Recombinant strain for producing farnesene as well as construction method and application of recombinant strain

Abstract

The invention discloses a recombinant strain for producing farnesene as well as a construction method and application thereof. The construction method comprises the following steps: preparing a pFII plasmid containing farnesene synthase, farnesyl pyrophosphate synthase and isopentene diphosphate isomerase; taking the pFII plasmid as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by utilizing the primer 1 and the primer 2 to obtain a fragment 1; taking the pFII plasmid as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by utilizing a primer 3 and a primer 4 to obtain a fragment 2; taking the pFII plasmid as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by utilizing a primer 5 and a primer 6 to obtain a fragment 3; carrying out seamless connection on the fragment 1, the fragment 2 and the fragment 3 to obtain a plasmid 1; introducing a plasmid 1, a plasmid 2 and a plasmid 3 into escherichia coli to obtain a recombinant strain for producing farnesene; the farnesene yield of the recombinant strain 1 constructed by the invention is 3.3 g / L within 96 hours, and technical support and data reference are provided for large-scale preparation of biological fuel farnesene by a biological method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation engineering, and particularly relates to a recombinant strain for producing farnesene, a construction method thereof, and an application thereof. Background Art

[0002] Terpene synthases are an enzyme family produced by organisms and involved in the biosynthetic pathway of terpenoid compounds. Terpenoid compounds are compounds composed of isoprene units (i.e., C5 units), widely distributed in nature, and are the main components of natural products such as fragrances, resins, and pigments. These enzymes use isoprenyl diphosphate substrates, such as geranyl diphosphate and farnesyl diphosphate, to generate terpenoid compounds through different reaction mechanisms. There is a lack of sequence-function relationship within the terpene synthase family, which makes it challenging to predict the product-result solely from the sequence.

[0003] Farnesene synthase belongs to class I terpene synthases and exhibits different activities in different hosts. Both protein activity and expression level may affect the yield of farnesene. Currently, farnesene synthase can be modified by protein fusion and overexpression to obtain a higher yield of farnesene. In yeast, fusing farnesene synthase and farnesyl diphosphate synthase and overexpressing them can increase the farnesene yield by 3 to 4 times compared to the control group. However, there are still challenges in achieving high-yield farnesene through genetic modification of farnesene synthase. Overexpression of farnesene synthase can cause toxicity to the host organism. In addition, the use of fusion proteins can lead to a decrease in expression level or a change in enzyme activity. Therefore, further research is needed to optimize the genetic modification of farnesene synthase to achieve efficient preparation of farnesene.

[0004] In many studies, it has been shown that recombinant fusion enzymes exhibit higher catalytic efficiency than the corresponding mixture of single enzymes. Therefore, there is great potential in using fusion enzymes for metabolic engineering at the branch points of biosynthetic pathways. Currently, there is no report on fusing the farnesene synthase gene with the farnesyl pyrophosphate synthase gene and expressing it in Escherichia coli for farnesene production. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a recombinant strain for producing farnesene.

[0006] The second object of the present invention is to provide a construction method of a recombinant strain for producing farnesene.

[0007] The third object of the present invention is to provide an application of a recombinant strain for producing farnesene in fermentative production of farnesene.

[0008] The technical solution of the present invention is outlined as follows:

[0009] A construction method of a recombinant strain for producing farnesene, comprising the following steps:

[0010] 1) Prepare the pFII plasmid containing farnesene synthase, farnesyl diphosphate synthase, and isopentenyl diphosphate isomerase;

[0011] 2) Using the pFII plasmid obtained in step 1) as a template, perform PCR amplification with primers 1 and 2 to obtain fragment 1;

[0012] Using the pFII plasmid obtained in step 1) as a template, perform PCR amplification with primers 3 and 4 to obtain fragment 2;

[0013] Using the pFII plasmid obtained in step 1) as a template, perform PCR amplification with primers 5 and 6 to obtain fragment 3;

[0014] The nucleotide sequence of primer 1 is shown in SEQ ID No.4;

[0015] The nucleotide sequence of primer 2 is shown in SEQ ID No.5;

[0016] The nucleotide sequence of primer 3 is shown in SEQ ID No.6;

[0017] The nucleotide sequence of primer 4 is shown in SEQ ID No.7;

[0018] The nucleotide sequence of primer 5 is shown in SEQ ID No.8;

[0019] The nucleotide sequence of primer 6 is shown in SEQ ID No.9;

[0020] 3) Perform seamless ligation of fragment 1, fragment 2, and fragment 3 to obtain plasmid 1 containing the fusion gene of farnesene synthase and farnesyl diphosphate synthase and the isopentenyl diphosphate isomerase gene;

[0021] The nucleotide sequence of plasmid 1 is shown in SEQ ID No.1;

[0022] 4) Introduce plasmid 1, plasmid 2, and plasmid 3 into Escherichia coli to obtain a recombinant strain producing farnesene;

[0023] The nucleotide sequence of plasmid 2 is shown in SEQ ID No.2;

[0024] The nucleotide sequence of plasmid 3 is shown in SEQ ID No.3.

[0025] The recombinant strain producing farnesene constructed by the above method.

[0026] The application of the above recombinant strain in the fermentation production of farnesene.

[0027] Advantages of the present invention:

[0028] 1. Optimize the farnesene synthesis pathway in Escherichia coli using a fusion gene, and a recombinant strain is obtained, providing new ideas and technical means for the high efficiency and controllability of farnesene biosynthesis.

[0029] 2. The recombinant strain constructed in the present invention has a farnesene yield as high as 3.3 g / L, providing technical support and data reference for the large-scale preparation of farnesene by biological methods.

[0030] 3. Aiming at the complexity of the farnesene biosynthesis pathway, the recombinant strain constructed in the present invention contains a fusion gene of farnesene synthase and farnesyl pyrophosphate synthase, realizing the improvement of farnesene yield. Description of the Drawings

[0031] Figure 1 It is the farnesene yield of the recombinant strain 1 for producing farnesene of the present invention at different times during shake flask fermentation. Detailed Embodiments

[0032] The technical solutions of the present invention will be further described in detail below through specific examples and drawings. The following examples are only descriptive, and the scope of protection of the present invention is not limited thereto.

[0033] The raw materials used in the present invention are all conventional commercially available products without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0034] Example 1

[0035] Construction of plasmid 1 containing a fusion gene of farnesene synthase and farnesyl pyrophosphate synthase and isopentenyl diphosphate isomerase gene:

[0036] 1) Prepare the pFII plasmid containing farnesene synthase, farnesyl pyrophosphate synthase and isopentenyl diphosphate isomerase by the method disclosed in Chinese Patent CN109722403A;

[0037] 2) Using the pFII plasmid obtained in step 1 as a template, perform PCR amplification with primers 1 and 2 to obtain fragment 1;

[0038] Using the pFII plasmid obtained in step 1 as a template, perform PCR amplification with primers 3 and 4 to obtain fragment 2;

[0039] Using the pFII plasmid obtained in step 1 as a template, perform PCR amplification with primers 5 and 6 to obtain fragment 3;

[0040] Table 1

[0041]

[0042] 3) The fragments 1, 2 and 3 were seamlessly ligated using seamless cloning technology to obtain plasmid 1 containing the farnesene synthase and farnesyl pyrophosphate synthase fusion gene and the isopentenyl diphosphate isomerase gene; the nucleotide sequence of plasmid 1 is shown as SEQ ID No.1.

[0043] Example 2

[0044] Construction of a recombinant strain producing farnesene

[0045] Plasmid 1, plasmid 2 (SEQ ID No.2) and plasmid 3 (SEQ ID No.3) constructed in Example 1 were introduced into Escherichia coli BL21(DE3) to obtain recombinant strain 1 producing farnesene.

[0046] Plasmid 2 was constructed according to the steps disclosed in CN109722403A and is plasmid pMEVT containing the genes of acetyl-CoA thiolase, HMG-CoA synthase and HMG-CoA reductase.

[0047] Plasmid 3 was constructed according to the steps disclosed in CN109722403A and is plasmid pMBIS containing the genes of mevalonate kinase, phosphomevalonate kinase and hydroxymethylglutaryl pyrophosphate decarboxylase.

[0048] Example 3

[0049] Preparation of farnesene by shake flask fermentation of recombinant strain 1 producing farnesene

[0050] The seed culture of recombinant strain 1 producing farnesene constructed in Example 2 was inoculated into a shake flask for fermentation to prepare farnesene:

[0051] Prepare the fermentation medium: Take 16 g of peptone, 10 g of yeast extract and 5 g of NaCl, add water to 1 L, and sterilize.

[0052] Take 300 mL and divide it equally into three 500 mL conical flasks (three parallel samples).

[0053] Under sterile conditions, add 1.57 ml of sterilized glycerol, 100 ul of chloramphenicol solution with a concentration of 34 g / L, 100 ul of kanamycin solution with a concentration of 50 g / L and 100 ul of ampicillin solution with a concentration of 100 g / L to each conical flask.

[0054] Inoculate the seed culture of recombinant strain 1 and culture it at 37 °C and 220 rpm until OD 600Between 0.6 and 0.8, the fermentation broth was obtained. 100 μL of IPTG solution with a concentration of 24 g / L, 1 mL of L-arabinose solution with a concentration of 300 g / L and 20 mL of n-decane were added to the fermentation broth, and induction expression was carried out at 33 °C and 220 rpm. 100 μL of the organic phase was taken every 24 h for gas chromatography detection of the farnesene concentration. The farnesene yield of the recombinant strain 1 was 3.3 g / L after 96 h of fermentation, as shown in Figure 1 .

Claims

1. Method for constructing recombinant strain for producing farnesene, characterized in that It includes the following steps: 1) Prepare the pFII plasmid containing farnesene synthase, farnesyl pyrophosphate synthase, and isopentenyl diphosphate isomerase; 2) Using the pFII plasmid as a template, perform PCR amplification with primer 1 and primer 2 to obtain fragment 1; using the pFII plasmid as a template, perform PCR amplification with primer 3 and primer 4 to obtain fragment 2; using the pFII plasmid as a template, perform PCR amplification with primer 5 and primer 6 to obtain fragment 3; The nucleotide sequence of the primer 1 is as shown in SEQ ID No.4; The nucleotide sequence of the primer 2 is as shown in SEQ ID No.5; The nucleotide sequence of the primer 3 is as shown in SEQ ID No.6; The nucleotide sequence of the primer 4 is as shown in SEQ ID No.7; The nucleotide sequence of the primer 5 is as shown in SEQ ID No.8; The nucleotide sequence of the primer 6 is as shown in SEQ ID No.9; 3) Perform seamless ligation of fragment 1, fragment 2, and fragment 3 to obtain plasmid 1 containing the fused gene of farnesene synthase and farnesyl pyrophosphate synthase and the isopentenyl diphosphate isomerase gene; The nucleotide sequence of the plasmid 1 is as shown in SEQ ID No.1; 4) Introduce plasmid 1, plasmid 2, and plasmid 3 into Escherichia coli to obtain a recombinant strain producing farnesene; The nucleotide sequence of the plasmid 2 is as shown in SEQ ID No.2; The nucleotide sequence of the plasmid 3 is as shown in SEQ ID No.

3.

2. A recombinant strain producing farnesene constructed by the method of claim 1.

3. Use of the recombinant strain of claim 2 for fermentative production of farnesene.

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