1, and the primer sequences used for knocking out genes are shown in Table 2.

[0028] Table 1 All strains and plasmids of the present application

[0029]

[0030]

[0031]

[0032] Table 2 Primers designed for knocking out genes in the present application

[0033]

[0034]

[0035]

[0036]

[0037] The present application quantitatively detects glucose, succinic acid and acetic acid by high performance liquid chromatography: 1 mL of fermentation broth is centrifuged at 12000 rpm for 5 min, and the supernatant is passed through a 0.22 μm filter membrane. The glucose detector is a differential detector, and the detection of succinic acid and acetic acid uses an ultraviolet detector (210 nm). The detection parameters are: column temperature 50℃, mobile phase 5mM dilute sulfuric acid, flow rate 0.6 mL·min -1 , injection volume 10 μL.

[0038] Calculation of succinic acid conversion rate: 1 mol of glucose is theoretically converted to 1 mol of succinic acid in aerobic fermentation, i.e., 8 g / L of initial sugar, 5.24 g / L of theoretical succinic acid production, and a theoretical conversion rate of 100%; the actual conversion rate is the percentage value of the actual yield of succinic acid to the theoretical yield, expressed as follows:

[0039] Succinic acid conversion rate (%) = (succinic acid yield / 5.24)*100

[0040] Example 1: Construction of recombinant strains

[0041] (I) Construction of E. coli engineering bacteria B1, B2, B3, B4, B5 and B6

[0042] 1. Construction of E. coli engineering bacteria B1

[0043] Three pairs of N20 sequences of sdhA gene were designed, and pTarget-F was used as a template. After PCR using sdhAn201-F / R, sdhAn202-F / R and sdhAn203-F / R, respectively, the gel was recovered, and then DPNI enzyme was used for digestion. The product was then transformed into E. coli JM109, and after sequencing verification, the plasmid was extracted to obtain pTarget-sdhAn201, pTarget-sdhAn202 and pTarget-sdhAn203 plasmids.

[0044] According to the sequence of the genome, 500 bp upstream and downstream of the gene to be knocked out were selected, primers sdhAup-F / R and sdhAdown-F / R were designed, and the E. coli BL21(DE3) genome was used as a template for PCR. The products sdhAup and sdhAdown were recovered from the gel after PCR, and then primers sdhAup-F and sdhAdown-R were used for fusion PCR. The sdhA-template for recombination was obtained after gel recovery.

[0045] The pCas plasmid was transformed into the knockout chassis cell BL21(DE3) to form BL21(DE3)-pCas. A single colony was picked from a kana-resistant LB solid culture plate and inoculated into 10-20 mL of LB medium, which was cultured at 30°C and 250 rpm for 8-12 h. The culture was transferred to 50 mL of LB medium, which was cultured at 30°C and 250 rpm until the OD 600 was 0.2, and then 10 mM of arabinose was added for induction. The induction was performed for at least 1 h, and the OD 600 was 0.6, and the electrocompetent cells were prepared.

[0046] The pTarget-sdhAn201, pTarget-sdhAn202, and pTarget-sdhAn203 plasmids were respectively electrotransformed with the sdhA-template at a ratio of 1:3 into the electrocompetent BL21(DE3)-pCas, and plated on Kana and Str plates and incubated in a 30°C incubator for 48 h. After single colonies grew, colony PCR was performed using the 500 bp upstream primer sdhAup-F and the 500 bp downstream primer sdhA-down-R of the gene to be knocked out. If the knockout was successful, the size of the PCR fragment was 1000 bp.

[0047] Elimination of the pTarget-sdhAn20 plasmid: a single colony with successful knockout was picked and inoculated into 10-20 mL of LB seed medium, which was cultured at 30°C and 250 rpm for 24 h. The culture was transferred to 50 mL of LB medium, which was cultured at 30°C and 250 rpm until the OD 600 was 0.4-0.6, and 1 mM IPTG was added for continued culture for 24 h. The sgRNA-pMB1 sequence was induced to be located on the pTarget-sdhAn20, the pTarget-sdhAn20 plasmid was eliminated, and single colonies were picked from Kana plates to obtain the strain with the pTarget-sdhAn20 plasmid eliminated.

[0048] Elimination of pCas plasmid: pick the single colony of successful elimination of pTarget-sdhAn20 plasmid, inoculate into 10-20 mL of LB seed medium, 42°C, 250 rpm for 24 h, coat the non-resistant plate and pick the single colony to obtain the pCas-eliminated strain. The obtained E. coli engineering strain BL21(DE3)△sdhA with deleted sdhA gene is named as B1.

[0049] 2. Construction of E. coli engineering strain B2

[0050] Using the above gene knockout method, the gene sdhB in E. coli BL21(DE3)△sdhA is knocked out, and the obtained E. coli engineering strain BL21(DE3)△sdhA△sdhB with deleted sdhB gene is named as B2.

[0051] 3. Construction of E. coli engineering strain B3

[0052] Using the above gene knockout method, the gene iclR in E. coli BL21(DE3)△sdhA is knocked out, and the obtained E. coli engineering strain BL21(DE3)△sdhA△iclR with deleted iclR gene is named as B3.

[0053] 4. Construction of E. coli engineering strain B4

[0054] Using the above gene knockout method, the gene icd in E. coli BL21(DE3)△sdhA△iclR is knocked out, and the obtained E. coli engineering strain BL21(DE3)△sdhA△iclR△icd with deleted icd gene is named as B4.

[0055] 5. Construction of E. coli engineering strain B5

[0056] Using the above gene knockout method, the gene poxB in E. coli BL21(DE3)△sdhA△iclR△icd is knocked out, and the obtained E. coli engineering strain BL21(DE3)△sdhA△iclR△icd△poxB with deleted poxB gene is named as B5.

[0057] 6. Construction of E. coli engineering strain B6

[0058] Using the above gene knockout method, the gene ptsG in E. coli BL21(DE3)△sdhA△iclR△icd△poxB is knocked out, and the obtained E. coli engineering strain BL21(DE3)△sdhA△iclR△icd△poxB△ptsG with deleted ptsG gene is named as B6.

[0059] The primers related to gene knockout are listed in Table 2.

[0060] (ii) Construction of E. coli engineering strains B6-gltA, B6-aceA and B6-gltA-aceA

[0061] 1. Construction of recombinant plasmids pJUN-gltA, pJUN-aceA and pJUN-gltA-aceA

[0062] Table 3 Primers designed for construction of plasmids

[0063]

[0064] Using primers F-gltA-F / R, F-aceA-F / R, the target genes gltA and aceA were amplified from the genome of E. coli BL21(DE3) and named as F-gltA and 1F-aceA, respectively. Using primer F-PUTRinfC-rplT-F / R, the constitutive promoter F-PUTRinfC-rplT was amplified from the genome of E. coli BL21(DE3). Using primers V-pCDF-F / R, the vector 1V-pCDF was amplified from plasmid pCDFDuet-1.

[0065] The PCR products were purified using a kit, respectively, and ligated with 1V-pCDF, F-PUTRinfC-rplT and F-gltA using a one-step cloning enzyme at 50°C for 15 min, and then transformed into E. coli JM109 and verified by sequencing to obtain plasmid pJUN-gltA.

[0066] The PCR products were purified using a kit, respectively, and ligated with 1V-pCDF, F-PUTRinfC-rplT and F-gltA using a one-step cloning enzyme at 50°C for 15 min, and then transformed into E. coli JM109 and verified by sequencing to obtain plasmid pJUN-gltA.

[0067] Using primers F-gltA-aceA-F / R, the target gene aceA was amplified from pJUN-aceA and named as 2F-aceA. Using primers V-gltA-aceA-F / R, the product 2V-pCDF was amplified from pJUN-gltA. Using a one-step cloning enzyme, 2V-pCDF and 2F-aceA were ligated at 50°C for 15 min, and then transformed into E. coli JM109 and verified by sequencing to obtain plasmid pJUN-gltA-aceA. The primers for constructing the plasmid are listed in Table 3.

[0068] 2. Construction of E. coli engineering strains B6-gltA, B6-aceA and B6-gltA-aceA

[0069] The recombinant plasmid pJUN-gltA and pJUN-aceA were transformed into B6 respectively to obtain the E. coli engineering bacteria B6-gltA and B6-aceA, and the recombinant plasmid pJUN-gltA-aceA was transformed into B6 to obtain the E. coli engineering bacteria B6-gltA-aceA.

[0070] Example 2: Shake flask fermentation of E. coli engineering bacteria B1, B2, B3, B4, B5, B6, B6-gltA, B6-aceA and B6-gltA-aceA

[0071] Shake flask fermentation and product detection method: single colony on the plate was inoculated into 20 mL LB medium, 37℃, 250 r / min culture overnight to obtain shake flask fermentation seed liquid, and the seed liquid was inoculated into 50 mL M9 modified medium, the final concentration OD 600 was 0.2, 37℃, 250 r / min culture, and the succinic acid yield was detected by HPLC at 24h and 48h during fermentation.

[0072] Result analysis: from Figure 2 it can be seen that E. coli BL21(DE3) will not accumulate succinic acid under aerobic conditions, and knocking out sdhA can block the tricarboxylic acid cycle to make it accumulate succinic acid under aerobic conditions, and the yield of B1 reaches 0.47 g / L after 48h fermentation; knocking out sdhB on the basis of knocking out sdhA will not make the succinic acid yield increase, so sdhB is not knocked out subsequently; B3 knocking out iclR accumulates 0.54 g / L of succinic acid after 48h fermentation under aerobic conditions, which is 14.8% higher than that of B1; B4 knocking out icd accumulates 0.92 g / L of succinic acid after 48h fermentation under aerobic conditions, which is 95.7% higher than that of B1. At this time, the by-product in the fermentation broth is mainly acetic acid, in order to reduce the acetic acid content, poxB is knocked out on the basis of the recombinant bacteria B4, and the accumulation of succinic acid of the recombinant bacteria B5 after 48h fermentation is almost unchanged, and the acetic acid content is reduced by 7.64%, at this time, we speculate that the content of intracellular phosphoenolpyruvate (PEP) is less, and the content of oxaloacetate (OAA) which supplements the tricarboxylic acid cycle is less, which leads to the blockage of the tricarboxylic acid cycle, and the large amount of acetic acid production, therefore, ptsG is further knocked out to obtain B6, which increases the content of intracellular PEP, at this time, the accumulation of succinic acid after 48h fermentation reaches 3.75 g / L, and the by-product acetic acid content decreases by 63.7%, reaching 1.05 g / L.

[0073] To further improve succinic acid production and strengthen metabolic flux to target product, first, overexpressed aceA to strengthen metabolic flux to glyoxylate cycle, recombinant strain B6-aceA under aerobic conditions for 48 h fermentation succinic acid production reached 3.85 g / L, increased by 2.7%, compared with the recombinant bacteria B6 acetic acid accumulation decreased by 33.3%; In addition, overexpressed gltA to strengthen metabolic flux to the tricarboxylic acid cycle, compared with the recombinant strain B6-gltA under aerobic conditions for 48 h fermentation succinic acid production reached 4.25 g / L, increased by 13.3% compared with the recombinant bacteria B6, acetic acid accumulation decreased by 38.1% compared with the recombinant bacteria B6; Further, the recombinant strain B6-gltA-aceA under aerobic conditions for 48 h fermentation succinic acid production reached 4.49 g / L, reached 85.6% of the theoretical conversion rate, increased by 19.7% compared with the recombinant bacteria B6, increased by 855.3% compared with the genetic engineering bacteria B1, at this time, acetic acid accumulation decreased to 0.24 g / L, decreased by 77.1% compared with the recombinant bacteria B6, decreased by 94.0% compared with the genetic engineering bacteria B1.

[0074] Table 4 E. coli engineering bacteria fermentation succinic acid and acetic acid content

[0075]

[0076] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore, the protection scope of the present application should be defined by the claims.

Claims

1. An engineered Escherichia coli strain for producing succinic acid with reduced byproducts, characterized in that, The engineered Escherichia coli strain had the following genes knocked out: succinate dehydrogenase gene sdhA, transcription factor gene iclR, isocitrate dehydrogenase gene icd, pyruvate dehydrogenase gene poxB, and pyruvate dehydrogenase gene ptsG. It also overexpressed citrate synthase gene gltA and isocitrate lyase gene aceA. The nucleotide sequence of the succinate dehydrogenase gene sdhA is shown in SEQ ID NO.1; the nucleotide sequence of the transcription factor gene iclR is shown in SEQ ID NO.3; the nucleotide sequence of the isocitrate dehydrogenase gene icd is shown in SEQ ID NO.4; the nucleotide sequence of the pyruvate dehydrogenase gene poxB is shown in SEQ ID NO.5; the nucleotide sequence of the pyruvate dehydrogenase gene ptsG is shown in SEQ ID NO.6; and the nucleotide sequence of the citrate synthase gene gltA is shown in SEQ ID NO.

7. The nucleotide sequence of the isolimonite lyase gene aceA is shown in SEQ ID NO.8; The host of the Escherichia coli is Escherichia coli BL21(DE3) or MG1655; The constitutive promoter PUTRinfc-rplt shown in SEQ ID NO.9 was linked to the citrate synthase gene gltA and the isocitrate lyase gene aceA into the vector pCDFDuet-1.

2. A method for constructing the engineered Escherichia coli strain of claim 1, characterized in that, The method includes the following steps: (1) Genetically engineered bacteria with the above genes knocked out were obtained by knocking out the sdhA, iclR, icd, poxB and ptsG genes in Escherichia coli BL21(DE3) by CRISPR / CAS9. (2) The constitutive promoter PUTRINFC-rplt shown in SEQ ID NO.9, the gene fragment of citrate synthase gene gltA shown in SEQ ID NO.7, and the gene fragment of isocitrate lyase gene aceA shown in SEQ ID NO.8 were ligated into the pCDFDuet-1 vector to obtain a recombinant vector, and the recombinant vector was transferred into the genetically engineered bacteria of step (1).

3. A method for producing succinic acid under aerobic conditions, wherein the method comprises inoculating the engineered Escherichia coli of claim 1 into a fermentation medium for fermentation.

4. The method according to claim 3, characterized in that, The fermentation medium contains, by g / L, 6-8 Na2HPO4, 2-4 KH2PO4, 0.5-1.5 NH4Cl, 0.5-1 NaCl, 0.15-0.3 MgSO4, 0.1-0.2 CaCl2, 7-8 tryptone, 2-3 yeast extract, and 8-9 glucose.

5. The method according to claim 3 or 4, characterized in that, The fermentation temperature is 30–40°C, and the fermentation time is 20–50 hours.

6. The method according to claim 5, characterized in that, The fermentation speed is 200-250 r / min.

7. The use of the engineered Escherichia coli of claim 1 or the method of any one of claims 3 to 6 in the preparation of succinic acid or products containing succinic acid.

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