Nucleic acid molecules, expression cassettes, recombinant strains and their applications

Abstract

The present invention relates to the field of bioengineering, and in particular to nucleic acid molecules, expression cassettes, recombinant strains and their applications. The present invention provides a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 1. The present invention discloses a coenzyme Q10-producing genetic engineering strain, which comprises replacing the endogenous COQ1 gene of Saccharomyces cerevisiae with Paracoccus denitrificans The DPP synthesis gene from the source was further overexpressed in the endogenous genes COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20 and IDI1 of Saccharomyces cerevisiae, as well as UbiC and UbiN, and the shake flask yield was increased to 100 mg / L. The present invention realizes the production of coenzyme Q10 by Saccharomyces cerevisiae for the first time, has the potential to be applied in industrial production, and is suitable for practical promotion and application.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to nucleic acid molecules, expression boxes, recombinant strains and applications thereof. Background Art

[0002] Coenzyme Q (CoQ), also known as ubiquinone, is a class of fat-soluble quinone compounds that participate in oxidative phosphorylation and ATP regeneration. The side chain of Coenzyme Q is composed of multiple isoprene units, and the number of isoprene units is divided into CoQ6-CoQ10. Coenzyme Q10 can be produced through three pathways: chemical synthesis, semi-chemical synthesis, and microbial synthesis. At present, microbial fermentation is the most feasible method for producing Coenzyme Q10. In industry, Coenzyme Q10 is mainly produced through large-scale fermentation of Rhodobacter sphaeroides.

[0003] As the application of Coenzyme Q10 in the pharmaceutical and cosmetic industries continues to expand, the demand for Coenzyme Q10 is also growing. Therefore, there is an urgent need to increase the Coenzyme Q10 production of these biological producers to meet the growing demand. Compared with Rhodobacter sphaeroides, Saccharomyces cerevisiae has the potential to be transformed into a Coenzyme Q10 producer due to its advantages such as stable fermentation and short fermentation cycle. At present, some people have achieved heterologous production of Coenzyme Q10 in Escherichia coli and Corynebacterium glutamicum through genetic modification, but there is no report on the production of Coenzyme Q10 in Saccharomyces cerevisiae. Summary of the invention

[0004] In view of this, the present invention provides a nucleic acid molecule, an expression cassette, a recombinant strain and its application. The present invention discloses a coenzyme Q10-producing genetic engineering strain, which comprises replacing the endogenous COQ1 gene of Saccharomyces cerevisiae with Paracoccus denitrificans The DPP synthesis gene from Saccharomyces cerevisiae was further overexpressed with endogenous genes COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20 and IDI1, as well as Escherichia coli Source: UbiC, Rhodobacter capsulatus The UbiN from the source increases the shake flask yield to 100 mg / L. The present invention realizes the production of coenzyme Q10 by brewer's yeast for the first time, has the potential to be applied to industrial production, and is suitable for practical promotion and application.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a nucleic acid molecule having:

[0007] (1) The nucleotide sequence shown in SEQ ID NO: 1;

[0008] (2) A nucleotide sequence obtained by modifying, replacing, deleting and / or adding one or more bases of the nucleotide sequence shown in (1);

[0009] (3) a sequence having at least 80% homology with the nucleotide sequence shown in (1);

[0010] (4) A complementary sequence of the sequence shown in (1), (2) or (3).

[0011] The present invention also provides a plasmid, comprising: the above nucleic acid molecule.

[0012] The present invention also provides a strain, and the plasmid is transformed and / or transfected into a chassis strain.

[0013] In some embodiments of the present invention, in the above-mentioned strains, the chassis strain includes: yeast.

[0014] In some embodiments of the present invention, among the above strains, the chassis strain comprises: Saccharomyces cerevisiae ( Saccharomyces cerevisiae )、Kluyveromyces lactis( Kluyveromyces lactis )、Issatchenia orientalis( Issatchenkia orientalis ), Yarrowia lipolytica ( Yarrowia lipolytica ) and Pichia pastoris ( Pichia pastoris )

[0015] The present invention also provides the use of the above nucleic acid molecule, the above plasmid and / or the above strain in the preparation and / or production of coenzyme Q10.

[0016] The present invention also provides the use of overexpressing endogenous genes and / or exogenous genes in the preparation and / or production of coenzyme Q10;

[0017] The endogenous genes include: Saccharomyces cerevisiae One or more of the COQ3 gene, COQ5 gene, COQ6 gene, COQ7 gene, COQ2 gene, tHMG1 gene, ERG20 gene and IDI1 gene of the source;

[0018] The exogenous genes include: Rhodobacter capsulatus UbiN gene from source and / or Escherichia coli The UbiC gene of origin.

[0019] The present invention also provides an expression cassette, comprising: an endogenous gene and / or an exogenous gene and an acceptable gene element;

[0020] The endogenous genes include: Saccharomyces cerevisiaeOne or more of the COQ3 gene, COQ5 gene, COQ6 gene, COQ7 gene, COQ2 gene, tHMG1 gene, ERG20 gene and IDI1 gene of the source;

[0021] The exogenous genes include: Rhodobacter capsulatus UbiN gene from source and / or Escherichia coli The UbiC gene of origin.

[0022] In some embodiments of the present invention, the above-mentioned expression cassette includes: one or more of expression cassette 1 to expression cassette 5;

[0023] The expression cassette 1 includes: UbiN gene and UbiC gene;

[0024] The expression cassette 2 includes: COQ3 gene and COQ5 gene;

[0025] The expression cassette 3 includes: COQ6 gene and COQ7 gene;

[0026] The expression cassette 4 includes: COQ2 gene and IDI1 gene;

[0027] The expression cassette 5 includes: tHMG1 gene and ERG20 gene.

[0028] The present invention also provides a recombinant plasmid, comprising: the above expression cassette.

[0029] The present invention also provides a recombinant strain, and the recombinant plasmid is used to transform and / or transfect the strain.

[0030] In some embodiments of the present invention, the above-mentioned recombinant strain has a deposit number of: CCTCC NO: M2024615.

[0031] The present invention also provides the use of the above expression cassette, the above recombinant plasmid and / or the above recombinant strain in any of the following items:

[0032] (I) Preparation and / or production of coenzyme Q10; and / or

[0033] (II) preparing and / or producing products containing coenzyme Q10; and / or

[0034] (III) Increase the production of coenzyme Q10.

[0035] The present invention also provides a product, comprising: the above nucleic acid molecule, the above plasmid, the above strain, the above expression cassette, the above recombinant plasmid and / or the above recombinant strain.

[0036] The present invention also provides a method for preparing coenzyme Q10, which comprises inoculating and fermenting the above-mentioned recombinant strain to obtain the coenzyme Q10.

[0037] In some embodiments of the present invention, in the above-mentioned preparation method, the inoculation time is 20 to 24 hours.

[0038] In some embodiments of the present invention, in the above-mentioned preparation method, the inoculation adopts an inoculation medium, and the inoculation medium comprises: 10-25 g / L peptone, 5-15 g / L yeast powder and 20-30 g / L glucose.

[0039] In some embodiments of the present invention, in the above preparation method, the inoculation culture medium comprises: 20 g / L peptone, 10 g / L yeast powder and 20 g / L glucose.

[0040] In some embodiments of the present invention, in the above preparation method, the fermentation time is 48 to 84 hours.

[0041] In some embodiments of the present invention, in the above-mentioned preparation method, the fermentation adopts a fermentation medium, and the fermentation medium includes: 10~60g / L of glucose or glycerol, 2~3g / L of potassium dihydrogen phosphate, 2.5~3.0g / L of dipotassium hydrogen phosphate, 10~28g / L of yeast powder and 10~20g / L of yeast peptone.

[0042] In some embodiments of the present invention, in the above preparation method, the fermentation medium includes: 20 g / L glucose, 2.2 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 10 g / L yeast powder and 20 g / L yeast peptone.

[0043] The invention discloses a coenzyme Q10 producing genetic engineering strain, which comprises replacing the endogenous COQ1 gene of Saccharomyces cerevisiae with

[0044] Paracoccus denitrificans

[0045] The DPP synthesis gene from Saccharomyces cerevisiae was further overexpressed with endogenous genes COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20 and IDI1, as well as

[0046] Escherichia coli

[0047] Source: UbiC,

[0048] Rhodobacter capsulatus

[0049] The UbiN from the source increases the shake flask yield to 100 mg / L. The present invention realizes the production of coenzyme Q10 by brewer's yeast for the first time, has the potential to be applied to industrial production, and is suitable for practical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0051] Figure 1 The biosynthesis pathway of coenzyme Q10 in the engineered yeast of the present invention is shown;

[0052] Figure 2 The genome map of yeast COQ1 gene replaced with PdDDSA is shown;

[0053] Figure 3 The genomic map of the yeast genome with integrated UbiC and UbiN is shown;

[0054] Figure 4 The figure shows the accumulation of coenzyme Q10 after the recombinant yeast was cultured in a shake flask for 96 hours.

[0055] Biological Deposit Description

[0056] Saccharomyces cerevisiae Saccharomyces cerevisiae CQ14), the deposit date is April 3, 2024, the deposit number is CCTCC No: M 2024615, the name of the depository is: China Center for Type Culture Collection, and the address of the depository is: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0057] The invention discloses nucleic acid molecules, expression boxes, recombinant strains and applications thereof.

[0058] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0059] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0060] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0061] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0062] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0063] The present invention provides a method for constructing a recombinant strain producing coenzyme Q10, which comprises replacing the endogenous HexPP gene produced by saccharomyces cerevisiae with a synthetic gene producing DPP, thereby realizing the production of coenzyme Q10 in saccharomyces cerevisiae.

[0064] Furthermore, the nucleotide sequence of the DPP synthesis gene is shown in SEQ ID NO.1.

[0065] Furthermore, the recombinant yeast strain overexpressed 5 genes of the coenzyme Q10 synthesis pathway and 3 genes of the mevalonate pathway, namely COQ3 (SGD: S000005456), COQ5 (SGD: S000004578), COQ6 (SGD: S000003487), COQ7 (SGD: S000005651), COQ2 (SGD: S000005324), tHMG1 (genbank: AJS90608.1 (expression starts from the 531st amino acid)), ERG20 (SGD: S000003703), and IDI1 (SGD: S000006038), all of which were derived from Saccharomyces cerevisiae .

[0066] Furthermore, the recombinant yeast overexpresses the gene UbiN responsible for decarboxylation and hydroxylation and the gene UbiC responsible for the synthesis of pHBA in the head. UbiN is derived from Rhodobacter capsulatus (genbank: WP_013068139.1), UbiC is derived from Escherichia coli (genbank: CAD6022848.1).

[0067] The present invention also provides a method for producing coenzyme Q10, wherein the engineered bacteria transformed by the above synthetic biology are used to produce coenzyme Q10 on a large scale by fermentation, and the method comprises the following steps:

[0068] The aforementioned recombinant strain is inoculated into a seed culture medium and cultured for 20 to 24 hours, the seed solution is inoculated into a fermentation culture medium, fermented and cultured for 48 to 84 hours, and then separated and purified to obtain the product;

[0069] Furthermore, the formula of the seed culture medium is: peptone 20g / L, yeast powder 10g / L, glucose 20g / L;

[0070] The formula of the fermentation medium is 20 g / L glucose or glycerol, 2.2 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 10 g / L yeast powder, and 20 g / L peptone.

[0071] The culture was performed under shaking at a temperature of 30° C. and a rotation speed of 200 rpm.

[0072] Sequence information involved in the present invention:

[0073]

[0074] Primer information involved in the present invention:

[0075] Table 1 Nucleotide sequences of primers

[0076]

[0077] In Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0078] The present invention will be further described below in conjunction with embodiments:

[0079] Example 1 Construction of the coenzyme Q10-producing Saccharomyces cerevisiae strain of the present invention

[0080] coding Paracoccus denitrificans The DDSA gene from the source was first codon optimized and then chemically synthesized by Shanghai Qingke Biotechnology Co., Ltd.; PdDDSA-COQ1-F and TCYC1-COQ1-R were used as primers to amplify the DDSA gene by PCR, and the PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by a clean-up kit;

[0081] S accharomyces cerevisiae CEN.PK2-1C or BY4741 genome was used as a template, and PCR products of homology arms COQ1-UP and COQ1-Dn were obtained by PCR using primers COQ1-UF / COQ1-PdDDSA-UR and COQ1-TCYC1-DF / COQ1-DR. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0082] The purified products of the homology arms COQ1-UP and COQ1-Dn and the purified products of the DDSA gene were edited using CRISPR / Cas9 technology. Saccharomyces cerevisiae The COQ1 gene of CEN.PK2-1C was replaced with the DDSA gene to obtain the yeast engineered strain CQ01 ( Figure 2 ).

[0083] Example 2 The present invention integrates genes of the coenzyme Q synthesis pathway into the genome

[0084] Chemical synthesis (Shanghai Qingke Biotechnology Co., Ltd.) coding Escherichia coli Source: UbiC, Rhodobacter capsulatus UbiN from the source; using UbiC-F / UbiC-R and UbiN-F / UbiN-R as primers, PCR amplification of UbiC and UbiN genes was performed, the PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified by a clean-up kit;

[0085] The target plasmid pDS01 was double-digested with BamHI and HindIII, and the digestion products were detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by gel excision; then the purified UbiC gene fragment was ligated with the linearized plasmid pDS01 using the Novozymes one-step cloning kit (37°C, 30 min); the ligation product was transformed into E .coli DH5α, and obtain the transformation product; the transformation product is spread on LB solid medium (containing ampicillin at a final concentration of 100 mg / L), and after obtaining a single clone, the culture is shaken in a flask at 37°C and 220 rpm for 8-12 hours, and then the plasmid is extracted for sequencing verification. If the verification is correct, the plasmid pDS01-UbiC expressing the UbiC gene is obtained;

[0086] The target plasmid pDS01-UbiC was double-digested with NotI and BcuI, and the digestion products were detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by gel excision; then the purified UbiN gene fragment was ligated with the linearized plasmid pDS01-UbiC using the Novozymes one-step cloning kit (37°C, 30 min); the ligation product was transformed into E .coli DH5α, and obtain the transformation product; the transformation product is spread on LB solid medium (containing ampicillin at a final concentration of 100 mg / L), and after obtaining a single clone, the culture is shaken at 37°C and 220 rpm for 8-12 hours, and then the plasmid is extracted for sequencing verification. If the verification is correct, the expression plasmid pDS01-UbiC-UbiN of the UbiC gene and the UbiN gene is obtained;

[0087] by Saccharomyces cerevisiae Using CEN.PK2-1C or BY4741 genome as template, PCR products of homology arms int10-UP and int10-Dn were obtained by PCR using primers int10-UF / int10-UR and int10-DF / int10-DR. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0088] Using plasmid pDS01-UbiC-UbiN as template, PCR products of UbiC and UbiN expression cassettes were obtained by PCR using primers int10-F / int10-R. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0089] The purified products of homology arms int10-UP and int10-Dn, and the purified products of UbiC and UbiN expression cassettes were integrated into the int10 locus of the engineered strain CQ01 using CRISPR / Cas9 gene editing technology to obtain the yeast engineered strain CQ02 ( Figure 3 ).

[0090] Taking the construction method of plasmid pDS01-UbiC-UbiN as an example, Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome was used as a template and plasmid pDS01 was used as a vector to construct expression plasmids for five genes COQ3, COQ5, COQ6, COQ7, COQ2 in the coenzyme Q pathway and three genes tHMG1, ERG20, and IDI1 in the MVA pathway, namely pDS02-COQ3-COQ5, pDS03-COQ6-COQ7, pDS04-COQ2-IDI, and pDS05-tHMG1-ERG20, respectively.

[0091] Taking the construction method of yeast engineering strain CQ02 as an example, the expression plasmids pDS02-COQ3-COQ5, pDS03-COQ6-COQ7, pDS04-COQ2-IDI, and pDS05-tHMG1-ERG20 of the above-mentioned five genes of the coenzyme Q pathway and three genes of the MVA pathway were used as templates, PCR products were obtained by PCR, and the corresponding expression cassettes of the five genes of the coenzyme Q pathway and the three genes of the MVA pathway were integrated into the genome of the yeast engineering strain CQ02 by using CRISPR / Cas9 gene editing technology to obtain the yeast engineering strain CQ14.

[0092] Example 3 Fermentation of the recombinant yeast strain producing coenzyme Q10 of the present invention

[0093] The recombinant Saccharomyces cerevisiae engineered strain CQ14 constructed in Example 2 was inoculated into a seed culture medium, and cultured on a shaker at 30°C and 200 rpm for 24 hours. The seed solution was inoculated into a fermentation medium at 1% (v / v), and cultured on a shaker at 30°C and 200 rpm for 96 hours. The fermentation broth was centrifuged to obtain bacterial cells, and the cells were broken by a freezing grinder, and then extracted with an organic solvent ethyl acetate, filtered and quantified by GC, and the yield of coenzyme Q10 in the fermentation broth of the Saccharomyces cerevisiae engineered strain CQ14 reached more than 100 mg / L ( Figure 4 ).

[0094] The formula of the seed culture medium is: peptone 20g / L, yeast powder 10g / L, glucose 20g / L;

[0095] The formula of the fermentation medium is 40 g / L glucose, 2.2 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 10 g / L yeast powder, and 20 g / L peptone.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant strain, characterized in that: Transform the recombinant plasmid into the strain; The recombinant plasmid contains: endogenous genes and exogenous genes and acceptable gene elements; The endogenous genes include: Saccharomyces cerevisiae COQ3 gene, COQ5 gene, COQ6 gene, COQ7 gene, COQ2 gene, tHMG1 gene, ERG20 gene and IDI1 gene of origin; The exogenous genes include: Rhodobacter capsulatus UbiN gene from Escherichia coli UbiC gene of origin; In the strain, the endogenous COQ1 gene of Saccharomyces cerevisiae for producing HexPP is replaced with a synthetic gene for producing DPP, and the sequence of the synthetic gene for DPP is shown in SEQ ID NO:

1.

2. The recombinant strain according to claim 1, characterized in that Its deposit number is: CCTCC NO: M2024615.

3. Use of the recombinant strain according to claim 1 or 2 in the preparation and / or production of coenzyme Q10.

4. A method for preparing coenzyme Q10, characterized in that: The recombinant strain according to claim 1 or 2 is inoculated and fermented to obtain the coenzyme Q10.

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