Engineering strain for synthesizing squalene and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an engineering strain for synthesizing squalene and application of the engineering strain, the engineering strain is obtained by transforming an expression vector into bacillus subtilis, the expression vector comprises a squalene synthase gene tll1096, the transformed engineering strain can synthesize squalene, and the engineering strain can be used for synthesizing squalene. And on the basis, an engineering strain for more efficiently producing squalene can be obtained by dynamically regulating and controlling a TCA way, and the synthesis amount of squalene can reach 12.47 mg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineering strain for synthesizing squalene and application thereof. Background Art

[0002] Squalene is a straight-chain unsaturated olefin formed by the end-to-end connection of six isoprene units. It is an important intermediate in the synthesis of many biologically active triterpenoid compounds, such as hopane compounds and sterol compounds that play important cellular functions in organisms. In addition, squalene itself has a variety of physiological activities, such as antioxidant effects, reducing the risk of cancer and enhancing the immune system, which makes it widely used in the medical drugs, food and cosmetics industries as a dietary supplement, vaccine adjuvant, moisturizer, cardioprotectant, anti-tumor agent and natural antioxidant.

[0003] Squalene was originally extracted from shark livers. With the sharp decline in shark populations and the release of relevant animal protection regulations, squalene can no longer be produced through this channel. However, with the increasing demand for squalene, extracting squalene from plants has become a new research direction in order to meet market demand. However, the squalene content in plants is very low, the extraction and purification process is complicated, and the seasonal production of raw materials and intensive labor requirements make it difficult to meet large-scale market demand, limiting its potential as an alternative source. In addition, some researchers have achieved large-scale production of squalene through chemical synthesis. At present, more than a dozen chemical synthesis methods and pathways have been reported. However, the chemical synthesis method of squalene is relatively difficult, the reaction conditions are generally harsh, and there are problems such as high pollution and by-products, resulting in high downstream separation and purification costs, which is difficult to expand to application areas such as food and health products. Therefore, establishing a new green manufacturing route for squalene has become an urgent problem to be solved in the industrial production of squalene. Microorganisms have the advantages of fast growth, easy cultivation, and strong metabolic plasticity, and are considered to be a potential choice to meet these challenges.

[0004] With the rapid development of synthetic biology and metabolic engineering, the efficient, simple, environmentally friendly and sustainable biosynthesis of squalene can be achieved by constructing microbial cell factories. Bacillus subtilis, a high isoprene producer, is a safe microorganism classified as food grade. It has the advantages of simple and clear genome structure, easy genetic modification, high cell growth density, short cycle, easy cultivation, simple fermentation conditions, and strong protein secretion ability. In addition, as a representative of triterpenoid compounds, the synthesis of squalene requires the key intermediates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as precursor compounds, just like the biosynthetic pathways of other terpenoid compounds. There are currently two main pathways for the synthesis of IPP and DMAPP: the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway; the MVA pathway mainly exists in eukaryotic organisms, such as plants and fungi; the MEP pathway mainly exists in prokaryotes, such as Bacillus subtilis. Therefore, from the above we can know that although Bacillus subtilis cannot synthesize squalene, it has the potential to synthesize squalene after modification. Summary of the invention

[0005] The present invention provides an engineered strain of Bacillus subtilis, which can be obtained by transferring the squalene synthase gene tll1096, the farnesyl pyrophosphate synthase gene or the key gene of the MEP pathway into wild Bacillus subtilis; and on this basis, an engineered strain for efficiently producing squalene can also be obtained by dynamically regulating the TCA pathway.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an engineered strain for synthesizing squalene, which is obtained by transforming an expression vector into Bacillus subtilis, wherein the expression vector comprises a sequence as shown in SEQ ID NO:1.

[0008] Preferably, the above expression vector also includes the sequence shown in SEQ ID NO:2.

[0009] Preferably, the above expression vector also includes key genes of the MEP pathway.

[0010] Preferably, the above-mentioned key genes of the MEP pathway include one or more of the dxs, dxr, ispd or isph genes; the sequences of the dxs, dxr, ispd and isph genes are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

[0011] In another aspect, the present invention provides an engineered strain with dynamic regulation capability, which is obtained by replacing the pdhA gene promoter in the engineered strain of the present invention with a logarithmic phase-dependent promoter.

[0012] Preferably, the logarithmic phase dependent promoter is selected from P abrB Promoter, P valS Promoter or P hag One or more of the promoters; P abrB Promoter, P valS Promoter and P hag The nucleotide sequence of the promoter may include the sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0013] The present invention also provides a fermentation system for squalene synthesis, comprising the engineered strain of the present invention and / or the engineered strain with dynamic regulation capability of the present invention.

[0014] Preferably, the fermentation system for squalene synthesis further comprises a Bacillus subtilis culture medium.

[0015] In another aspect, the present invention provides an application of the engineered strain of the present invention and / or the engineered strain with dynamic regulation capability of the present invention and / or the fermentation system for squalene synthesis of the present invention in synthesizing squalene.

[0016] The beneficial effects of the present invention include at least:

[0017] (1) The Bacillus subtilis engineered strain heterologously expressing the squalene synthase gene tll1096 provided by the present invention can synthesize squalene, and the yield can reach 3.2 mg / L.

[0018] (2) The Bacillus subtilis engineered strain provided by the present invention that heterologously expresses the squalene synthase gene tll1096 and overexpresses the farnesyl pyrophosphate synthase gene can synthesize squalene with a yield of up to 4.3 mg / L, which is nearly 35% higher than that of the engineered strain that only transduced the tll1096 gene.

[0019] (3) The Bacillus subtilis engineered strain provided by the present invention, which heterologously expresses the squalene synthase gene tll1096 and overexpresses the farnesyl pyrophosphate synthase gene and the key genes of the MEP pathway, can synthesize squalene, and the synthesis amount can reach 10.82 mg / L, which is about 238.1% higher than the yield of the engineered strain integrated with only the tll1096 gene; and about 151.6% higher than the yield of the engineered strain integrated with the tll1096 gene and the farnesyl pyrophosphate synthase gene.

[0020] (4) The Bacillus subtilis engineered strain provided by the present invention, which heterologously expresses the squalene synthase gene tll1096, overexpresses the farnesyl pyrophosphate synthase gene and the key genes of the MEP pathway, and replaces the pdhA gene promoter with a logarithmic phase-dependent promoter, can synthesize squalene, and the synthesis yield can reach 12.47 mg / L, which is about 289.7% higher than the yield of the engineered strain that only integrates the tll1096 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of dynamic regulation of TCA;

[0022] Figure 2 pHT01-P 43 Vector construction strategy and schematic diagram;

[0023] Figure 3 The construction strategy and schematic diagram of squalene synthase expression vector;

[0024] Figure 4 The construction strategy and schematic diagram of the recombinant vector for overexpressing ispA;

[0025] Figure 5 The strategy and schematic diagram for constructing MEP multi-gene overexpression vector;

[0026] Figure 6 The construction strategy and schematic diagram of the pdhA gene promoter replacement cassette;

[0027] Figure 7 To dynamically regulate the effects on cell growth;

[0028] Figure 8 This is a comparison chart of squalene production of engineered strains. DETAILED DESCRIPTION

[0029] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0030] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0031] The embodiment of the present invention provides an engineered strain for synthesizing squalene, which is obtained by transforming an expression vector into Bacillus subtilis, wherein the expression vector includes a sequence as shown in SEQ ID NO:1.

[0032] It should be noted that the sequence shown in SEQ ID NO: 1 in the present invention may be the squalene synthase gene tll1096 from thermophilic cyanobacteria (T. elongatus). The engineered strain constructed in the present invention can synthesize squalene, and the yield can reach 3.2 mg / L; indicating that the engineered strain obtained by transforming the squalene synthase gene tll1096 into Bacillus subtilis can synthesize squalene.

[0033] It should also be noted that in the present invention, the key gene tll1096 for squalene synthesis is constructed into the pHT01 plasmid, and the key genes of ispA and MEP pathway are overexpressed to obtain the recombinant plasmid, which is then transformed into the SCK6 strain to achieve heterologous expression of squalene synthase and overexpression of ispA and MEP pathway genes, reconstruct the SCK6 metabolic pathway, and enable Bacillus subtilis that cannot synthesize squalene to synthesize squalene.

[0034] In some specific examples, the expression vector may also include a sequence as shown in SEQ ID NO:2.

[0035] It should be noted that the sequence shown in SEQ ID NO: 2 may be derived from the nickyl pyrophosphate synthase gene (ispA gene), which is constructed into an expression vector and transformed into Bacillus subtilis, so that the overexpression of the nickyl pyrophosphate synthase gene can increase the synthesis of squalene. Specifically, the overexpression of the farnesyl pyrophosphate synthase gene (ispA gene) by the engineering strain of the present invention is conducive to the synthesis of terpenoids, and the integration of the ispA gene into the above-mentioned engineering strain for overexpression can significantly increase the synthesis of squalene, and the yield can reach 4.3 mg / L, which is nearly 35% higher than that of the above-mentioned engineering strain.

[0036] In some specific examples, the above expression vector may also include key genes of the MEP pathway.

[0037] It should be noted that after the key genes of the MEP pathway are constructed into the above-mentioned expression vector and transformed into Bacillus subtilis, the overexpression of the key genes of the MEP pathway can further increase the synthesis of squalene; specifically, the MEP pathway can increase the precursor substances such as IPP and DMAPP for the synthesis of squalene. After overexpressing the key genes of the MEP pathway (such as dxs, dxr, ispd or isph), the engineered strain can increase the synthesis of squalene, and the synthesis amount can reach 10.82 mg / L, which is about 238.1% higher than the yield of the engineered strain integrated with the tll1096 gene; the yield is increased by about 151.6% compared with the engineered strain integrated with the tll1096 gene and the farnesyl pyrophosphate synthase gene.

[0038] In some specific examples, the above-mentioned key genes of the MEP pathway include one or more of the dxs, dxr, ispd or isph genes; the sequences of the dxs, dxr, ispd and isph genes are shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, respectively.

[0039] It should be noted that the key genes of the MEP pathway of the present invention are well known in the art, including but not limited to dxs, dxr, ispd or isph genes. One of the genes listed above can be constructed, or multiple genes can be constructed into the expression vector at the same time, which can further increase the synthesis of squalene.

[0040] When integrated into any of the above-mentioned engineered strains, one of the genes listed above can be integrated, or multiple genes can be integrated at the same time; in addition, the nucleotides of the dxs, dxr, ispd and isph genes can include the sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0041] In some specific examples, the Bacillus subtilis is Bacillus subtilis SCK6 strain.

[0042] It should be noted that the Bacillus subtilis in the present invention may be any Bacillus subtilis known in the present invention, preferably Bacillus subtilis SCK6.

[0043] The embodiment of the present invention further provides an engineering strain with dynamic regulation capability, which is obtained by replacing the pdhA gene promoter in the engineering strain of the present invention with a logarithmic phase-dependent promoter.

[0044] In some specific examples, the logarithmic phase-dependent promoter is selected from P abrB Promoter, P valS Promoter or P hag One or more of the promoters; P abrB Promoter, P valS Promoter and P hag The nucleotide sequence of the promoter may include the sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0045] It should be noted that in the present invention, by replacing the promoter of the pdhA gene (pyruvate dehydrogenase E1 subunit) in the engineered strain with a logarithmic phase-dependent promoter, the engineered strain for efficiently producing squalene can be obtained by dynamically regulating the TCA pathway (a schematic diagram of dynamically regulating TCA is shown in FIG. Figure 1 As shown in the figure, in the cell growth stage, the pyruvate dehydrogenase complex is expressed. When the cell enters the late stage and reaches the product accumulation stage, the expression of the pyruvate dehydrogenase complex is weakened, thereby weakening the metabolic flux entering the TCA cycle, so that more carbon sources flow to the direction of squalene synthesis. Through this strategy, cells can reasonably allocate carbon sources and separate cell growth from production. The problems of slow cell growth and metabolic imbalance in the process of microbial synthesis of squalene are solved; the squalene synthesis yield of the engineered strain after replacing the promoter can reach 12.47 mg / L, which is about 289.7% higher than the yield of the engineered strain integrated with the tll1096 gene.

[0046] It should also be noted that the traditional metabolic engineering transformation method is to statically optimize the target metabolic pathway by designing and transforming genetic coding components. The biggest feature of this transformation method is orientation and irreversibility. The transformed cells do not have the ability to sense specified signals and make specific adjustments during the growth process; this single reduction or increase in the expression of key proteins may have irreversible side effects on cell growth, and face many challenges in the later scale-up production. Therefore, in order to timely balance the relationship between the gene expression required for product synthesis and the global metabolic level, the introduction of the dynamic regulation gene circuit of the present invention can respond to metabolic signals in real time, and timely feedback regulation, so as to adapt to changes in the host's internal metabolism or environment, stably synthesize the required products, and improve the stability and scalability of fermentation culture.

[0047] The embodiment of the present invention further provides a fermentation system for squalene synthesis, comprising the engineered strain of the present invention and / or the engineered strain with dynamic regulation capability of the present invention.

[0048] It should be noted that any of the above-mentioned engineered strains can be prepared into a fermentation system for synthesizing squalene, and a large amount of squalene can be synthesized, thus solving the problem that Bacillus subtilis cannot synthesize squalene.

[0049] In some specific examples, the fermentation system for squalene synthesis further includes a Bacillus subtilis culture medium.

[0050] It should be noted that the other substances prepared into the fermentation system with the engineered strain can be any culture medium suitable for the growth of Bacillus subtilis (i.e., Bacillus subtilis culture medium); of course, in addition to these, other substances may also be included, such as additives that promote growth.

[0051] The embodiments of the present invention also provide an application of the engineered strain of the present invention and / or the engineered strain with dynamic regulation capability of the present invention and / or the fermentation system for squalene synthesis of the present invention in synthesizing squalene.

[0052] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0053] In the following examples, the primer pair sequences used are shown in Table 1.

[0054] Table 1 Primer pairs used in the embodiments of the present invention

[0055]

[0056] In the following example, strain SCK6: originated from B. subtilis 168, ErmR, 1A751 derived, lacA::PxylA-comK, constructed according to Zhang XZ, Zhang YHP. Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. [J]. Microbial Biotechnology, 2011, 4(1): 98-105. DOI: 10.1111 / j.1751-7915.2010.00230.x. provided by Professor Shi Ting from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

[0057] 1. Construction of recombinant plasmid for squalene synthesis

[0058] (I) Construction of recombinant plasmid pHT01-P43

[0059] Replace the inducible promoter P on plasmid pHT01 grac The constitutive promoter P 43 :The recombinant vector pHT01-P43 was constructed by seamless cloning. The construction diagram is shown in Figure 2 ; The specific method is as follows:

[0060] (1) Using pHT01 plasmid as the starting plasmid, pHP13-P 43 The plasmid was used as a template and pHT01-P43-F / pHT01-P43-R as primers (see Table 1) were used for PCR amplification to obtain the PCR amplification product (promoter P 43 , sequence as shown in SEQ ID NO: 10), PCR system as shown in Table 2, amplification program as shown in Table 3 below;

[0061] Table 2 Preparation of PCR system (50 μL)

[0062] Reagents Dosage PrimeSTARMaxPremix (2×) 25μL Upstream primer 2μL Downstream primer 2μL DNA template 2μL Double distilled water 19μL

[0063] Table 3 PCR amplification program

[0064] 1) 94℃, 3min - 2)98℃，10s - 3)55℃，15s - 4) 72℃, 5s / kb 2), 3), 4) Repeat 35 times 5) 72℃, 5min -

[0065] (2) Purify the PCR product using a PCR product purification kit (Shanghai Sangon Biotechnology Co., Ltd.) to obtain the purified target gene;

[0066] (3) The purified target gene was ligated to the pHT01 plasmid double-digested with KpnI and BamHI using T4 DNA ligase (Takara) to obtain a recombinant plasmid (pHT01-P 43 ); wherein, the double enzyme digestion system is shown in Table 4 below, the reaction system for connecting the target gene to the vector is shown in Table 5, and the reaction procedure is: react at 4°C overnight.

[0067] Table 4 Preparation of double enzyme digestion system (20 μL)

[0068]

[0069]

[0070] Table 5 Preparation of the connection system (20 μL)

[0071] Reagents Dosage T4 Ligase 2μL T4DNALigaseBuffer 1μL pHT01 plasmid fragment 2μg Purpose fragment 4 μg Double distilled water up to 20 μL

[0072] (4) The recombinant plasmid was transformed into Escherichia coli DH5α by the heat shock method, and the engineered strain was spread on LB medium agar plates containing 100 μg / mL ampicillin and incubated at 37°C for 12 h. A single colony was picked and placed in 5 mL of LB medium containing ampicillin (Sangon Biotechnology (Shanghai) Co., Ltd.), and shake-cultured at 37°C for 12 h. After that, plasmid DNA was extracted to obtain the pHT01-P43 recombinant plasmid, which was then sent to a sequencing company for sequencing verification.

[0073] (II) Construction of pHT01-P43 -TeSQS recombinant plasmid

[0074] In plasmid pHT01-P 43 The codon-optimized squalene synthase gene (tll1096) from thermophilic cyanobacteria was heterologously expressed to construct pHT01-P 43 -TeSQS recombinant plasmid, and transformation details are as follows:

[0075] (1) Using the Java codon adaptation basis, the squalene synthase gene sequence from T. Elongatus in Bacillus subtilis (B. subtilis168) was optimized to prevent rare codons from blocking gene expression; restriction sites such as XbaI, SfiI, and XmaI were avoided in the codon-optimized sequence; the optimized gene sequence was flanked by an upstream restriction endonuclease, an RBS (AAAGGAGG), and a downstream restriction endonuclease; finally, the whole gene was synthesized by Qingke Biotechnology Co., Ltd. and constructed on the pUC57 plasmid;

[0076] (2) The recombinant plasmid was constructed by restriction enzyme ligation, specifically including: designing primer pair Tesqs-F / Tesqs-R according to the optimized TeSQS sequence (SEQ ID NO: 1), amplifying the tll1096 (EMBL: BAC08649) gene (the amplification system and amplification procedure are shown in Table 2 and Table 3 above, respectively), purifying the PCR product using a PCR product purification kit, and ligating the purified target gene to pHT01-P double-digested with XbaI and XmaI. 43 Recombinant plasmid pHT01-P 43 -TeSQS;

[0077] (3) The recombinant plasmid was transformed into Escherichia coli DH5α by heat shock method, and the engineered strain was spread on LB medium agar plate containing 100 μg / mL ampicillin and incubated at 37°C for 12 h; a single colony was picked and placed in 5 mL LB medium containing ampicillin, and shake-cultured at 37°C for 12 h, after which plasmid DNA was extracted to obtain pHT01-P 43 -TeSQS recombinant plasmid; plasmid PCR verification of Tesqs-F / PHT-R was performed using primers (amplification system and amplification program are shown in Table 2 and Table 3 above, respectively); schematic diagram of heterologous squalene synthase recombinant plasmid construction is shown in Figure 3 .

[0078] (III) Construction of recombinant plasmid pHT01-p43-ispA-TeSQS

[0079] Overexpression of farnesyl pyrophosphate synthase gene (ispA) to construct recombinant plasmid pHT01-p43-ispA-TeSQS, specifically including: 43 -TeSQS recombinant plasmid to construct pHT01-P43-ispA-tSQS recombinant vector; the pHT01-P43-tSQS plasmid was double-digested with SfiI and XmaI, and the digested fragments were recovered using a gel recovery kit; the primer pair ispa-F / ispa-R was designed using the gene sequence of ispA (NCBIGene ID: 938652), and the ispA gene was amplified using B. subtilis Z2 (CCTCC NO: M2020002) genomic DNA as a template (the amplification system and amplification procedure are shown in Tables 2 and 3 above, respectively), and then double-digested with Sfi I and Xma I and ligated to the pHT01-P43-tSQS plasmid that had been digested with the same enzymes. 43 -tSQS vector; and the positive transformants were selected by the above method and plasmid DNA was extracted to obtain pHT01-p43-ispA-TeSQS recombinant plasmid; the ispA gene overexpression vector construction strategy is shown in Figure 4 .

[0080] (IV) Construction of recombinant plasmid pHT01-P 43 -MEP-ispA-TeSQS

[0081] Overexpression of key genes in the MEP pathway to construct the recombinant plasmid pHT01-P 43 -MEP-ispA-TeSQS, in pHT01-P 43 -ispA-tSQS recombinant vector to construct pHT01-P 43 -MEP-ispA-TeSQS recombinant vector;

[0082] (1) Using B. subtilis Z2 genomic DNA as a template, four genes, dxs, dxr, ispd, and isph, were amplified (the primers for amplifying dxs were P 43 S-F2 and P 43 S-R2; the primer for amplifying dxr is P 43 SR-F and P 43 SR-R; the primer for amplifying ispd is P 43 D-F2 and P 43 D-R2; the primer for amplifying isph is P 43 DH-F and P 43 DH-R; the amplification system and amplification procedure are shown in Table 2 and Table 3 above, respectively);

[0083] (2) Obtaining the recombinant plasmid pHT01-P overexpressing the MEP pathway by overlapping extension PCR and seamless cloning 43 -MEP-ispA-TeSQS (MEP genes are dxs, dxr, ispd and isph): First, use primer P 43 S-F1 and P 43 S-R1 amplified P 43 ; Then use primer P 43 S-F2 and P 43 S-R2 amplified dxs (the amplification system and amplification procedure are shown in Table 2 and Table 3 above, respectively); then, without adding primers, the template (the P amplified above) was 43 After mixing with dxs) in a ratio of 1:1, the amplification was performed using the following procedures: 1) 94°C for 3 min; 2) 98°C for 10 s; 3) 55°C for 15 s; 4) 72°C for 5 s / kb, and step 2) was repeated 15 times; 3) 72°C for 5 min; primers were then added and the amplification was performed 25 times under the same conditions; the overlap extension PCR products were then ligated to pHT01-P by seamless cloning. 43 -ispA-TeSQS plasmid to obtain the recombinant plasmid

[0084] pHT01-P 43 -dxs-dxr-P 43 -ispA-TeSQS: Use primer P 43 SR-F and P 43 SR-R amplified dxr (amplification system and amplification procedure are shown in Table 2 and Table 3 above, respectively); then connected to it by seamless cloning

[0085] pHT01-P 43 -dxs-P 43 -ispA-TeSQS to obtain the recombinant plasmid pHT01-P 43 -dxs-dxr-P 43 -ispA-TeSQS; pHT01-P 43 -dxs-dxr-ispd-P 43 -ispA-TeSQS recombinant plasmid and

[0086] pHT01-P 43 -dxs-dxr-ispd-isph-P 43 -ispA-TeSQS recombinant plasmid (i.e. pHT01-P 43 -MEP-ispA-TeSQS) (wherein the primer pairs used are: P 43 (P 43 D-F1, P 43D-R1), ispd(P 43 D-F2, P 43 D-R2); isph(P 43 DH-F、P 43 DH-R));

[0087] (3) Using the above method (the method of (ii) (3) above) to select positive transformants, plasmid DNA was extracted to obtain pHT01-P 43 -MEP-ispA-TeSQS recombinant plasmid; vector construction schematic diagram see Figure 5 .

[0088] 2. Construction of engineered strains BS-1, BS-2 and BS-3 for squalene synthesis

[0089] pHT01-P 43 -TeSQS recombinant plasmid, pHT01-P 43 -ispA-TeSQS recombinant plasmid and pHT01-P 43 -MEP-ispA-TeSQS recombinant plasmid was transformed into SCK6 strain to obtain engineered strains BS-1, BS-2 and BS-3; the details are as follows:

[0090] The Bacillus subtilis SCK6 strain was inoculated into 20 mL LB medium containing 1 μg / mL erythromycin in a test tube, and cultured overnight (about 12 h) at 37°C and 200 rpm with shaking to obtain a culture; the culture was diluted to OD 0.05 in fresh LB medium containing 1% (w / v) xylose. 600 =1.0, and then grow for 2 hours; divide the obtained cell culture into equal parts and store at -80°C with 10% glycerol; add 1 μL of the target gene fragment (recombinant plasmid) to the EP tube containing 200 μL of competent cells, flick it to mix, and shake and culture at 37°C 200rpm for 90 minutes; spread 200 μL of bacterial solution on solid LB medium containing 5 μg / mL Chl, and culture at 37°C overnight to obtain single colonies; pick several single colonies in 10 μL of sterile water, and use 2×T5 SuperPCRMix for colony PCR verification; positive colonies are inoculated in liquid LB medium containing 5 μg / mL Chl, and shake and culture at 37°C overnight; further verify by bacterial solution PCR (amplification system and amplification program are shown in Table 2 and Table 3 above, respectively); PCR products are sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0091] 3. Construction of an engineered bacterium that dynamically regulates squalene synthesis

[0092] (I) Construction of the pdhA gene promoter replacement cassette

[0093] The Cre / loxp scarless editing method was used to integrate the promoter replacement cassette into the SCK6 genome, specifically including:

[0094] (1) Using the SCK6 genome as a template, primer pairs pdhA upper-F / pdhA upper-R and pdhA lower-P abrB -F(pdhA under -P hag -F, pdhA down -P vals -F) / pdhA-R PCR amplification (amplification system and amplification program are shown in Table 2 and Table 3 above, respectively) to obtain 900 bp of homology arms upstream and downstream of the insertion site (upstream promoter of pdhA gene);

[0095] (2) Plasmid p7z6 was used as a template and primer pair lox-F / lox-R was used to PCR amplify the gene fragment lox71-BleoR-lox66 containing bleomycin and lox sites;

[0096] (3) Then, using the B. subtilis 168 genome as a template, primer pairs P abrB -F / P abrB -R, P hag -F / P hag -R, P vals -F / P vals -R were used to perform PCR amplification (the amplification system and amplification procedure are shown in Table 2 and Table 3 above, respectively) to obtain P abrB Promoter, P valS Promoter and P hag Promoter;

[0097] (4) Fusion PCR was used to fuse the fragments obtained by the above amplification to obtain pdhA-lox71-BleoR-lox66-P abrB -pdhA lower promoter replaces the integration frame, pdhA upper-lox71-BleoR-lox66-P valS -pdhA lower promoter replaces the integration cassette and pdhA upper -lox71-BleoR-lox66-P hag -pdhA promoter replaces the integration cassette.

[0098] (5) Then, the integration frame was transformed into SCK6 competent cells by the above method (the method of (ii) (3) above), and screened in LB medium containing bleomycin. After the transformants were verified to be correct by bacterial liquid PCR and sequencing, they were stored in a -80°C refrigerator with 15% glycerol;

[0099] (6) To eliminate the resistance gene in the strain, the plasmid PDG148-Cre was transformed into the strain and screened on an LB plate containing bleomycin and ampicillin. After PCR verification, the transformant was inoculated into an LB medium containing 0.2 mmol / L IPTG to induce the expression of Cre recombinase, thereby starting site-specific recombination to eliminate the resistance gene. Subsequently, the strain was transferred to an LB antibiotic-free medium and cultured at 50°C and 220 rpm for 12 h. The culture solution was then streaked onto an LB antibiotic-free solid medium. When a single colony grew on the plate, it was spotted on the corresponding antibiotic plate to verify whether the resistance gene had been eliminated. The engineered strain BS-4 (P abrB ), BS-5(P ValS ) and BS-6(P hag ); construction diagram see Figure 6 .

[0100] 4. Verification of the synthetic effect of squalene

[0101] (I) Fermentation of engineered strains

[0102] Bacillus subtilis SCK6, BS-1, BS-2, BS-3, BS-4, BS-5 and BS-6 preserved in frozen glycerol tubes were streaked onto LB solid plates and cultured overnight in an incubator at 37°C; a single colony on the solid plate was picked and placed in 50 mL of fermentation medium (17 g / L peptone, 3 g / L soytone, 2.5 g / L glucose, 5 g / L NaCl, 2.5 g / L K2HPO4; 115°C, 30 min high pressure liquid sterilization), and cultured at 37°C 180 rpm for 24 h to obtain a seed culture solution; the seed culture solution was inoculated into 100 mL of fermentation medium at a volume ratio of 1:100, and shake flask fermentation was carried out at 37°C, 180 rpm for aerobic culture for 48 h.

[0103] (II) Extraction and detection of squalene in engineered strains

[0104] ① Extraction of squalene: Take 50 mL of fermentation broth after 48 h of shake flask fermentation, centrifuge at 8000 rpm at 4 ° C for 5 min, discard the supernatant, add 10 mL of PBS (pH 7.2-7.4) to the precipitate and vortex to break it up, centrifuge at 8000 rpm at 4 ° C for 5 min, discard the supernatant, wash the bacteria twice and resuspend them with 5 mL of 1% sodium hydroxide methanol, and perform ultrasonic disruption on ice, set to a power of 300 W, work for 10 s, rest for 10 s, and ultrasonicate for 30 min; after fully disrupting the cells, add 5 mL of n-hexane, vortex at 2000 rpm for 10 min to extract the target product; centrifuge at 13000 rpm for 5 min, collect the upper organic phase in a clean EP tube, blow dry and redissolve it with an appropriate amount of n-hexane, filter it with a 0.22 μm organic filter membrane, and place it in a brown gas phase injection bottle.

[0105] ② Detection and identification of squalene: Squalene was detected by gas chromatography, and the gas phase conditions were as follows: DB-5 capillary column (30m×0.25mm×0.25μm); injection port temperature was 280℃; detector temperature was 300℃; carrier gas was nitrogen, and the flow rate was 1.5mL / min; injection volume was 1μL, and the split ratio was 10:1; the detector was FID detector: the initial column temperature was 150℃, maintained for 2min, increased to 200℃ at a rate of 20℃ / min, maintained for 2min, and then increased to 260℃ at a rate of 20℃ / min, and maintained for 3min. Subsequently, it was increased to 310℃ at a rate of 25℃ / min and maintained for 8min.

[0106] The results are as follows Figure 8 As shown, the de novo synthesis of squalene from glucose was successfully achieved in Bacillus subtilis through the heterologous expression of squalene synthase; in the engineered strain BS-1 that heterologously expressed TeSQS, squalene production was preliminarily detected, and the squalene production reached 3.2 mg / L.

[0107] In addition, the overexpression of the ispA gene contributes to the synthesis of terpenoids, which has important guiding significance for the further transformation of engineered strains. Therefore, on the basis of heterologous expression of TeSQS, the endogenous farnesyl pyrophosphate synthase gene (ispA) of B. subtilis was integrated into the plasmid for overexpression, and the squalene production of the obtained engineered strain BS-2 reached 4.3 mg / L, which was nearly 35% higher than that of BS-1.

[0108] In addition, the MEP pathway can increase the precursor substances such as IPP and DMAPP for the synthesis of squalene. After overexpressing the key genes of the MEP pathway, the yield of the engineered strain BS-3 reached 10.82 mg / L, which was about 238.1% higher than that of BS-1.

[0109] In addition, if Figure 7 As shown, compared with the engineered strain BS-3, knocking out the gene pdhA in the TCA cycle pathway significantly affected cell growth ( Figure 7 △pdhA in the figure means that only the pdhA gene was knocked out without replacement), and the growth rate of the engineered strains BS-4 and BS-5 after the TCA cycle was dynamically regulated by the period-dependent promoter was not significantly affected under shake flask conditions (the growth of the BS-6 strain was affected compared with the control group); from the perspective of squalene production (see Figure 8), BS-4, BS-5 (11.41 mg / L) and BS-6 (11.13 mg / L) all increased the production of squalene to varying degrees, among which BS-4 had the highest squalene production, reaching 12.47 mg / L. This shows that this strategy can effectively improve the ability of Bacillus subtilis to synthesize squalene without affecting cell growth.

[0110] In summary, the wild-type SCK6 strain cannot synthesize squalene, but the pHT01-P 43 -TeSQS engineered strains BS-1, pHT01-P 43 -ispA-TeSQS engineered strains BS-2 and pHT01-P 43 -MEP-ispA-TeSQS engineered strain BS-3 can synthesize squalene; in addition, the engineered strain using a dynamic regulation strategy based on a period-dependent promoter increased the yield by 2.9 times compared with the engineered strain BS-1 to 12.47 mg / L without affecting cell growth.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An engineered strain for synthesizing squalene, characterized in that: It is obtained by transforming an expression vector into Bacillus subtilis, wherein the expression vector includes the sequence shown in SEQ ID NO:

1.

2. The engineered strain according to claim 1, characterized in that The expression vector also includes the sequence shown in SEQ ID NO:

2.

3. The engineered strain according to claim 1 or 2, characterized in that The expression vector also includes key genes of the MEP pathway.

4. The engineered strain according to claim 3, characterized in that The key genes of the MEP pathway include one or more of the dxs, dxr, ispd or isph genes; the sequences of the dxs, dxr, ispd and isph genes are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

5. The engineered strain according to claim 1, 2 or 4, characterized in that: The Bacillus subtilis is the Bacillus subtilis SCK6 strain.

6. An engineered strain with dynamic regulation capability, characterized in that: The pdhA gene promoter in the engineered strain according to any one of claims 1 to 5 is replaced with a logarithmic phase-dependent promoter.

7. The engineered strain according to claim 6, characterized in that The logarithmic phase-dependent promoter was selected from P abrB Promoter, P valS Promoter or P hag Any of the promoters; P abrB Promoter, P valS Promoter and P hag The nucleotide sequence of the promoter may include the sequences shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

8. A fermentation system for squalene synthesis, characterized in that: It includes the engineered strain according to any one of claims 1 to 5 and / or the engineered strain with dynamic regulation ability according to claim 6 or 7.

9. The fermentation system according to claim 8, characterized in that: The fermentation system also includes a Bacillus subtilis culture medium.

10. Use of the engineered strain according to any one of claims 1 to 5 and / or the engineered strain with dynamic regulation capability according to claim 6 or 7 and / or the fermentation system for squalene synthesis according to claim 8 or 9 in the synthesis of squalene.

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