Electron transport chain subunit acCYTB with low-temperature activity and application thereof

Abstract

The invention discloses an electron transport chain subunit acCYTB with low-temperature activity and application of the electron transport chain subunit acCYTB, and relates to the technical field of gene engineering and the technical field of enzyme engineering. The electron transport chain subunit provided by the invention is any one of the following (1), (2) and (3): (1) acCYTB protein, the amino acid sequence of which is as shown in 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 acCYTB protein to obtain a protein which has more than 90% of identity with the acCYTB protein and has the same biological function with the acCYTB 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 present invention relates to the technical fields of gene engineering and enzyme engineering, and in particular to an electron transport chain subunit acCYTB with low-temperature activity and an application thereof. Background Art

[0002] In low-temperature environments, metabolic activity is inhibited, which can lead to a slowdown or even stagnation of physiological functions. To adapt to cold environments, organisms must adjust their biochemical pathways to maintain basic physiological activities and energy production. These adaptive adjustments help organisms remain active in low temperatures and carry out normal growth, reproduction, and other life activities.

[0003] The electron transport chain (ETC) is a key component of energy production in cells, involving proteins encoded by multiple genes, such as NDUFA12, SDHD, and CYTB. CYTB encodes cytochrome b, the core protein of complex III. Cytochrome b is responsible for receiving electrons from coenzyme QH2 and transferring them to cytochrome c via the coenzyme Q cycle, catalyzing the electron transport process and promoting ATP production. These proteins work synergistically to generate ATP within mitochondria through redox reactions. The efficiency of the ETC may decrease under low temperature conditions, so enhancing the low-temperature activity of the enzymes encoded by these genes is crucial for improving energy metabolism and survival in cold environments.

[0004] In some industrial fermentation processes, low temperatures can reduce side reactions and improve product purity and quality. This is particularly important in beer brewing and dairy fermentation. Low-temperature fermentation also reduces 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 yields, reducing costs, and enhancing product quality.

[0005] At the same time, some industrial fermentation fields also require the development of high-temperature-active microbial strains. For example, many industrial reactions require high temperatures to accelerate reaction rates or achieve specific chemical transformations. Traditional low-temperature-active microbial strains are easily inactivated in such environments, resulting in low catalytic efficiency. Developing 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 cost-effectiveness of industrial production. Summary of the Invention

[0006] The present invention aims to provide an electron transport chain subunit, acCYTB, with low-temperature activity and its application to address the aforementioned problems of the prior art. This electron transport chain subunit can improve the low-temperature tolerance of organisms, thereby finding applications in 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) acCYTB 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 acCYTB protein to obtain a protein having more than 90% identity with the acCYTB protein and having the same biological function as the acCYTB 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 genes encoding the above-mentioned electron transport chain subunits.

[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 the substances (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-mentioned 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 above-mentioned electron transport chain subunit, comprising the steps of mutating the amino acid at position 304 of the electron transport chain subunit into an aliphatic amino acid, mutating the amino acid at position 333 into an aliphatic amino acid, and / or mutating the amino acid at position 375 into an amino acid with stronger basic properties; the aliphatic amino acid is preferably leucine; and the amino acid with stronger basic properties is preferably arginine.

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

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

[0029] The present invention also modifies the optimal enzyme activity temperature by mutating key amino acid sites. This method is simple and rapid; it even allows for the improvement of growth and production efficiency of organisms under different temperature environments by editing key sites in the organism's own homologous genes without the need for exogenous gene transfer. This method can be widely applied to improve the temperature tolerance of organisms, providing 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0031] Figure 1The figure shows the activity test results of acCYTB protein at 30°C. 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 acCYTB protein mutant; and the original genotype refers to the group transfected with the recombinant plasmid carrying the gene encoding the acCYTB protein.

[0032] Figure 2 The figure shows the activity test results of acCYTB protein under 4°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 acCYTB protein mutant; and the original genotype refers to the group transfected with the recombinant plasmid carrying the gene encoding the acCYTB 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 a low-temperature-active cytochrome B gene from a species of the Actinoscyphiidae family. The present invention names the gene acCYTB. The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the acCYTB protein encoded by the gene is shown in SEQ ID NO. 2.

[0040] SEQ ID NO.1:

[0041]

[0042] SEQ ID NO.2:

[0043] MVQFSKQNPVLSIVNGMVIDLPAPANLSYMWNFGSLLGVCLVMQIATGMFLAMHYCADVSLAFASVDHIMRDVNYGFLLSYFHANGASMFFLCLYIHVGRGLYYGSYSKIEVWNVGVVLFMLTMATAFIGYVLPWGQMSFWGATVITNLLSAIPYMGTDVVQWVWGGFSVSNATLNRFFSLHYLFPFLLAA LAIVHLICLHVDGSNNPIGVSSDLDKVAFHVYYTSKDWYGMVAFAVFFCSLVYLAPNLLGDPENFIQANPLVTPVHIQPEWYFLFAYAILRSIPNKLGGVVAMFSSLLMLFLIPWLHSSRLSGLTFRPLARIAFWFLAADFFLLTWIGSQPVEEPFILVGQLASIFYFSYFLVISPLLGYLENHLLFPKKG.

[0044] A mutant of the acCYTB protein was constructed by site-directed mutation of amino acid Ile (I) at position 304 of the acCYTB protein to leucine (Leu), amino acid Phe (F) at position 333 to leucine (Leu), and amino acid His (H) at position 375 to arginine (Arg). This mutant exhibited an increased optimal activity temperature. The amino acid sequence of this mutant is shown in SEQ ID NO. 3.

[0045] SEQ ID NO.3:

[0046] MVQFSKQNPVLSIVNGMVIDLPAPANLSYMWNFGSLLGVCLVMQIATGMFLAMHYCADVSLAFASVDHIMRDVNYGFLLSYFHANGASMFFLCLYIHVGRGLYYGSYSKIEVWNVGVVLFMLTMATAFIGYVLPWGQMSFWGATVITNLLS AIPYMGTDVVQWVWGGFSVSNATLNRFFSLHYLFPFLLAALAIVHLICLHVDGSNNPIGVSSDLDKVAFHVYYTSKDWYGMVAFAVFCSLVYLAPNLLGDPENFIQANPLVTPVHIQPEWYFLFAYAILRSIPNKLGGVVAMFSSLLMLFL LPWLHSSRLSGLTFRPLARIAFWFLAADF L LLTWIGSQPVEEPFILVGQLASIFYFSYFLVISPLLGYLEN R LLFPKKG, the underlined part is the mutation site.

[0047] Example 2

[0048] 1. Experimental Materials

[0049] YPD medium: 20 g / L glucose, 20 g / L tryptone, and 10 g / L yeast extract.

[0050] 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 Lu, 0.1 g / L His, and 0.1 g / L Trp. Solid medium was prepared by adding 1.5% (w / v) agar.

[0051] The pRS416 vector is a Saccharomyces cerevisiae expression vector and can be purchased from biological companies (such as Shanghai Zeye Biotechnology Co., Ltd., etc.).

[0052] 2. Construction of recombinant yeast strains

[0053] The coding genes for the acCYTB protein and its mutant from 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. These recombinant plasmids were then transformed into Saccharomyces cerevisiae using lithium acetate (LiOAc)-mediated yeast transformation, with the empty plasmid pRS416 serving as a control. The specific steps were as follows:

[0054] A fresh single colony was cultured at 30°C overnight. The culture was then diluted to an OD 600 The culture medium was added to 5 mL of fresh YPD medium and cultured at 30 °C until the OD 600 About 0.6. Collect the cells, wash with 1 mL of sterile ddH2O and 500 μL of 0.1M LiOAc, and then resuspend in 100 μL of 0.1M LiOAc. Next, 20 μL of cells were mixed with 80 μL of transformation buffer (58.6 μL of 50% PEG3350, 7.7 μL of 1M LiOAc, 9.0 μL of DMSO, 4.7 μL of ssDNA) and the recombinant plasmid. Gently pipette the mixture evenly, incubate at 30°C for 35 minutes, and then heat shock treat at 42°C for 15 minutes. Centrifuge to remove the supernatant, resuspend the cells in 200 μL of sterile ddH2O, spread on selective solid culture medium, and culture at 30°C for 3 days.

[0055] 3. Yeast Mitochondria Extraction

[0056] Different positive transformants were identified and resuspended in 1× PBS buffer (1 mL, P1020, Solebro) and washed by centrifugation at 5000 × g for 1 minute at room temperature. After two washes, relatively pure yeast cells were obtained. Yeast mitochondria were then extracted using a yeast mitochondrial extraction kit (EX2900, Solebro) for subsequent experiments.

[0057] 4. Electron Transport Chain Subunit Activity Assay

[0058] The mitochondria obtained in the previous step were ultrasonically disrupted (power 200 W, ultrasonication for 5 seconds, intervals of 10 seconds, repeated 15 times). The respiratory chain complexes obtained from the lysed mitochondria were quantified using the BCA protein assay kit (23225, Thermo Fisher). Subsequently, the activity of the electron transport chain subunit acCYTB protein was detected using the mitochondrial respiratory chain complex I activity detection kit (BC0515, Solebo) according to the manufacturer's kit instructions. The protein activity was detected at 30°C and 4°C according to the experimental group. Finally, the enzyme activity of each mitochondrial electron transport chain complex was measured at the corresponding temperature using a multifunctional microplate reader; 3 technical replicates were set for each group of tests to ensure the reliability of the results. Finally, the complex activity value 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.

[0059] The results of the activity test of the electron transport chain subunit acCYTB protein are shown in Figure 1 and Figure 2 The results showed that the activity signal of acCYTB protein under 4°C treatment conditions was significantly stronger than that of the mutant and the control. Under 30°C treatment conditions, the enzyme activity of the acCYTB mutant was significantly stronger than that of acCYTB and the control. These results indicate that acCYTB protein has good catalytic activity under low temperature conditions and can be used as a potential low-temperature breeding gene resource for the genetic improvement of low-temperature tolerance of organisms such as yeast. In addition, the 304th, 333rd and 375th amino acids of the acCYTB protein are the key determining sites of its active temperature. After mutating the 304th amino acid to Leu (L), the 333rd amino acid to Leu (L), and the 375th amino acid to Arg (R), the active temperature of the acCYTB protein can be effectively increased, which has a wide range of application scenarios in improving the tolerance of organisms to different temperatures.

[0060] The codon-optimized sequence of the gene encoding acCYTB protein (SEQ ID NO. 4):

[0061]

[0062] The codon-optimized sequence of the gene encoding the acCYTB protein mutant (SEQ ID NO. 5):

[0063]

[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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) acCYTB 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 acCYTB protein to obtain a protein having more than 90% identity with the acCYTB protein and having the same biological function as the acCYTB 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 steps of mutating the amino acid at position 304 of the electron transport chain subunit into an aliphatic amino acid, mutating the amino acid at position 333 into an aliphatic amino acid, and / or mutating the amino acid at position 375 into an amino acid with stronger alkaline properties.

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