Fusion protein and application thereof in succinic acid production

Abstract

The invention discloses a fusion protein and application thereof in succinic acid production. Fusion expression of fumarase and fumaric acid reductase is respectively carried out in escherichia coli and saccharomyces cerevisiae by utilizing different connecting peptides, and the fusion protein in which the enzyme activity of fumarase is stronger than that of independent fumarase and the genetically engineered bacterium for expressing the fusion protein are obtained. When the genetically engineered bacterium is used for fermentation production of succinic acid, the yield of succinic acid is higher than that of succinic acid which does not express the fusion protein. The specific connecting peptide is utilized to improve the transfer efficiency of the fumaric acid, the efficient catalysis of the fumaric acid is realized, an effective fusion protein is provided for the efficient production of the succinic acid, meanwhile, more efficient genetically engineered bacteria are provided for the production of the succinic acid by utilizing microbial fermentation, and the production efficiency and yield of the succinic acid are favorably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and enzyme engineering. More particularly, it relates to a fusion protein and its application in succinic acid production. BACKGROUND

[0002] The tricarboxylic acid (TCA) cycle is the metabolic hub of nutrients such as sugars. Under anaerobic or microaerobic conditions, the reverse reduction TCA pathway will be activated to synthesize important raw materials needed by cells. The reduction TCA pathway is the main pathway for microorganisms to synthesize succinic acid. The pathway starts with phosphoenolpyruvate synthesized by glycolysis, and generates oxaloacetate under the action of corresponding enzymes. Then oxaloacetate is converted into malate, and malate is converted into fumaric acid under the action of fumarase. Finally, fumaric acid is converted into succinic acid under the catalysis of fumarate reductase.

[0003] In the production of succinic acid, the concentration of the intermediate product fumaric acid is low, the spatial distance between fumarase and fumarate reductase is large, and the transfer efficiency of fumaric acid is low, which will limit the reaction rate to some extent, thereby limiting the yield of succinic acid. A linker peptide is a polypeptide that can covalently connect the functional domains of two proteins, and the length is usually 3-50 amino acids. By using a linker peptide to reasonably control the distance between two enzymes, or to achieve spatial proximity effect in vivo, the transfer efficiency of fumaric acid can be improved, and efficient catalysis of fumaric acid can be achieved.

[0004] Although there are reports that the use of a linker peptide to fuse alcohol dehydrogenase and NAD(P)H oxidase can improve the activity of the two enzymes and improve their catalytic properties, there is no report on the use of a linker peptide to improve the enzyme activity of fumarase and fumarate reductase. For different enzymes, the linker peptide required for fusion expression to improve the enzyme activity is not the same, and the length of the linker peptide, the composition and sequence of the amino acids in the linker peptide, etc. will affect the enzyme activity of the obtained fusion protein and affect its catalytic properties. It is not easy to obtain a fusion protein of fumarase and fumarate reductase with improved enzyme activity compared with fumarase or fumarate reductase alone. SUMMARY

[0005] The present application provides a fusion protein to solve the above technical problems, and provides the application of the fusion protein in succinic acid production.

[0006] The first object of the present application is to provide a fusion protein.

[0007] The second object of the present application is to provide a gene encoding the fusion protein.

[0008] The third object of the present application is to provide a recombinant vector containing the gene.

[0009] A fourth object of the present application is to provide a genetically engineered bacterium.

[0010] A fifth object of the present application is to provide a composition.

[0011] A sixth object of the present application is to provide the use of the fusion protein, the genetically engineered bacterium or the composition in succinic acid production.

[0012] A seventh object of the present application is to provide the use of the fusion protein, the gene, the recombinant vector, the genetically engineered bacterium or the composition in preparing a preparation for producing succinic acid.

[0013] An eighth object of the present application is to provide a method for producing succinic acid.

[0014] The above objects of the present application are achieved by the following technical solutions. The present application discloses a fusion protein of fumarase and fumarate reductase connected by a specific linker peptide, which has higher enzyme activity than fumarase alone, and the production of succinic acid in Saccharomyces cerevisiae expressing the fusion protein is significantly improved. Therefore, the present application claims the fusion protein and its use in succinic acid production.

[0015] The present application provides a fusion protein comprising fumarase and fumarate reductase connected by a linker peptide; the linker peptide is L2 or L3; wherein the amino acid sequence of L2 is shown in SEQ ID NO. 2, and the amino acid sequence of L3 is shown in SEQ ID NO. 3.

[0016] Specifically, the fumarase can catalyze the dehydration of malic acid to generate fumaric acid, and the fumarate reductase can catalyze the conversion of fumaric acid to succinic acid.

[0017] Specifically, the fumarase is derived from Rhizopus oryzae ( Rhizopus oryzae ), and the fumarate reductase is derived from Trypanosoma brucei ( Trypanosoma brucei ).

[0018] Preferably, the linker peptide in the fusion protein is L2. The enzyme activity of the fusion protein obtained by fusing fumarase and fumarate reductase with L2 is the highest.

[0019] The present application also provides a construction method of the fusion protein, which connects fumarase (FUM) and fumarate reductase (FRD) in a certain order by using a linker peptide (L2 or L3).

[0020] The construction method of the fusion protein is well known to those skilled in the art, for example, the following steps can be used: S1. connecting a gene encoding a target protein (FUM and FRD) with a gene encoding a linker peptide (L2 or L3) in series to construct a fusion gene encoding the fusion protein; wherein the method of series connection comprises: designing appropriate primers, obtaining the fusion gene by polymerase chain reaction (PCR) method, or directly synthesizing the required fusion gene by artificial synthesis method; S2. inserting the fusion gene obtained in S1 into the multiple cloning site region of an expression vector to obtain a recombinant vector containing the fusion gene; S3. transforming the host cell with the recombinant vector obtained in S2 to obtain a transformed host cell; S4. culturing the transformed host cell, inducing the expression of the fusion protein and separating to obtain the fusion protein.

[0021] In a specific embodiment of the present application, the fumarase used for constructing the fusion protein is derived from Rhizopus oryzae (Rhizopus oryzae) and is named RoFUM, and its sequence number in NCBI is P55250.1; the fumarate reductase used is derived from Trypanosoma brucei (Trypanosoma brucei) and is named TbFRD, and its sequence number in NCBI is AAN40014.1. Rhizopus oryzae Trypanosoma brucei

[0022] Specifically, when a prokaryotic expression system is used for the construction and expression of the fusion protein, the gene sequence encoding the target protein needs to be codon-optimized according to the host cell (bacterium) used. In a specific embodiment of the present application, the fusion protein is recombinantly expressed by using Escherichia coli; wherein the genes encoding RoFUM and TbFRD are codon-optimized, the nucleotide sequence of the optimized gene encoding RoFUM is shown in SEQ ID NO. 4, and the nucleotide sequence of the optimized gene encoding TbFRD is shown in SEQ ID NO. 5.

[0023] The gene encoding the linker peptide is not particularly limited as long as the corresponding linker peptide can be expressed by translation.

[0024] Alternatively, the nucleotide sequence of the gene encoding the linker peptide L2 is shown in SEQ ID NO. 7.

[0025] Alternatively, the nucleotide sequence of the gene encoding the linker peptide L3 is shown in SEQ ID NO. 8.

[0026] In addition, the present application does not have a particular limitation on the connection order of the linker peptide and the fumarase and the fumarate reductase, and the connection order can be fumarase-linker peptide-fumarate reductase or fumarate reductase-linker peptide-fumarase.

[0027] ​​Preferably, the connection sequence is fumarase-connection peptide-fumarate reductase. The genetically engineered bacteria expressing the fusion protein of this connection sequence has relatively higher succinic acid yield.

[0028] The present application also claims a gene encoding the fusion protein of the present application.

[0029] The present application also claims a recombinant vector containing the above-mentioned gene.

[0030] In particular, the recombinant vector is a recombinant expression vector.

[0031] In particular, the recombinant expression vector includes a recombinant prokaryotic expression vector and a recombinant eukaryotic expression vector.

[0032] Optionally, the expression vector used for constructing the recombinant prokaryotic expression vector is pET-30a.

[0033] Optionally, the expression vector used for constructing the recombinant eukaryotic expression vector is pYEp352.

[0034] The present application also claims a genetically engineered bacteria capable of expressing the fusion protein of the present application.

[0035] In particular, the genetically engineered bacteria contains the gene encoding the fusion protein of the present application or the recombinant vector.

[0036] Optionally, when the fusion protein is expressed by a prokaryotic expression system, the starting strain of the genetically engineered bacteria is Escherichia coli (E. coli) BL21 (DE3). Escherichia coli ).

[0037] Optionally, when the fusion protein is expressed by a eukaryotic expression system, the starting strain of the genetically engineered bacteria is Saccharomyces cerevisiae (S. cerevisiae) BY4741-MP. Saccharomyces cerevisiae ).

[0038] Optionally, the E. coli is DE3 strain.

[0039] Optionally, the S. cerevisiae is BY4741-MP strain. The strain is a S. cerevisiae strain capable of synthesizing malate, i.e. a strain carrying expression plasmid pUG34-MDH-PYC.

[0040] Optionally, when the fusion protein is expressed, the promoter used is PCCW12, and its nucleotide sequence is shown in SEQ ID NO. 9; the terminator used is tFBA1, and its nucleotide sequence is shown in SEQ ID NO. 10.

[0041] The present application also claims a composition containing the fusion protein of the present application or the genetically engineered bacteria, and adjuvants.

[0042] The fusion protein, the genetically engineered bacteria or the composition can be used to catalyze the production of succinic acid from fumaric acid, and improve the yield of succinic acid. Therefore, the application also protects the use of the fusion protein, the genetically engineered bacteria or the composition in the production of succinic acid.

[0043] The application also protects the use of the fusion protein, the gene, the recombinant vector, the genetically engineered bacteria or the composition in the preparation of a preparation for the production of succinic acid.

[0044] The application also provides a method for producing succinic acid, which comprises fermenting the genetically engineered bacteria and a carbon source.

[0045] Alternatively, the carbon source is at least one carbohydrate; the carbohydrate is selected from monosaccharides, disaccharides, polysaccharides and mixtures thereof; the monosaccharide is selected from glucose, fructose, xylose, galactose and mixtures thereof; the disaccharide is selected from lactose, sucrose, maltose, cellobiose and mixtures thereof; and the polysaccharide is selected from starch, glycogen, cellulose and mixtures thereof.

[0046] The application has the following advantages: The application uses different connecting peptides (connecting peptides L2 or L3) to perform fusion expression of fumarase and fumarate reductase in E. coli and S. cerevisiae, respectively, to obtain fusion proteins with stronger enzyme activity of fumarase than the single fumarase, and genetically engineered bacteria expressing the fusion proteins. When the genetically engineered bacteria are used to ferment and produce succinic acid, the yield is also higher than that of the control. That is, the application uses specific connecting peptides to improve the transfer efficiency of fumaric acid, realizes efficient catalysis of fumaric acid, provides effective fusion proteins for efficient production of succinic acid, and also provides more efficient genetically engineered bacteria for the production of succinic acid by microbial fermentation, which is conducive to improving the production efficiency and yield of succinic acid. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The figure is the fumarase enzyme activity determination result of the six fusion proteins obtained by IPTG induction expression in Example 1. p <0.05.

[0048] Figure 2 The figure is the structure diagram of each fusion fragment containing the PCCW12 promoter and the tFBA1 terminator constructed in Example 2.

[0049] Figure 3 The figure is the concentration of succinic acid obtained by fermenting each S. cerevisiae expressing different fusion proteins. p <0.01. DETAILED DESCRIPTION

[0050] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Among them, the reagents related to PCR amplification reaction are purchased from Beijing Zison Company; the restriction endonucleases are purchased from New England BioLabs; the T4 DNA ligase is purchased from Takara; the plasmid extraction kit is purchased from Omega Company; the purification kit for PCR products and enzyme digestion products is purchased from Shanghai Huashun Company.

[0052] Example 1 Recombinant expression of fusion protein in Escherichia coli and determination of its enzyme activity In this example, different linker peptides (L1-L3) are used to fuse fumarase (FUM) and fumarate reductase (FRD) for expression, and different fusion proteins are obtained. The amino acid sequences of the different linker peptides are as follows: Linker peptide L1: EKKKEKKKEKKK (shown in SEQ ID NO. 1) Linker peptide L2: CRDTSDADQGTSYLF (shown in SEQ ID NO. 2) Linker peptide L3: GGGGSGGGGSGGGGS (shown in SEQ ID NO. 3) The fumarase is derived from Rhizopus oryzae ( Rhizopus oryzae ), named RoFUM, and the sequence number of the fumarase in NCBI is P55250.1. The fumarate reductase is derived from Trypanosoma brucei ( Trypanosoma brucei ), named TbFRD, and the sequence number of the fumarase in NCBI is AAN40014.1.

[0053] The construction strategy of the fusion protein is as follows: the genes encoding RoFUM and TbFRD are linked together and then constructed on a plasmid to obtain a recombinant plasmid, specific primers are designed, and the designed primers are used for PCR amplification of the recombinant plasmid, and the linker peptide is added between the two target protein genes. The nucleotide sequences of the primers used for linking the genes encoding RoFUM and TbFRD together are shown in Table 1.

[0054] Table 1 Primers used for linking RoFUM and TbFRD

[0055] The specific process is as follows: 1. Obtaining of plasmid backbones pET1 and pET2 Taking pET-30a plasmid as a template, primer pairs Z-F1 / R1 and Z-F2 / R1 (nucleotide sequences of the primers are shown in Table 1) are used for PCR amplification to obtain plasmid backbones pET1 and pET2.

[0056] The reaction system and reaction procedure for PCR amplification are as follows: The reaction system is: 2xPhanta Max Buffer buffer (Vazyme) 25 μL, dNTP (10 mM of each dNTP) mixture 1 μL, DNA template 20 ng, primers (10 μM) 2 μL each, Phanta Max Super Fidelity DNA polymerase (2.5 U / μL) 1 μL, and distilled water to 50 μL. When performing fusion PCR, the DNA template is each fragment added to the system in a molar ratio of 1:1.

[0057] The reaction procedure is: 95°C pre-denaturation for 3 minutes (1 cycle); 95°C denaturation for 15 seconds, 56°C annealing for 15 seconds, and 72°C extension for 1 minute (30 cycles); and 72°C extension for 5 minutes (1 cycle).

[0058] The reaction system and reaction procedure for each PCR amplification described later are all referred to this, and the extension time in the reaction procedure is adjusted according to the length of the amplified sequence and the extension efficiency of the enzyme.

[0059] 2. Concatenation of RoFUM and TbFRD Beijing Qianke Biological Technology Co., Ltd. is commissioned to code optimize and synthesize the genes encoding RoFUM and TbFRD, to obtain the optimized gene fragment encoding RoFUM, represented as RoFUM , the nucleotide sequence of which is shown as SEQ ID NO. 4, and the optimized gene fragment encoding TbFRD, represented as TbFRD , the nucleotide sequence of which is shown as SEQ ID NO. 5.

[0060] Taking the synthesized gene fragments encoding RoFUM and TbFRD as templates, respectively, primer pair RoFUM-F1 / R1 is used for PCR amplification to obtain RoFUM1 gene fragment, and primer pair TbFRD-F2 / R1 is used for PCR amplification to obtain TbFRD1 gene fragment. The gene fragments obtained by PCR amplification are purified, and after purification and recovery, are used as templates for the next step. RoFUM1 , and TbFRD1 ​The mixture of the gene fragments (molar ratio 1:1) was used as a template, and primers RoFUM-F1 and TbFRD-R1 were used for PCR fusion amplification to obtain a FUM-FRD fusion fragment; and the synthesized gene fragment was used as a template, and primer pair RoFUM-F2 / R1 was used for PCR amplification to obtain RoFUM2 a gene fragment. The gene fragment obtained by PCR amplification was purified, and after purification and recovery, the TbFRD2 gene fragment was used as a template, and primer pair TbFRD-F1 / R1 was used for PCR amplification to obtain RoFUM2 a gene fragment. The mixture of the gene fragments (molar ratio 1:1) was used as a template, and primers TbFRD-F1 and RoFUM-R1 were used for PCR fusion amplification to obtain a FRD-FUM fusion fragment. TbFRD2 The amplification product (plasmid backbone pET1 and pET2) of pET-30a plasmid was purified, and then digested by endonuclease

[0061] I, and then the whole system was incubated at 80 ℃ for 20 min. After digestion, the fusion fragment FUM-FRD and pET1 were mixed, and FRD-FUM and pET2 were mixed, and the reaction system was prepared according to the one-step cloning kit instructions (Vazyme) and incubated at 50 ℃ for 15 min for homologous recombination connection; 10 μL of the recombination product was added to 100 μL of DH5α competent cells, which were placed on ice for 30 min, and then heated at 42 ℃ for 45 s; 1 mL of LB liquid medium was added and incubated at 37 ℃ for 1 h; the obtained bacterial liquid was spread on LB solid medium containing antibiotic Amp, and incubated at 37 ℃ until single colonies grew; 5 single colonies were selected and incubated in LB liquid medium, and PCR detection was performed for positive and sequencing confirmation, and then the plasmid was extracted and stored, and named as pET-FUM-FRD and pET-FRD-FUM. Dpn The digestion reaction system was: 10×digestion buffer (TAKARA) 5 μL,

[0062] I 1 μL, purified DNA product 20 μL, and distilled water to 50 μL; the digestion reaction conditions were: 37 ℃ incubation for 3 h. The one-step cloning reaction system was: 2×ClonExpress Mix 5 μL, linearized vector 1.5 μL, and total insert 3.5 μL, with a total volume of 10 μL. Dpn 2, Linker peptide inserted into FUM-FRD / FRD-FUM fusion fragment

[0063] ​The gene encoding the linker peptide is inserted into the two proteins by PCR amplification using the constructed pET-FUM-FRD or pET-FRD-FUM as a template to obtain the corresponding recombinant plasmid. The nucleotide sequences of the genes encoding the linker peptides L1-L3 are shown in SEQ ID NO. 6-8, respectively, and the nucleotide sequences of the primer pairs used for inserting the linker peptides are shown in Table 2. Among them, primer pairs 1-3 are used to insert the linker peptides L1-L3 between FUM and FRD of pET-FUM-FRD, respectively; and primer pairs 4-6 are used to insert the linker peptides L1-L3 between FRD and FUM of pET-FRD-FUM, respectively.

[0064] Table 2 Primers used for inserting linker peptides

[0065] Here, the insertion of the linker peptide L1 between FUM and FRD to obtain the recombinant plasmid pET-FUM-L1-FRD is taken as an example for detailed description.

[0066] PCR amplification is performed using primer pair 1 shown in Table 2 and pET-FUM-FRD as a template, and the amplification product is detected by agarose gel electrophoresis to obtain a single target band pET-FUM-L1-FRD, which is purified and recovered.

[0067] The purified and recovered target band pET-FUM-L1-FRD is digested with endonuclease Dpn I (the reaction system is the same as before), and the whole system is incubated at 80°C for 20 min; 10 μL of the product is added to 100 μL of DH5α competent cells, which are placed on ice for 30 min, and then heat shocked at 42°C for 45 s, followed by the addition of 1 mL of LB liquid medium, which is incubated at 37°C for 1 h; the obtained bacterial solution is spread on LB solid medium containing antibiotic Amp, and incubated at 37°C until single colonies grow; 5 single colonies are selected and incubated in LB liquid medium, and PCR detection and sequencing confirmation are performed to obtain the pET-FUM-L1-FRD plasmid.

[0068] By a similar method, the recombinant plasmids pET-FUM-L2-FRD and pET-FUM-L3-FRD are constructed using primer pairs 2 and 3 and pET-FUM-FRD as a template. The recombinant plasmids pET-FRD-L1-FUM, pET-FRD-L2-FUM and pET-FRD-L3-FUM are constructed using primer pairs 3-6 and pET-FRD-FUM as a template.

[0069] 3. Recombinant expression of fusion proteins and enzyme activity determination The recombinant plasmids constructed in step 2 were transformed into *E. coli* DE3 and cultured in LB liquid medium (containing 30 mg / mL kanaciline) for 8 h to obtain seed culture. The seed culture was inoculated into TB liquid fermentation medium (containing 30 mg / mL kanaciline) at a 5% inoculation rate and cultured at 37°C on a shaker for 2 h. IPTG (final concentration 0.1 mM) was then added, and the culture was transferred to a shaker at 25°C for another 24 h. The fermentation broth was centrifuged at 12000 rpm for 10 min at 4°C, and the bacterial cells were collected. 50 mL of PPS buffer (25 mM, pH 7.0) was added to the bacterial cells to fully resuspend them. The cells were then disrupted using an ultrasonic cell disruptor, centrifuged at 10000 rpm for 5 min, and the supernatant was collected for enzyme activity assay. Simultaneously with the construction and expression of the recombinant expression plasmid of the fusion protein, this invention also constructed and expressed a separate recombinant plasmid for RoFUM. Using recombinant RoFUM as a control, and the obtained fusion proteins as experimental groups, their FUM enzyme activities were measured, and the relative enzyme activity (%) was calculated according to the relative enzyme activity calculation formula. FUM enzyme activity detection: L-malic acid was used as a substrate, and the reaction was carried out at its optimal reaction temperature for 10 min. The absorbance at 290 nm was measured, and the enzyme activity was calculated.

[0070] Formula for calculating relative enzyme activity: Relative enzyme activity (%) = [Control enzyme activity - Experimental group enzyme activity] / Control enzyme activity × 100%.

[0071] The fumarate enzyme activity assay results of the six fusion proteins obtained by IPTG-induced expression in this embodiment are as follows: Figure 1 As shown. By Figure 1 It can be seen that after using different linker peptides to fuse FUM and FRD, the enzyme activity of FUM in the fusion protein linked by L1 linker peptide decreased significantly, while the enzyme activity of FUM in the fusion protein linked by L2 and L3 linker peptides increased. This indicates that linker peptides such as L2 and L3 are more suitable for the fusion expression of FUM and FRD enzymes.

[0072] Example 2 Recombinant expression of fusion protein in Saccharomyces cerevisiae The primers used for PCR amplification in this embodiment are shown in Table 3.

[0073] Table 3 Primers used to construct recombinant expression plasmids of Saccharomyces cerevisiae

[0074] 1. Construction of recombinant expression plasmids of Saccharomyces cerevisiae With brewer's yeast ( Saccharomyces cerevisiaeThe genomic DNA was used as a template, and the sequences of the promoter PCCW12 (nucleotide sequence as shown in SEQ ID NO. 9) and the terminator tFBA1 (nucleotide sequence as shown in SEQ ID NO. 10) were amplified by using primer pairs PCCW12-F / R and tFBA1-F / R (as shown in Table 3) respectively and purified and recovered.

[0075] The optimized genes encoding RoFUM and TbFRD synthesized in Example 1 were used as templates, and the sequences of RoFUM-3 and TbFRD-3 were amplified by using primer pairs RoFUM-F3 / R2 and TbFRD-F3 / R2 (as shown in Table 3) respectively and purified and recovered.

[0076] A mixture of the promoter PCCW12, the terminator tFBA1 and the RoFUM-3 fragment was used as a template (molar ratio 1:1:1), and primer PCCW12-F and tFBA1-R were used for PCR fusion amplification to obtain the PCCW12-RoFUM-tFBA1 fusion fragment; another mixture of the promoter PCCW12, the terminator tFBA1 and the TbFRD-3 fragment was used as a template, and primer PCCW12-F2 and tFBA1-R were used for PCR fusion amplification to obtain the PCCW12-TbFRD-tFBA1 fusion fragment. The structures of the PCCW12-RoFUM-tFBA1 fusion fragment and the PCCW12-TbFRD-tFBA1 fusion fragment are shown in Figure 2 , which are marked as FUM and FRD, respectively. Figure 2

[0077] The pYEp352 Saccharomyces cerevisiae plasmid was digested according to the digestion reaction system and digestion conditions described in Example 1, and the products after digestion of pYEp352 were mixed with the PCCW12-RoFUM-tFBA1 fusion fragment and the PCCW12-TbFRD-tFBA1 fusion fragment, and the reaction system was prepared according to the instructions of the one-step cloning kit (Vazyme) and incubated at 50°C for 15 min for homologous recombination and ligation; 10 μL of the recombination product was added to 100 μL of DH5α competent cells, which were placed on ice for 30 min, and then heat shocked at 42°C for 45 s, and then 1 mL of LB liquid medium was added and incubated at 37°C for 1 h, and then spread on LB solid medium containing antibiotic Amp and incubated at 37°C until single colonies grew; 5 single colonies were selected and cultured in LB liquid medium, and PCR detection was performed, and after positive and sequencing confirmation, the plasmid was extracted and stored, and the recombinant Saccharomyces cerevisiae expression plasmid p352-F2 was obtained.

[0078] ​The DNA fragments of FUM-L1-FRD, FUM-L2-FRD and FUM-L3-FRD were amplified from the recombinant plasmids pET-FUM-L1-FRD, pET-FUM-L2-FRD and pET-FUM-L3-FRD respectively using primers RoFUM-F2 and TbFRD-R2. The obtained DNA fragments were mixed with the promoter PCCW12 and terminator tFBA1 fragments respectively, and the mixture was used as a template for PCR fusion amplification using primers PCCW12-F and tFBA1-R, to obtain the fusion fragments PCCW12-FUM-L1-FRD-tFBA1, PCCW12-FUM-L2-FRD-tFBA1 and PCCW12-FUM-L3-FRD-tFBA1. The structure of the fusion fragments is shown in Fig. 2. Figure 2

[0079] Similarly, the DNA fragments of FRD-L1-FUM, FRD-L2-FUM and FRD-L3-FUM were amplified from pET-FRD-L1-FUM, pET-FRD-L2-FUM and pET-FRD-L3-FUM respectively using primers TbFRD-F2 and RoFUM-R2. The obtained DNA fragments were mixed with the promoter PCCW12 and terminator tFBA1 fragments respectively, and the mixture was used as a template for PCR fusion amplification using primers PCCW12-F and tFBA1-R, to obtain the fusion fragments PCCW12-FRD-L1-FUM-tFBA1, PCCW12-FRD-L2-FUM-tFBA1 and PCCW12-FRD-L3-FUM-tFBA1. The structure of the fusion fragments is shown in Fig. 3. Figure 2

[0080] The expression plasmid pYEp352 of S. cerevisiae was amplified using primer pair Z352-F / R, and was fused with the above fragments respectively by seamless cloning, to obtain the recombinant expression plasmids p352-FUM-L1-FRD, p352-FUM-L2-FRD, p352-FUM-L3-FRD, p352-FRD-L1-FUM, p352-FRD-L2-FUM and p352-FRD-L3-FUM of S. cerevisiae fusion proteins.

[0081] 2. Transformation of the recombinant expression plasmid of S. cerevisiae ​​The seven recombinant plasmids constructed above were transformed into Saccharomyces cerevisiae BY4741-MP by LiAc transformation method. The obtained transformation liquid was spread on SD-U plates and placed in a 30°C incubator for inverted culture for 2-4 days. Positive transformants were screened and subjected to fermentation verification. The successfully transformed Saccharomyces cerevisiae monoclonal colonies were named as MPF2 (containing plasmid p352-F2), MPR1 (containing plasmid p352-FUM-L1-FRD), MPR2 (containing plasmid p352-FUM-L2-FRD), MPR3 (containing plasmid p352-FUM-L3-FRD), MPR4 (containing plasmid p352-FRD-L1-FUM), MPR5 (containing plasmid p352-FRD-L2-FUM) and MPR6 (containing plasmid p352-FRD-L3-FUM), respectively.

[0082] Example 3 Fermentation production of succinic acid Each of the successfully transformed Saccharomyces cerevisiae monoclonal colonies in Example 2 was inoculated into a sterilized 10 mL YPD medium (5 wt% glucose, 2 wt% peptone, 1 wt% yeast extract and the balance of water, pH 7.0) and cultured at 30°C and 200 rpm for 12-18 h. The obtained bacterial liquid was inoculated into a 50 mL YPD medium at a concentration of OD600=0.1 (each group of experiments was repeated for 3 times) and cultured at 30°C and 200 rpm. At the 48th hour of culture, samples were taken and subjected to detection of succinic acid production. The sample was obtained by centrifuging 1 mL of the bacterial liquid at 12000 rpm for 5 min, diluting 100 μL of the supernatant with a mobile phase (2.43 mM dilute sulfuric acid) by 10 times, removing impurities with a 0.22 μM filter membrane and detecting the sugar content and succinic acid production by high performance liquid chromatography. 600

[0083] The specific process of high performance liquid chromatography detection was as follows: an automatic sampler was set to automatically inject 10 μL of sample, the flow rate of the mobile phase was maintained at 0.6 mL / min, the column temperature was maintained at 40°C and the analysis time was 25 min. The detector worked at 40°C.

[0084] The succinic acid concentrations obtained by fermentation of each Saccharomyces cerevisiae expressing different fusion proteins were as shown in Table 1. Figure 3 As can be seen from Table 1, the succinic acid production increased after fusion expression of FUM and FRD, indicating that the transfer efficiency of fumaric acid between the two enzymes was improved. Among them, the L2 linker peptide had the best effect and the combination of FUM-L2-FRD was the best. Figure 3

[0085] ​​From the above results, it can be seen that the fumarase and fumarate reductase are fused and expressed in Saccharomyces cerevisiae by using the connecting peptide, so that the yield of succinic acid in the production of succinic acid by fermentation of Saccharomyces cerevisiae is improved.

[0086] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises fumarate enzyme and fumarate reductase linked by a linker peptide; the linker peptide is L2 or L3, the amino acid sequence of L2 is shown in SEQ ID NO.2, and the amino acid sequence of L3 is shown in SEQ ID NO.

3.

2. A gene characterized in that, The gene encodes the fusion protein of claim 1.

3. A recombinant vector, characterized in that, It contains the gene described in claim 2.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are able to express the fusion protein of claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacteria contain the gene of claim 2 or the recombinant vector of claim 3.

6. A composition, characterized in that, It contains the fusion protein of claim 1 or the genetically engineered bacteria of claim 4 or 5, and excipients.

7. The use of the fusion protein of claim 1, the genetically engineered bacteria of claim 4 or 5, or the composition of claim 6 in the production of succinic acid.

8. The use of the fusion protein of claim 1, the gene of claim 2, the recombinant vector of claim 3, the genetically engineered bacteria of claim 4 or 5, or the composition of claim 6 in the preparation of a formulation for the production of succinic acid.

9. A method for producing succinic acid, characterized in that, Obtained by fermentation using the genetically engineered bacteria and carbon source described in claim 4 or 5.

10. The method according to claim 9, characterized in that, The carbon source is at least one carbohydrate.

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