CN118531016B

CN118531016B - Nucleic acid molecules, expression cassettes, recombinant strains and their applications

Info

Publication number: CN118531016B
Application number: CN202410759816.6A
Authority: CN
Inventors: 李论; 郭晓晶; 李�根
Original assignee: New Carrier Biotech Hainan Co ltd
Current assignee: New Carrier Biotech Hainan Co ltd
Priority date: 2024-06-13
Filing date: 2024-06-13
Publication date: 2025-04-04
Anticipated expiration: 2044-06-13
Also published as: CN118531016A

Abstract

The invention relates to the field of bioengineering, in particular to a nucleic acid molecule, an expression cassette, a recombinant strain and application thereof. The invention provides a nucleic acid molecule which has a nucleotide sequence as shown in SEQ ID NO. 1. The invention discloses a coenzyme Q10-producing genetic engineering strain which comprises the steps of replacing a Paracoccus denitrificans-source DPP (dipeptidyl peptidase) synthetic gene of an endogenous COQ1 gene of saccharomyces cerevisiae, further overexpressing endogenous genes of the saccharomyces cerevisiae, such as COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20, IDI1 and UbiC and UbiN, and improving shake flask yield to 100mg/L. The invention realizes the production of coenzyme Q10 by Saccharomyces cerevisiae for the first time, has potential for industrial production, and is suitable for practical popularization and application.

Description

Nucleic acid molecule, expression cassette, recombinant strain and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to a nucleic acid molecule, an expression cassette, a recombinant strain and application thereof.

Background

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 consists of a number of isoprene units, which in turn are divided into CoQ6-CoQ10. Coenzyme Q10 can be produced by three pathways, chemical synthesis, semi-chemical synthesis and microbial synthesis. At present, microbial fermentation is the most feasible method for producing coenzyme Q10, and the industry mainly produces coenzyme Q10 by large-scale fermentation of rhodobacter sphaeroides.

As the use of coenzyme Q10 in the pharmaceutical and cosmetic industries continues to expand, so does the demand for coenzyme Q10. Thus, there is an urgent need to increase the production of coenzyme Q10 by these biological producers to meet the increasing demand. Compared with rhodobacter sphaeroides, the saccharomyces cerevisiae has the potential of being transformed into coenzyme Q10 producing bacteria due to the advantages of stable strain fermentation, short fermentation period and the like. At present, heterologous production of coenzyme Q10 in Escherichia coli and Corynebacterium glutamicum has been achieved by genetic engineering, but production of coenzyme Q10 in Saccharomyces cerevisiae has not been reported.

Disclosure of Invention

In view of this, the invention provides nucleic acid molecules, expression cassettes, recombinant strains and uses thereof. The invention discloses a coenzyme Q10-producing genetic engineering strain which comprises the steps of replacing a Paracoccus denitrificans-source DPP (dipeptidyl peptidase) synthetic gene of an endogenous COQ1 gene of saccharomyces cerevisiae, further overexpressing endogenous genes of the saccharomyces cerevisiae, such as COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20, IDI1 and a ESCHERICHIA COLI-source UbiC and Rhodobacter capsulatus-source UbiN, and improving shake flask yield to 100mg/L. The invention realizes the production of coenzyme Q10 by Saccharomyces cerevisiae for the first time, has potential for industrial production, and is suitable for practical popularization and application.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides a nucleic acid molecule having:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) A nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequence shown in (1);

(3) A sequence having at least 80% homology to the nucleotide sequence shown in (1);

(4) The complement of the sequence shown in (1), (2) or (3).

The invention also provides plasmids comprising the nucleic acid molecules.

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

In some embodiments of the invention, the chassis strain comprises a yeast.

In some embodiments of the invention, the chassis strain comprises one or more of Saccharomyces cerevisiae (Saccharomyces cerevisiae), kluyveromyces lactis (Kluyveromyces lactis), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), yarrowia lipolytica (Yarrowia lipolytica) and Pichia pastoris.

The invention also provides the application of the nucleic acid molecules, the plasmids and/or the strains in preparing and/or producing coenzyme Q10.

The invention also provides application of the over-expressed endogenous gene and/or exogenous gene in preparing and/or producing coenzyme Q10;

The endogenous genes comprise one or more of Saccharomyces cerevisiae-derived COQ3 genes, saccharomyces cerevisiae genes, COQ6 genes, COQ7 genes, COQ2 genes, tHMG1 genes, ERG20 genes and IDI1 genes;

The exogenous genes comprise UbiN genes from Rhodobacter capsulatus sources and/or UbiC genes from ESCHERICHIA COLI sources.

The invention also provides an expression cassette comprising an endogenous gene and/or an exogenous gene and an acceptable genetic element;

In some embodiments of the invention, the expression cassette comprises one or more of expression cassettes 1-5;

the expression cassette 1 comprises UbiN genes and UbiC genes;

The expression cassette 2 comprises a COQ3 gene and a COQ5 gene;

the expression cassette 3 comprises a COQ6 gene and a COQ7 gene;

The expression cassette 4 comprises COQ2 gene and IDI1 gene;

the expression cassette 5 comprises a tHMG1 gene and an ERG20 gene.

The invention also provides a recombinant plasmid, which comprises the expression cassette.

The invention also provides a recombinant strain, and the recombinant plasmid is transformed and/or transfected into the strain.

In some embodiments of the present invention, the recombinant strain has a preservation number of CCTCC NO: M2024615.

The invention also provides the application of the expression cassette, the recombinant plasmid and/or the recombinant strain in any of the following;

(I) Preparation and/or production of coenzyme Q10, and/or

(II) preparation and/or production of a product comprising coenzyme Q10, and/or

(III) improving the yield of coenzyme Q10.

The invention also provides products comprising the above nucleic acid molecules, the above plasmids, the above strains, the above expression cassettes, the above recombinant plasmids and/or the above recombinant strains.

The invention also provides a preparation method of the coenzyme Q10, which comprises the steps of inoculating the recombinant strain, fermenting and obtaining the coenzyme Q10.

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

In some embodiments of the invention, the inoculation adopts an inoculation culture medium, wherein the inoculation culture medium comprises 10-25 g/L of peptone, 5-15 g/L of yeast powder and 20-30 g/L of glucose.

In some embodiments of the present invention, the inoculation medium comprises 20g/L peptone, 10g/L yeast powder and 20g/L glucose.

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

In some embodiments of the invention, in the preparation method, fermentation medium is adopted for fermentation, wherein the fermentation medium comprises 10-60 g/L of glucose or glycerol, 2-3 g/L of monopotassium phosphate, 2.5-3.0 g/L of dipotassium phosphate, 10-28 g/L of yeast powder and 10-20 g/L of yeast peptone.

In some embodiments of the present invention, the fermentation medium comprises 20g/L glucose, 2.2g/L potassium dihydrogen phosphate, 2.9g/L dipotassium hydrogen phosphate, 10g/L yeast powder and 20g/L yeast peptone.

The invention discloses a genetic engineering strain for producing coenzyme Q10, which comprises the replacement of an endogenous COQ1 gene of saccharomyces cerevisiae

Paracoccus denitrificans

The DPP synthetic gene is derived and further overexpresses Saccharomyces cerevisiae endogenous genes COQ3, COQ5, COQ6, COQ7, COQ2, tHMG1, ERG20 and IDI1 and

Escherichia coli

UbiC of the source of the liquid,

Rhodobacter capsulatus

UbiN from the source, the shake flask yield was increased to 100mg/L. The invention realizes the production of coenzyme Q10 by Saccharomyces cerevisiae for the first time, has potential for industrial production, and is suitable for practical popularization and application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the biosynthetic pathway of coenzyme Q10 in an engineered Saccharomyces cerevisiae of the invention;

FIG. 2 shows a genomic map of a yeast COQ1 gene replaced with PdDDSA;

FIG. 3 shows a genomic map of yeast genomic integration UbiC and UbiN;

FIG. 4 shows the accumulation of coenzyme Q10 after 96h of cultivation of recombinant yeasts on shake flasks.

Description of biological preservation

Saccharomyces cerevisiae (Saccharomyces cerevisiaeCQ) with a preservation date of 2024, 04, 03, a preservation number of CCTCC No. M2024615, a preservation unit name of China center for type culture Collection, and a preservation center address of China university of Wuhan.

Detailed Description

The invention discloses a nucleic acid molecule, an expression cassette, a recombinant strain and application thereof.

It should be understood that one or more of the expressions ". The expressions" individually include each of the objects recited after the expressions and various combinations of two or more of the recited objects unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Furthermore, the numerical ranges and parameters setting forth the present invention are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

The invention provides a construction method of a recombinant strain for producing coenzyme Q10, which comprises the step of replacing a HexPP gene produced by an endogenous Saccharomyces cerevisiae with a synthetic gene for producing DPP, so that the coenzyme Q10 can be produced in the Saccharomyces cerevisiae.

Further, the nucleotide sequence of the DPP synthesis gene is shown as SEQ ID NO. 1.

Further, the recombinant yeast strain overexpresses 5 genes of the coenzyme Q10 synthesis pathway and 3 genes of the mevalonate pathway, COQ3（SGD:S000005456）、COQ5（SGD:S000004578）、COQ6（SGD:S000003487）、COQ7（SGD:S000005651）、COQ2（SGD:S000005324）、tHMG1（genbank：AJS90608.1（ from the 531 th amino acid), ERG20 (SGD: S000003703), IDI1 (SGD: S000006038), all derived from Saccharomyces cerevisiae.

Further, the recombinant yeast over-expresses a gene UbiN responsible for decarboxylation and hydroxylation, a gene Ubic responsible for synthesis of head parahydroxybenzoic acid (pHBA), ubin derived from Rhodobacter capsulatus (genbank: WP_ 013068139.1), ubiC derived from ESCHERICHIA COLI (genbank: CAD 6022848.1).

The invention also provides a method for producing coenzyme Q10, which is to produce coenzyme Q10 on a large scale by fermenting the engineering bacteria subjected to the synthetic biological transformation, and comprises the following steps:

Inoculating the recombinant strain to a seed culture medium, culturing for 20-24 hours, inoculating a seed solution to a fermentation culture medium, fermenting and culturing for 48-84 hours, and separating and purifying to obtain the recombinant strain;

further, the seed culture medium comprises 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;

The formula of the fermentation medium comprises 20g/L of glucose or glycerol, 2.2g/L of monopotassium phosphate, 2.9g/L of dipotassium phosphate, 10g/L of yeast powder and 20g/L of peptone.

The culture is shaking culture, the temperature is 30 ℃, and the rotating speed is 200rpm.

Sequence information related to the present invention:

Nucleotide sequence optimized by DDSA codon (SEQ ID NO:1)：ATGGGTATGAATGAAAATGTTTCTAAACCATTGGATAGATTGTCTGTTGAATTGGCTGGTGATATGGATAGAGTTAATGCTTTGATCAGAGAAAGAATGGCTTCTAGACATGCTCCAAGAATTCCTGAAGTTACTGCTCATTTGGTTGAAGCTGGTGGTAAAAGATTGAGACCAATGTTGGTTTTGGCTGCAGCTAGATTATGTGGTTATCAAGGTAATTCTCATGTTTTGTTGGCTGCAGCTGTTGAATTTATTCATACTGCTACTTTGTTGCATGATGATGTTGTTGATGAATCTCAACAAAGAAGAGGTAGACCAACTGCTAATTTGTTGTGGGATAATAAATCTTCTGTTTTGGTTGGTGATTATTTGTTTGCTAGATCTTTTCAATTGATGGCTGATACTGAATCTATGCAAGTTATGAGAATTTTGGCTAATGCATCTGCTACTATTGCTGAAGGTGAAGTTTTGCAATTGACTGCTGCTCAAGATGTTTCTACTACTGAAGATACTTATATTCAAATTGTTAGAGGTAAAACTGCTGCTTTGTTTTCTGCTGCTACTGAAGCTGGTGCTGTTGTTGCTGGTGCTGATCCTGCTGTTCAACAAGCTTTGTTTGATTATGGTGATGCTTTGGGTATTGCTTTTCAAATTGTTGATGATTTGTTGGATTATGGTGGTTCTACTACAACTATTGGTAAAAATGTTGGTGATGATTTCAGAGAGAGAAAATTAACTTTGCCTGTTATTAAAGCTATTGCTAGAGCTGACGAAGCTGAAAGAGCTTTTTGGGAAAGAACTATTGGTCAAGGTAGACAAGATGAAGCTGATTTAGCTACTGCTTTGGAAATTTTGAGAAGGAGAGAAGCTTTGGAAGCTGCTAGAGCAGATGCAATTGCTTGGGCTGGTAGAGCTAAAGCTGCTTTGCAAGCTGCTCCTGATCAACCATTGAGAAGGATTTTGGCAGATTTGGCTGACTTTGTTGTTTCTAGATTGTCTTAA.

Primer information related to the invention:

TABLE 1 nucleotide sequences of primers

In examples 1 to 3 of the present invention, all the raw materials and reagents used were commercially available.

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of coenzyme Q10-producing Saccharomyces cerevisiae Strain of the invention

The DDSA gene from Paracoccus denitrificans source is first codon optimized and then synthesized chemically by Shanghai qinghao biological science and technology Co., ltd, pdDDSA-COQ1-F and TCYC1-COQ1-R are used as primer to amplify the DDSA gene, the PCR product is detected by 1.0% agarose gel electrophoresis and the clean-up kit is used to purify the gene fragment;

The PCR products of the homology arms COQ1-UP and COQ1-Dn were obtained BY PCR using primers COQ1-UF/COQ1-PdDDSA-UR, COQ1-TCYC1-DF/COQ1-DR with Saccharomyces cerevisiae EN.PK2-1C or BY4741 genome as a template, and the PCR products were detected BY 1.0% agarose gel electrophoresis and the gene fragment was purified BY using a clean-UP kit;

The purified products of the homologous arms COQ1-UP and COQ1-Dn and the purified product of the DDSA gene were replaced with the DDSA gene by using CRISPR/Cas9 gene editing technology, and the Saccharomyces cerevisiae EN.PK2-1C COQ1 gene was replaced with the DDSA gene to obtain the yeast engineering strain CQ01 (FIG. 2).

EXAMPLE 2 integration of coenzyme Q synthetic pathway genes into genome according to the invention

Combining and synthesizing (Shanghai qing department Biotechnology Co., ltd.) UbiC from code ESCHERICHIA COLI and UbiN from Rhodobacter capsulatus, respectively using UbiC-F/UbiC-R, ubiN-F/UbiN-R as primer, PCR amplifying UbiC and UbiN genes, detecting PCR product by 1.0% agarose gel electrophoresis and purifying gene fragment by clean-up kit;

Double enzyme digestion is carried out on a target plasmid pDS01 by two enzymes, namely BamHI and HindIII, enzyme digestion products are detected by 1.0% agarose gel electrophoresis, gel digestion is carried out, the linearized vector fragment is recovered and purified, then a one-step cloning kit of Norprazid is adopted to connect the purified UbiC gene fragment with the linearized plasmid pDS01 (37 ℃ for 30 min), the connection product is converted into E. coliDH α, the conversion product is obtained, the conversion product is coated on LB solid medium (containing 100mg/L ampicillin) to obtain single clone, shake flask culture is carried out for 8-12 h under the conditions of 37 ℃ and 220rpm, then plasmid extraction is carried out for sequencing verification, and the plasmid pDS01-UbiC expressing UbiC gene is obtained after verification is correct;

Double enzyme digestion is carried out on a target plasmid pDS01-UbiC by using NotI and BcuI enzymes, enzyme digestion products are detected by using 1.0% agarose gel electrophoresis, and cut gel is recovered and purified to obtain linearized vector fragments, then a one-step cloning kit of Noruzan is adopted to connect the purified UbiN gene fragments with the linearized plasmid pDS01-UbiC (37 ℃ and 30 min), the connection products are converted into E. coliDH5 alpha to obtain conversion products, the conversion products are coated on LB solid culture medium (containing 100mg/L ampicillin) to obtain monoclone, shake flask culture is carried out for 8-12 h under the conditions of 37 ℃ and 220rpm, then plasmids are extracted for sequencing verification, and the expression plasmids pDS01-UbiC-UbiN of UbiC genes and UbiN genes are obtained after verification;

Using Saccharomyces cerevisiae EN.PK2-1C or BY4741 genome as template, obtaining PCR products of homology arms int10-UP and int10-Dn BY PCR using primers int10-UF/int10-UR, int10-DF/int10-DR, detecting the PCR products BY 1.0% agarose gel electrophoresis and purifying the gene fragment BY using clean-UP kit;

Using plasmid pDS01-UbiC-UbiN as a template, obtaining PCR products of UbiC and UbiN expression cassettes by PCR using a primer int10-F/int10-R, detecting the PCR products by 1.0% agarose gel electrophoresis and purifying the gene fragments by using a clean-up kit;

purified products of the homology arms int10-UP and int10-Dn, ubiC and UbiN expression cassettes, and pDS01-UbiC-UbiN were integrated into the int10 site of engineering strain CQ01 using CRISPR/Cas9 gene editing technology to obtain yeast engineering strain CQ02 (FIG. 3).

Taking the construction method of the plasmid pDS01-UbiC-UbiN as an example, taking Saccharomyces cerevisiae EN.PK2-1C or BY4741 genome as a template, taking the plasmid pDS01 as a vector to respectively construct expression plasmids of 5 genes COQ3, COQ5, COQ6, COQ7, COQ2 and 3 genes tHMG1, ERG20 and IDI1 of the coenzyme Q path, namely pDS02-COQ3-COQ5, pDS03-COQ6-COQ7, pDS04-COQ2-IDI and pDS05-tHMG1-ERG20.

Taking a construction method of the yeast engineering strain CQ02 as an example, respectively taking expression plasmids pDS02-COQ3-COQ5, pDS03-COQ6-COQ7, pDS04-COQ2-IDI and pDS05-tHMG1-ERG20 of the 5 genes of the coenzyme Q pathway and the 3 genes of the MVA pathway as templates, obtaining PCR products by PCR, and integrating the corresponding expression cassettes of the 5 genes of the coenzyme Q pathway and the 3 genes of the MVA pathway into a genome of the yeast engineering strain CQ02 by using a CRISPR/Cas9 gene editing technology to obtain the yeast engineering strain CQ14.

EXAMPLE 3 fermentation of recombinant coenzyme Q10-producing Yeast strains of the invention

The recombinant Saccharomyces cerevisiae engineering strain CQ14 constructed in example 2 was inoculated in a seed culture medium, shake-cultured at 30℃and 200rpm for 24 hours, the seed solution was inoculated in a fermentation culture medium according to 1% (v/v), and shake-cultured at 30℃and 200rpm for 96 hours. And centrifuging the fermentation liquor to obtain thalli, crushing cells by a freeze grinder, extracting by using an organic solvent ethyl acetate, filtering and quantifying by GC, so that the output of coenzyme Q10 in the fermentation liquor of the saccharomyces cerevisiae engineering strain CQ14 reaches more than 100mg/L (figure 4).

Wherein the seed culture medium comprises 20g/L peptone, 10g/L yeast powder and 20g/L glucose;

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

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Recombinant strain, characterized in that the recombinant plasmid is transformed into the strain;

The recombinant plasmid contains endogenous genes, exogenous genes and acceptable gene elements;

the endogenous genes comprise Saccharomyces cerevisiae-derived COQ3 genes, COQ5 genes, COQ6 genes, COQ7 genes, COQ2 genes, tHMG1 genes, ERG20 genes and IDI1 genes;

the exogenous genes comprise UbiN gene from Rhodobacter capsulatus and UbiC gene from ESCHERICHIA COLI;

The strain replaces the COQ1 gene for producing HexPP by Saccharomyces cerevisiae endogenously with a synthetic gene for producing DPP, and the synthetic gene sequence of the DPP is shown as SEQ ID NO. 1.

2. The recombinant strain of claim 1, wherein the recombinant strain has a preservation number of CCTCC NO: M2024615.

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

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