Engineered yeast for producing squalene and application thereof

Abstract

The invention relates to the technical field of bioengineering, and discloses a yeast engineering bacterium for producing squalene and application thereof, the yeast engineering bacterium comprises yeast chassis cells, a heterologous squalene biosynthesis module, an NADPH supply module, a precursor supply module and a grease body engineering module, the specific technological process comprises the following steps: integrating a key enzyme coding gene in a heterologous squalene biosynthesis module into a yeast chassis cell to obtain a yeast genome containing a squalene biosynthesis pathway; integrating the NADPH supply module, the precursor supply module and the grease body engineering module into a yeast genome to obtain the yeast engineering bacteria for producing squalene; and fermenting the yeast engineering bacteria by taking a YYG culture medium as a substrate to obtain squalene. The yeast engineering bacterium constructs and optimizes a biosynthesis integration module required by squalene, realizes adaptation of a squalene biosynthesis pathway and yeast chassis cells, can efficiently and stably produce squalene, and also has the characteristics of greenness and safety.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a yeast engineering bacterium for producing squalene and application thereof. Background Art

[0002] Squalene is an important polyunsaturated triterpene, widely used in food, cosmetics, pharmaceutical and other industries. As an open-ring triterpene compound, squalene is a key precursor for the synthesis of various unsaturated triterpenes and steroids. Squalene has excellent antioxidant properties due to its multiple conjugated unsaturated double bonds, and can be used to treat eczema and make anti-aging and anti-wrinkle cosmetics. In addition to its good antioxidant properties, squalene also has good anti-cardiovascular and anti-cancer activities. In addition, squalene can promote tissue repair and wound healing by stimulating the innate immune cells of the organism, and can also reduce the fatty acid content, thereby reducing blood sugar and blood lipids. Due to its strong stability and good biocompatibility, it is currently used in many vaccines and drug delivery emulsions.

[0003] At present, the main source of squalene is extracted from the liver of deep-sea sharks. This extraction method has led to the killing of a large number of sharks and the destruction of ecological balance. Another way to obtain squalene is to extract it from plants and their processed byproducts. Although it partially solves the problem of raw material sources, it also has shortcomings such as small planting area, difficult collection, and long cycle. Therefore, based on meeting the huge market demand for squalene in the future and protecting wild marine animals and the natural environment, it is urgent to develop an economical, green, and economical squalene manufacturing method to fill the huge market gap and benefit human health.

[0004] With the development of synthetic biology and metabolic engineering, constructing microbial cell factories to efficiently synthesize natural products has become an effective green production method. Yeast (Saccharomyces) is a unicellular fungus and an important model organism that combines multiple advantages such as clear genetic background, short fermentation cycle and high biosafety. Therefore, optimizing the squalene biosynthesis pathway in yeast and obtaining high-yield squalene microbial cell engineering is one of the important ways to achieve efficient, green and safe production of squalene. Summary of the invention

[0005] In view of this, the present invention provides a yeast engineering bacterium for producing squalene and its application, optimizes the squalene biosynthesis pathway, constructs a microbial cell factory, and realizes the efficient, green and safe production of squalene.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides a yeast engineering bacterium for producing squalene, wherein the yeast engineering bacterium is constructed by:

[0008] S1: Integrate the key enzyme encoding genes in the heterologous squalene biosynthesis module into yeast chassis cells to obtain a yeast genome containing the squalene biosynthesis pathway;

[0009] S2: Integrate the NADPH supply module, precursor supply module and lipid body engineering module into the yeast genome in S1 to obtain a yeast engineering strain that produces squalene;

[0010] Wherein, the yeast chassis cell is Yarrowia lipolytica Polf-Δku70;

[0011] The heterologous squalene biosynthesis module includes ScHMG1 gene and ERG9 gene;

[0012] The NADPH supply module includes a G6PD gene and a PGD gene;

[0013] The precursor supply module includes AtIPK gene and LhCHK gene;

[0014] The lipid body engineering module includes the DGA1 gene;

[0015] The nucleotide sequence of the ScHMG1 gene is the nucleotide sequence shown in Genbank Accession No. 854900, and / or the amino acid sequence of the protein encoded by the ScHMG1 gene is the amino acid sequence shown in Genbank Accession No. DAA09822.1;

[0016] The nucleotide sequence of the ERG9 gene is the nucleotide sequence shown in Genbank Accession No. 2906604, and / or the amino acid sequence of the protein encoded by the ERG9 gene is the amino acid sequence shown in Genbank Accession No. XP_499929.1;

[0017] The nucleotide sequence of the G6PD gene is the nucleotide sequence shown in Genbank Accession No. 2912994, and / or the amino acid sequence of the protein encoded by the G6PD gene is the amino acid sequence shown in Genbank Accession No. XP_504275.1;

[0018] The nucleotide sequence of the PGD gene is the nucleotide sequence shown in Genbank Accession No. 2906913, and / or the amino acid sequence of the protein encoded by the PGD gene is the amino acid sequence shown in Genbank Accession No. XP_500938.1;

[0019] The nucleotide sequence of the AtIPK gene is the nucleotide sequence shown in Genbank Accession No. 839204, and / or the amino acid sequence of the protein encoded by the AtIPK gene is the amino acid sequence shown in Genbank Accession No. NP_173986.2;

[0020] The nucleotide sequence of the LhCHK gene is the nucleotide sequence shown in Genbank Accession No. 41984014, and / or the amino acid sequence of the protein encoded by the LhCHK gene is the amino acid sequence shown in Genbank Accession No. XP_031005798.1;

[0021] The nucleotide sequence of the DGA1 gene is the nucleotide sequence shown in Genbank Accession No. 2911905, and / or the amino acid sequence of the protein encoded by the DGA1 gene is the amino acid sequence shown in Genbank Accession No. XP_504700.3.

[0022] Application of squalene-producing yeast engineering bacteria in the preparation of squalene production preparations.

[0023] The present invention provides a yeast engineering bacterium for producing squalene and its application. Compared with the prior art, its beneficial effects are:

[0024] The present invention constructs and optimizes the biosynthetic integration module required for squalene, creates a microbial cell factory, can efficiently produce squalene, and at the same time, has the characteristics of being green and safe;

[0025] The yeast engineering bacteria for producing squalene of the present invention can achieve the adaptation of the squalene biosynthesis pathway and yeast chassis cells, and achieve efficient and stable synthesis of squalene in yeast chassis cells by expressing an NADPH supply module, a precursor supply module and an oil body engineering module. DETAILED DESCRIPTION

[0026] Some embodiments of the present invention provide a yeast engineering bacterium for producing squalene, comprising yeast chassis cells, a heterologous squalene biosynthesis module, a NADPH supply module, a precursor supply module and a lipid body engineering module.

[0027] In some embodiments of the present invention, the yeast genome is integrated with heterologous squalene biosynthesis gene ScHMG1 and at least one G6PD gene that regulates squalene biosynthesis, and the yeast chassis cell is any one of the eukaryotic organisms of the genus Saccharomyces, such as Yarrowia lipolytica and Saccharomyces cerevisiae. Preferably, the yeast chassis cell is Yarrowia lipolytica, which has the characteristics of strong stress tolerance, broad substrate utilization spectrum, sufficient supply of acetyl-CoA and reducing power, etc., is an ideal chassis for biosynthesis, and is conducive to the production of squalene.

[0028] In some embodiments of the present invention, the heterologous squalene biosynthesis module includes the ScHMG1 encoding gene and the ERG9 encoding gene; the NADPH supply module includes the G6PD encoding gene and the PGD encoding gene; the precursor supply module includes the AtIPK encoding gene and the LhCHK encoding gene; and the lipid body engineering module includes the DGA1 encoding gene.

[0029] In some embodiments of the present invention, the NADPH supply module is composed of a gene encoding glucose-6-phosphate dehydrogenase (G6PD) (Genbank accession number is nucleotide sequence accession number 2912994 and / or the amino acid sequence accession number XP_504275.1 encoding the protein) and a gene encoding 6-phosphogluconate dehydrogenase (PGD) (Genbank accession number is nucleotide sequence accession number 2906913 and / or the amino acid sequence accession number XP_500938.1 encoding the protein); the precursor supply module is composed of a gene encoding isopentenyl phosphate kinase (AtIPK) (Genbank accession number is nucleotide sequence accession number 839204 and / or the amino acid sequence accession number NP_173986.2 encoding the protein) and a gene encoding choline kinase (Choline The lipid body engineering module is composed of a gene encoding a diacylglycerol acyltransferase (Diacylglycerol acyltransferase, DGA1) (Genbank accession number is a nucleotide sequence accession number of 2911905 and / or an amino acid sequence accession number of XP_504700.3).

[0030] In some embodiments of the present invention, the nucleotide sequence of the ScHMG1 encoding gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. 854900 and has the same function for squalene synthesis; the nucleotide sequence of the ERG9 encoding gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. 2906604 and has the same function for squalene synthesis; the nucleotide sequence of the G6PD encoding gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. 2912994 and has the same function for squalene synthesis; the nucleotide sequence of the PGD encoding gene is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. 29 06913 or a nucleotide sequence with a homology greater than 80% and having the same function for squalene synthesis; the nucleotide sequence of the AtIPK encoding gene is a nucleotide sequence with a Genbank accession number of 839204 or a nucleotide sequence with a homology greater than 80% and having the same function for squalene synthesis; the nucleotide sequence of the LhCHK encoding gene is a nucleotide sequence with a Genbank accession number of 41984014 or a nucleotide sequence with a homology greater than 80% and having the same function for squalene synthesis; the nucleotide sequence of the DGA1 encoding gene is a nucleotide sequence with a Genbank accession number of 2911905 or a nucleotide sequence with a homology greater than 80% and having the same function for squalene synthesis.

[0031] In some embodiments of the present invention, the amino acid sequence of ScHMG1 is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. DAA09822.1 and has an amino acid sequence with equivalent function for squalene synthesis; the amino acid sequence of ERG9 is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. XP_499929.1 and has an amino acid sequence with equivalent function for squalene synthesis; the amino acid sequence of G6PD is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. XP_504275.1 and has an amino acid sequence with equivalent function for squalene synthesis; the amino acid sequence of PGD is the same as or has a homology greater than 80% to the sequence shown in Genbank Accession No. XP_50093 8.1 is identical or has a homology greater than 80% to the sequence shown in Genbank Accession No. NP_173986.2 and has an amino acid sequence with equivalent function for squalene synthesis; the amino acid sequence of LhCHK is identical or has a homology greater than 80% to the sequence shown in Genbank Accession No. XP_031005798.1 and has an amino acid sequence with equivalent function for squalene synthesis; the amino acid sequence of DGA1 is identical or has a homology greater than 80% to the sequence shown in Genbank Accession No. XP_504700.3 and has an amino acid sequence with equivalent function for squalene synthesis.

[0032] Some other embodiments of the present invention provide a method for producing squalene by yeast engineering bacteria, the steps of which are as follows:

[0033] S1: The key enzyme ScHMG1 encoding gene in the heterologous squalene biosynthesis module was integrated into yeast chassis cells, combined with overexpression of the endogenous squalene synthase gene ERG9 in yeast chassis cells to obtain a yeast genome containing the squalene biosynthesis pathway;

[0034] S2: Integrate the NADPH supply module, precursor supply module and lipid body engineering module into the yeast genome in S1 to obtain a yeast engineering strain that produces squalene;

[0035] S3: fermenting the engineered yeast in S2 for 50-100 hours, preferably for 72 hours, using YYG medium containing 8-10 g / L yeast extract, 7-9 g / L yeast nitrogen source without amino acids and 50-60 g / L glucose as substrate to obtain squalene.

[0036] The nucleotide sequence of the ScHMG1 gene is the nucleotide sequence shown in Genbank Accession No. 854900, and / or the amino acid sequence of the protein encoded by the ScHMG1 gene is the amino acid sequence shown in Genbank Accession No. DAA09822.1;

[0037] The nucleotide sequence of the ERG9 gene is the nucleotide sequence shown in Genbank Accession No. 2906604, and / or the amino acid sequence of the protein encoded by the ERG9 gene is the amino acid sequence shown in Genbank Accession No. XP_499929.1;

[0038] The nucleotide sequence of the G6PD gene is the nucleotide sequence shown in Genbank Accession No. 2912994, and / or the amino acid sequence of the protein encoded by the G6PD gene is the amino acid sequence shown in Genbank Accession No. XP_504275.1;

[0039] The nucleotide sequence of the PGD gene is the nucleotide sequence shown in Genbank Accession No. 2906913, and / or the amino acid sequence of the protein encoded by the PGD gene is the amino acid sequence shown in Genbank Accession No. XP_500938.1;

[0040] The nucleotide sequence of the AtIPK gene is the nucleotide sequence shown in Genbank Accession No. 839204, and / or the amino acid sequence of the protein encoded by the AtIPK gene is the amino acid sequence shown in Genbank Accession No. NP_173986.2;

[0041] The nucleotide sequence of the LhCHK gene is the nucleotide sequence shown in Genbank Accession No. 41984014, and / or the amino acid sequence of the protein encoded by the LhCHK gene is the amino acid sequence shown in Genbank Accession No. XP_031005798.1;

[0042] The nucleotide sequence of the DGA1 gene is the nucleotide sequence shown in Genbank Accession No. 2911905, and / or the amino acid sequence of the protein encoded by the DGA1 gene is the amino acid sequence shown in Genbank Accession No. XP_504700.3.

[0043] In some embodiments of the present invention, the method for producing squalene by yeast engineering bacteria further comprises a multi-copy integration site upstream homology arm rDNAup, a multi-copy integration site downstream homology arm rDNAdown, promoters TEFp and FBAp, terminators XPR2t and LIP2t, and screening markers URA3 and LEU2.

[0044] In some embodiments of the present invention, in step S3, the yeast engineering bacteria are fermented with YYG-60 medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 60 g / L glucose as substrate, which can achieve effective accumulation of squalene in the fermentation broth, reaching more than 1600 mg / L, and has prospects for industrial application.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1 Construction of squalene biosynthesis module with optimized squalene synthesis pathway and green and efficient production of squalene by yeast engineering bacteria:

[0047] 1. Use high-fidelity enzyme amplification to construct the upstream homology arm rDNAup of the multi-copy integration site, the downstream homology arm rDNAdown of the multi-copy integration site, the promoters TEFp and FBAp, the ScHMG1 encoding gene (Genbank accession number is the nucleotide sequence accession number 854900 and / or the amino acid sequence accession number DAA09822.1 of the encoded protein), the ERG9 encoding gene ((Genbank accession number is the nucleotide sequence accession number 2906604 and / or the amino acid sequence accession number XP_499929.1 of the encoded protein), the G6PD encoding gene (Genbank accession number is the nucleotide sequence accession number 2912994 and / or the amino acid sequence accession number XP_504275.1 of the encoded protein), the PGD encoding gene (Genbank accession number is the nucleotide sequence accession number 2906913 and / or the amino acid sequence accession number XP_500938.1 of the encoded protein), the terminators XPR2t and LIP2t, and the screening markers URA3 and LEU2.

[0048] 2. Construct the integration vectors pUC19-ScHMG1, pUC19-ERG9, pUC19-G6PD, pUC19-PGD, pUC19-URA3 and pUC19-LEU2 respectively.

[0049] 3. The recovered functional module fragments M-ScHMG1 and M-ERG9 and the screening marker module fragment M-URA3 were co-transformed into Yarrowia lipolytica Polf-Δku70 to construct the yeast engineered strain YLSQ1; then the gene encoding glucose-6-phosphate dehydrogenase (G6PD) and the gene encoding 6-phosphogluconate dehydrogenase (PGD) in the pentose phosphate pathway were co-overexpressed.

[0050] 4. The recovered functional module fragments M-G6PD, M-PGD and the screening marker module fragment M-LEU2 were co-transformed into Yarrowia lipolytica to construct the yeast engineering strain YLSQ5.

[0051] 5. The squalene production of YLSQ5 reached 535.5 mg / L after fermentation for 72 h using YYG medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 50 g / L glucose as substrate.

[0052] Example 2 Construction of precursor supply module and lipid body engineering module and efficient production of squalene by yeast engineering bacteria:

[0053] 1. Use high-fidelity enzyme amplification to construct the upstream homology arm rDNAup of the multi-copy integration site, the downstream homology arm rDNAdown of the multi-copy integration site, the promoters TEFp and FBAp, the AtIPK coding gene (Genbank accession number is the nucleotide sequence accession number 839204 and / or the amino acid sequence accession number of the encoded protein NP_173986.2), the LhCHK coding gene (Genbank accession number is the nucleotide sequence accession number 41984014 and / or the amino acid sequence accession number of the encoded protein XP_031005798.1), the DGA1 coding gene (Genbank accession number is the nucleotide sequence accession number 2911905 and / or the amino acid sequence accession number of the encoded protein XP_504700.3), the terminators XPR2t and LIP2t, and the screening markers URA3 and LEU2.

[0054] 2. Construct the integration vectors pUC19-AtIPK, pUC19-LhCHK, pUC19-DGA1, pUC19-DGA2, pUC19-URA3 and pUC19-LEU2 respectively.

[0055] 3. The recovered functional module fragments M-AtIPK, M-LhCHK and the screening marker module fragment M-URA3 were co-transformed into Yarrowia lipolytica YLSQ5 to construct the engineered bacteria YLSQ6; then, the gene encoding diacylglycerol acyltransferase (DGA1) was overexpressed.

[0056] 4. The recovered functional module fragment M-DGA1 and the screening marker module fragment M-URA3 were co-transformed into Yarrowia lipolytica to construct the yeast engineering strain YLSQ7.

[0057] 5. The squalene production of YLSQ7 reached 1628.2 mg / L after fermentation for 72 h using YYG-60 medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 60 g / L glucose as substrate.

[0058] Example 3: Efficient production of squalene by yeast engineering bacteria:

[0059] This Example 3 is basically the same as Example 2, with the only difference being that in step 5, YYG medium containing 8 g / L yeast extract, 7 g / L yeast nitrogen source without amino acids and 50 g / L glucose is used as a substrate for fermentation for 50 hours to obtain squalene.

[0060] Example 4: Efficient production of squalene by yeast engineering bacteria:

[0061] This Example 4 is basically the same as Example 2, with the only difference being that in step 5, YYG medium containing 10 g / L yeast extract, 9 g / L yeast nitrogen source without amino acids and 60 g / L glucose is used as a substrate for fermentation for 100 h to obtain squalene.

[0062] The yield of squalene produced by the yeast engineering bacteria in the example using the culture medium as a substrate was tested, and the test results are shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] As can be seen from Table 1, the yeast engineering bacteria YLSQ5 of Example 1 is constructed only by the heterologous squalene biosynthesis module and the NADPH supply module, and the yield of squalene is relatively low. The yeast engineering bacteria YLSQ7 of Examples 2-4 further constructs a precursor supply module and an oil body engineering module on the basis of YLSQ5 and integrates them into YLSQ5, and the obtained yeast engineering bacteria YLSQ7 produces a higher yield of squalene; at the same time, it can be seen from Examples 2-4 that fermenting yeast engineering bacteria with YYG-60 medium containing 9 g / L yeast extract, 8 g / L yeast nitrogen source without amino acids and 60 g / L glucose as substrate can effectively achieve the accumulation of squalene in the fermentation broth and increase the yield of squalene.

[0067] It can be seen from the above embodiments that the present invention provides an engineered yeast for producing squalene and its application.

[0068] In summary, the squalene yield obtained by using the yeast engineering bacteria constructed and optimized by the present invention and the YYG-60 culture medium provided by the present invention is high, a microbial cell factory is created, and squalene can be produced efficiently, greenly and safely. Compared with the prior art, the yeast engineering bacteria for producing squalene of the present invention can achieve the adaptation of the squalene biosynthesis pathway and the yeast chassis cells, and realize the efficient and stable synthesis of squalene in the yeast.

[0069] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0070] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A yeast engineering bacterium for producing squalene, characterized in that: The construction method of the yeast engineering bacteria is: S1: Integrate the key enzyme encoding genes in the heterologous squalene biosynthesis module into yeast chassis cells to obtain a yeast genome containing the squalene biosynthesis pathway; S2: Integrate the NADPH supply module, precursor supply module and lipid body engineering module into the yeast genome in S1 to obtain a yeast engineering strain that produces squalene; Wherein, the yeast chassis cell is Yarrowia lipolytica Polf-Δku70; The heterologous squalene biosynthesis module includes ScHMG1 gene and ERG9 gene; The NADPH supply module includes a G6PD gene and a PGD gene; The precursor supply module includes AtIPK gene and LhCHK gene; The lipid body engineering module includes the DGA1 gene; The nucleotide sequence of the ScHMG1 gene is the nucleotide sequence shown in Genbank Accession No. 854900, and / or the amino acid sequence of the protein encoded by the ScHMG1 gene is the amino acid sequence shown in Genbank Accession No. DAA09822.1; The nucleotide sequence of the ERG9 gene is the nucleotide sequence shown in Genbank Accession No. 2906604, and / or the amino acid sequence of the protein encoded by the ERG9 gene is the amino acid sequence shown in Genbank Accession No. XP_499929.1; The nucleotide sequence of the G6PD gene is the nucleotide sequence shown in Genbank Accession No. 2912994, and / or the amino acid sequence of the protein encoded by the G6PD gene is the amino acid sequence shown in Genbank Accession No. XP_504275.1; The nucleotide sequence of the PGD gene is the nucleotide sequence shown in Genbank Accession No. 2906913, and / or the amino acid sequence of the protein encoded by the PGD gene is the amino acid sequence shown in Genbank Accession No. XP_500938.1; The nucleotide sequence of the AtIPK gene is the nucleotide sequence shown in Genbank Accession No. 839204, and / or the amino acid sequence of the protein encoded by the AtIPK gene is the amino acid sequence shown in Genbank Accession No. NP_173986.2; The nucleotide sequence of the LhCHK gene is the nucleotide sequence shown in Genbank Accession No. 41984014, and / or the amino acid sequence of the protein encoded by the LhCHK gene is the amino acid sequence shown in Genbank Accession No. XP_031005798.1; The nucleotide sequence of the DGA1 gene is the nucleotide sequence shown in Genbank Accession No. 2911905, and / or the amino acid sequence of the protein encoded by the DGA1 gene is the amino acid sequence shown in Genbank Accession No. XP_504700.

3.

2. Use of the yeast engineering bacteria for producing squalene as claimed in claim 1 in preparing squalene production preparations.

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