Electron transport chain subunit with low-temperature activity as well as coding gene and application of electron transport chain subunit

Abstract

The invention discloses an electron transport chain subunit with low-temperature activity as well as a coding gene and application thereof, and relates to the technical fields of gene engineering and enzyme engineering. The electron transport chain subunit is any one of the following (1), (2) and (3): (1) hoNDUFA12 protein, the amino acid sequence of which is shown as SEQ ID NO.2; (2) carrying out substitution, deletion and / or addition of one or more amino acid residues on the amino acid sequence of the hoNDUFA12 protein to obtain a protein which has more than 90% of identity with the hoNDUFA12 protein and has the same biological function with the hoNDUFA12 protein; and (3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (1) or (2). The electron transport chain subunit can improve the low-temperature resistance of organisms, so that the electron transport chain subunit is applied to low-temperature character improvement and industrial production.

Description

Technical Field

[0001] The invention relates to the technical fields of gene engineering and enzyme engineering, and in particular to an electron transport chain subunit with low-temperature activity, a coding gene thereof and an application thereof. Background Art

[0002] The metabolic activities of organisms in low temperature environments are inhibited, which may cause physiological functions to slow down or even stagnate. In order to adapt to cold environments, organisms must adjust their biochemical pathways to maintain basic physiological activities and energy production. These adaptive adjustments help organisms stay active in low temperatures and carry out normal growth, reproduction and other life activities.

[0003] The electron transport chain (ETC) is a key component for energy production in cells, involving proteins encoded by multiple genes, such as NDUFA12, SDHD, and CYTB. Among them, the NDUFA12 gene encodes a non-catalytic subunit of the mitochondrial electron transport chain complex I, which helps maintain the structural and functional stability of complex I, participates in the correct assembly of the complex, ensures the effective transfer of electrons, and may be involved in regulating the response of complex I to antioxidants. These proteins work synergistically to produce ATP in mitochondria through redox reactions. Under low temperature conditions, the efficiency of ETC may be reduced, so enhancing the low-temperature activity of enzymes encoded by these genes is essential for improving the energy metabolism and survival of organisms in cold environments.

[0004] In some industrial fermentation processes, low temperature conditions can reduce side reactions during fermentation and improve product purity and quality, which is particularly important in beer brewing and dairy fermentation. Low temperature fermentation can also reduce energy consumption, production costs, and the risk of microbial contamination. Therefore, cultivating microbial strains with low temperature activity is of great practical significance for increasing production, reducing costs, and improving product quality.

[0005] At the same time, some industrial fermentation fields also have the need to construct high-temperature active microbial strains. For example, many industrial reactions need to be carried out at high temperatures to accelerate the reaction rate or achieve specific chemical transformations. Traditional low-temperature active microbial strains are easily inactivated in such environments, resulting in low catalytic efficiency. The development of high-temperature active microbial strains can ensure stability and activity under high temperature conditions, thereby improving production efficiency and product quality. Therefore, cultivating electron transport chain subunits with different temperature activities can greatly improve the efficiency and economy of industrial production. Summary of the invention

[0006] The purpose of the present invention is to provide an electron transport chain subunit with low temperature activity and its encoding gene and application to solve the problems existing in the above-mentioned prior art. The electron transport chain subunit can improve the low temperature resistance of organisms, so that it can be applied to low temperature trait improvement and industrial production.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an electron transport chain subunit, which is any one of the following (1), (2) and (3):

[0009] (1) hoNDUFA12 protein, the amino acid sequence of which is shown in SEQ ID NO.2;

[0010] (2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the hoNDUFA12 protein to obtain a protein having more than 90% identity with the hoNDUFA12 protein and having the same biological function as the hoNDUFA12 protein;

[0011] (3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of (1) or (2).

[0012] The present invention also provides a gene encoding the above-mentioned electron transport chain subunit.

[0013] The present invention also provides a biomaterial for expressing the above-mentioned electron transport chain subunit, comprising the substance described in any one of (a1) to (a3):

[0014] (a1) a gene expression cassette containing a gene encoding the above-mentioned electron transport chain subunit;

[0015] (a2) a recombinant vector containing the gene expression cassette;

[0016] (a3) A recombinant microbial strain containing the recombinant vector.

[0017] The present invention also provides a mutant of an electron transport chain subunit, the amino acid sequence of which is shown in SEQ ID NO.3.

[0018] The present invention also provides a gene encoding the mutant.

[0019] The present invention also provides a biomaterial for expressing a mutant of a gene encoding an electron transport chain subunit, comprising any one of (b1) to (b3):

[0020] (b1) a gene expression cassette containing a gene encoding a mutant;

[0021] (b2) a recombinant vector containing the gene expression cassette;

[0022] (b3) A recombinant microbial strain containing the recombinant vector.

[0023] The present invention also provides the use of the above-mentioned electron transport chain subunits, the encoding genes of the electron transport chain subunits or the biological materials in improving the low temperature resistance of microbial strains.

[0024] The present invention also provides a method for improving the low temperature resistance of a microbial strain, comprising the steps of genetically transforming the above encoding gene into the microbial strain to construct a recombinant microbial strain that overexpresses the encoding gene.

[0025] The present invention also provides the use of the above-mentioned coding gene or biological material in preparing the above-mentioned electron transport chain subunit.

[0026] The present invention also provides a method for increasing the optimal activity temperature of the electron transport chain subunit, comprising the step of mutating the 22nd amino acid of the electron transport chain subunit to other more hydrophilic amino acids, wherein the other more hydrophilic amino acids are preferably tryptophan.

[0027] The present invention discloses the following technical effects:

[0028] The invention separates and clones an electron transport chain subunit with low temperature activity and its encoding gene from species of Hormathiidae, which can improve the low temperature resistance of organisms and thus be applied to low temperature trait improvement and industrial production.

[0029] At the same time, the present invention changes the optimal activity temperature of the enzyme by mutating the key amino acid sites. This method is simple and fast; it even allows the growth and production efficiency of organisms in different temperature environments to be improved by editing the key sites of the organism's own homologous genes without introducing exogenous genes. It can be widely used to improve the temperature tolerance of organisms, and provides an efficient, safe and environmentally friendly technical approach for agricultural production, industrial fermentation and biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1The figure shows the activity test results of the hoNDUFA12 protein under 30°C treatment conditions; the wild type refers to the group transfected with the empty plasmid pRS416; the mutant genotype refers to the group transfected with the recombinant plasmid carrying the gene encoding the mutant of the hoNDUFA12 protein; the original genotype refers to the group transfected with the recombinant plasmid carrying the gene encoding the hoNDUFA12 protein;

[0032] Figure 2 The figure shows the activity detection results of hoNDUFA12 protein under 4°C treatment conditions; the wild type refers to the group transfected with empty plasmid pRS416; the mutant genotype refers to the group transfected with recombinant plasmid carrying the gene encoding the hoNDUFA12 protein mutant; the original genotype refers to the group transfected with the recombinant plasmid carrying the gene encoding the hoNDUFA12 protein. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] The present invention isolates and clones an electron transport chain subunit gene with low-temperature activity from species of the Hormathiidae family, which is named hoNDUFA12 gene in the present invention. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the hoNDUFA12 protein encoded by it is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1:

[0041] ATGTCGAAGATTCAAAATTGGATGGGTTTCATGCGCCAAATTGGAGGCATCAGAGGTTCTTTT TTC AGATTTCTTAGAGAGGGTACAGCTCGAGTTGGCACTTGCGTTGGAGAAGACAAGTATGGGAATAAATATTACGAGAATAACTGGTACTTTTTCGGCCGAAATCGACACGTAGTTTACCCGTATGCAGGACGCTTGGAGCATGATGGTTCGCAAATTCCCGCTGAATGGCA CCGATGGATGCATTATATGACTGATGACACTCCTACTTTGGTTAAACCTGTACAGAGGAAGTTTCTTCTAGATCATGAAAGGAATTACACGGGCACTAAACAAGAATATGTTCCATATAGCACCACTAGACCAAAGATTGAGTCTTGGGAACCACAGAAAAGTTCATGA.

[0042] SEQ ID NO.2:

[0043] MSKIQNWMGFMRQIGGIRGSF F RFLREGTARVGTCVGEDKYGNKYYENNWYFFGRN RHVVYPYAGRLEHDGSQIPAEWHRWMHYMTDDTPTLVKPVQRKFLLDHERNYTGTKQEY VPYSTTRPKIESWEPQKSS.

[0044] Construction of a mutant of the hoNDUFA12 protein: After the site-directed mutation of the amino acid Phe (F) at position 22 of the hoNDUFA12 protein to Trp (W), the optimal activity temperature of the mutant increased. The amino acid sequence of the mutant is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0045] SEQ ID NO.3:

[0046] MSKIQNWMGFMRQIGGIRGSF W RFLREGTARVGTCVGEDKYGNKYYENNWYFFGRN RHVVYPYAGRLEHDGSQIPAEWHRWMHYMTDDTPTLVKPVQRKFLLDHERNYTGTKQEY VPYSTTRPKIESWEPQKSS, the underlined part indicates the mutation site.

[0047] SEQ ID NO.4:

[0048] ATGTCGAAGATTCAAAATTGGATGGGTTTCATGCGCCAAATTGGAGGCATCAGAGGTTCTTTT TGG AGATTTCTTAGAGAGGGTACAGCTCGAGTTGGCACTTGCGTTGGAGAAGACAAGTATGGGAATAAATATTACGAGAATAACTGGTACTTTTTCGGCCGAAATCGACACGTAGTTTACCCGTATGCAGGACGCTTGGAGCATGATGGTTCGCAAATTCCCGCTGAATGGCA CCGATGGATGCATTATATGACTGATGACACTCCTACTTTGGTTAAACCTGTACAGAGGAAGTTTCTTCTAGATCATGAAAGGAATTACACGGGCACTAAACAAGAATATGTTCCATATAGCACCACTAGACCAAAGATTGAGTCTTGGGAACCACAGAAAAGTTCATGA.

[0049] Example 2

[0050] 1. Experimental Materials

[0051] YPD medium: 20 g / L glucose, 20 g / L tryptone and 10 g / L yeast extract.

[0052] Selection medium (i.e., synthetic defective (SC) medium): made of 6.7 g / L yeast nitrogen base (YNB) without amino acids, 20 g / L glucose, 0.1 g / L Lue, 0.1 g / L His, and 0.1 g / L Trp. Solid medium was prepared by adding 1.5% (w / v) agar.

[0053] The pRS416 vector is a Saccharomyces cerevisiae expression vector and can be purchased from biological companies (such as Shanghai Zeye Biotechnology Co., Ltd., etc.).

[0054] 2. Construction of recombinant yeast strains

[0055] The coding genes of the hoNDUFA12 protein and its mutants in Example 1 were codon-optimized and cloned into the pRS416 vector, placed between the TEF1 promoter and the CYC1 terminator, to construct two recombinant plasmids. Then, the two recombinant plasmids were transformed into Saccharomyces cerevisiae using the lithium acetate (LiOAc)-mediated yeast transformation method, and the empty plasmid pRS416 was used as a control. The specific steps are as follows:

[0056] A fresh single colony was cultured overnight at 30°C. The culture was then diluted to OD 600 The culture medium was placed in 5 mL of fresh YPD medium and cultured at 30 °C until the OD 600 The cell density is about 0.6. Collect the cells and wash with 1 mL of sterile ddH 2 O and 500 μL 0.1M LiOAc, then resuspended in 100 μL 0.1M LiOAc. Next, 20 μL of cells were mixed with 80 μL transformation buffer (58.6 μL 50% PEG3350, 7.7 μL 1M LiOAc, 9.0 μL DMSO, 4.7 μL ssDNA) and recombinant plasmid. The mixture was gently pipetted evenly, incubated at 30°C for 35 minutes, and then heat-shocked at 42°C for 15 minutes. The supernatant was centrifuged and the cells were resuspended in 200 μL sterile ddH 2 O, and spread on selective solid culture medium and culture at 30°C for 3 days.

[0057] 3. Yeast Mitochondria Extraction

[0058] Different positive transformants were identified and resuspended in 1×PBS buffer (1 mL, P1020, Solebol) and washed by centrifugation at 5000×g for 1 minute at room temperature. After two washes, relatively pure yeast cells were obtained. Subsequently, yeast mitochondria were extracted using a yeast mitochondrial extraction kit (EX2900, Solebol) for subsequent experiments.

[0059] 4. Electron Transport Chain Subunit Activity Assay

[0060] The mitochondria obtained in the previous step were ultrasonically disrupted (power 200W, ultrasonic for 5 seconds, interval 10 seconds, repeated 15 times). The BCA protein assay kit (23225, Thermo Fisher) was used to quantify the protein of the respiratory chain complex obtained by lysing the mitochondria. Subsequently, the activity of the electron transport chain subunit hoNDUFA12 protein was detected using the mitochondrial respiratory chain complex I activity detection kit (BC0515, Solebao) according to the manufacturer's kit instructions. The protein activity was detected at 30°C and 4°C according to the experimental grouping. Finally, the enzyme activity of each mitochondrial electron transport chain complex was determined at the corresponding temperature by a multifunctional microplate reader; 3 technical replicates were set for each group of detection to ensure the reliability of the results. Finally, the activity value of the complex was normalized, that is, Δ measured value = actual measured value / mean of 3 technical replicates of the control group. The larger the Δ measured value, the higher the enzyme activity.

[0061] The results of the activity test of the electron transport chain subunit hoNDUFA12 protein are shown in Figure 1 and Figure 2 The results showed that the activity signal of hoNDUFA12 protein under 4°C treatment conditions was significantly stronger than that of mutants and controls. Under 30°C treatment conditions, the enzyme activity of the mutant of hoNDUFA12 was significantly stronger than that of hoNDUFA12 and the control. These results indicate that the hoNDUFA12 protein has good catalytic activity under low temperature conditions and can be used as a potential low-temperature breeding gene resource for genetic improvement of low-temperature tolerance in organisms such as yeast. In addition, the 22nd amino acid of the hoNDUFA12 protein is the key determining site for its active temperature. After mutating it to Trp (W), the active temperature of the hoNDUFA12 protein can be effectively increased, which has a wide range of application scenarios in improving the tolerance of organisms to different temperatures.

[0062] The sequence of the gene encoding the hoNDUFA12 protein after codon optimization (SEQ ID NO.5):

[0063] ATGTCCAAGATCCAAAACTGGATGGGTTTCATGAGACAAATCGGTGGTATCAGAGGTTCTTTC TTCAGATTCTTGAGAGAAGGTACTGCCAGAGTTGGTACCTGTGTCGGTGAAGACAAATACGGTAACAAGTACTACGAAAACAACTGGTACTTTTTCGGTCGTAATCGTCACGTTGTCTACCCATACGCTGGTAGATTGGAACACGATGGTTCCCAAATTCCAGCTGAATGGCA CAGATGGATGCATTACATGACTGATGACACTCCAACTTTGGTCAAGCCAGTTCAAAGAAAGTTCTTATTGGACCACGAAAGAAACTACACTGGCACCAAGCAAGAATACGTTCCTTATTCCACCACCAGACCAAAGATTGAATCTTGGGAACCACAAAAGTCTTCTTAG.

[0064] The sequence of the gene encoding the hoNDUFA12 protein mutant after codon optimization (SEQ ID NO.6):

[0065] ATGTCCAAGATCCAAAACTGGATGGGTTTCATGAGACAAATCGGTGGTATCAGAGGTTCTTTC TGG AGATTCTTGAGAGAAGGTACTGCCAGAGTTGGTACCTGTGTCGGTGAAGACAAATACGGTAACAAGTACTACGAAAACAACTGGTACTTTTTCGGTCGTAATCGTCACGTTGTCTACCCATACGCTGGTAGATTGGAACACGATGGTTCCCAAATTCCAGCTGAATGGCA CAGATGGATGCATTACATGACTGATGACACTCCAACTTTGGTCAAGCCAGTTCAAAGAAAGTTCTTATTGGACCACGAAAGAAACTACACTGGCACCAAGCAAGAATACGTTCCTTATTCCACCACCAGACCAAAGATTGAATCTTGGGAACCACAAAAGTCTTCTTAG.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An electron transport chain subunit, characterized in that The electron transport chain subunit is any one of the following (1), (2) and (3): (1) hoNDUFA12 protein, the amino acid sequence of which is shown in SEQ ID NO.2; (2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the hoNDUFA12 protein to obtain a protein having more than 90% identity with the hoNDUFA12 protein and having the same biological function as the hoNDUFA12 protein; (3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of (1) or (2).

2. A gene encoding an electron transport chain subunit as claimed in claim 1.

3. A biomaterial for expressing the electron transport chain subunit according to claim 1, characterized in that: A substance comprising any one of (a1) to (a3): (a1) a gene expression cassette containing the coding gene according to claim 2; (a2) a recombinant vector containing the gene expression cassette; (a3) A recombinant microbial strain containing the recombinant vector.

4. A mutant of an electron transport chain subunit, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

5. A gene encoding the mutant according to claim 4.

6. A biological material for expressing the mutant according to claim 4, characterized in that: A substance comprising any one of (b1) to (b3): (b1) a gene expression cassette containing the coding gene according to claim 5; (b2) a recombinant vector containing the gene expression cassette; (b3) A recombinant microbial strain containing the recombinant vector.

7. Use of the electron transport chain subunit according to claim 1, the encoding gene according to claim 2 or the biomaterial according to claim 3 in improving the low temperature resistance of a microbial strain.

8. A method for improving the low temperature resistance of microbial strains, characterized in that: The method comprises the steps of genetically transforming the encoding gene according to claim 2 into the microbial strain to construct a recombinant microbial strain that overexpresses the encoding gene.

9. Use of the coding gene according to claim 2 or the biological material according to claim 3 in preparing the electron transport chain subunit according to claim 1.

10. A method for increasing the optimal activity temperature of the electron transport chain subunit according to claim 1, characterized in that: The method comprises the step of mutating the 22nd amino acid of the electron transport chain subunit into other amino acids with stronger hydrophilicity.

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