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

Abstract

The application discloses an electron transfer chain subunit acCYTB with low-temperature activity and application thereof, and relates to the fields of genetic engineering and enzyme engineering technology. The electron transfer chain subunit provided by the application is any one of the following (1), (2) and (3): (1) an acCYTB protein, wherein the amino acid sequence is shown as SEQ ID NO. 2; (2) a protein obtained by substituting, deleting and / or adding one or more amino acid residues in the amino acid sequence of the acCYTB protein, wherein the protein has more than 90% identity with the acCYTB protein and has the same biological function as 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 transfer chain subunit can improve the low-temperature resistance of organisms, and thus can be applied to low-temperature trait improvement and industrial production.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, and in particular to an electron transport chain subunit acCYTB with low-temperature activity and its applications. Background Technology

[0002] Metabolic activity is inhibited in low-temperature environments, which can lead to a slowdown or even cessation 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, carrying out normal growth, reproduction, and other life activities.

[0003] The electron transport chain (ETC) is a crucial component of energy production in cells, involving proteins encoded by multiple genes, such as NDUFA12, SDHD, and CYTB. Among these, CYTB encodes cytochrome b, the core protein of complex III, which 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 produce ATP within mitochondria through redox reactions. Under low-temperature conditions, the efficiency of the ETC may decrease; therefore, enhancing the cryogenic activity of these gene-encoded enzymes is essential for improving energy metabolism and survival capabilities in cold environments.

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

[0005] Meanwhile, some industrial fermentation fields also have a need to construct microbial strains with high-temperature activity. For example, many industrial reactions need to be carried out at high temperatures to accelerate reaction rates or achieve specific chemical transformations. Traditional low-temperature active microbial strains are easily inactivated under such conditions, 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, culturing 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 this invention is to provide an electron transport chain subunit acCYTB with low-temperature activity and its applications, thereby addressing the problems existing in the prior art. This electron transport chain subunit can improve the low-temperature resistance of organisms, and thus can be applied to low-temperature trait improvement and industrial production.

[0007] To achieve the above objectives, the present invention provides the following solution:

[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) The amino acid sequence of the acCYTB protein is replaced, deleted and / or added by one or more amino acid residues to obtain a protein that has more than 90% identity with the acCYTB protein and has the same biological function as the acCYTB protein.

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

[0012] The present invention also provides the coding genes of the electron transport chain subunits described above.

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

[0014] (a1) A gene expression cassette containing the coding gene of the above-mentioned electron transport chain subunits;

[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 the coding gene of the above-described mutant.

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

[0020] (b1) Gene expression cassette containing the coding gene of the 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 application of the above-mentioned electron transport chain subunit, the coding gene of the electron transport chain subunit, or the biomaterial in improving the low temperature resistance of microbial strains.

[0024] The present invention also provides a method for improving the low-temperature resistance of microbial strains, comprising the step of genetically transforming the above-mentioned coding gene into the microbial strain to construct a recombinant microbial strain overexpressing the coding gene.

[0025] The present invention also provides the application of the above-mentioned coding gene or biological material in the preparation of 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 304th amino acid of the electron transport chain subunit to an aliphatic amino acid, the 333rd amino acid to an aliphatic amino acid, and / or the 375th amino acid to an amino acid with stronger basic properties; the aliphatic amino acid is preferably leucine; the amino acid with stronger basic properties is preferably arginine.

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

[0028] This invention isolates and clones a low-temperature active electron transport chain subunit and its encoding gene from species of the Venus flytrap family (Actinoscyphiidae), which can improve the low-temperature resistance of organisms and thus be applied to the improvement of low-temperature traits and industrial production.

[0029] Meanwhile, this invention alters the optimal activity temperature of enzymes by mutating key amino acid sites. This method is simple and rapid; it even allows for improvements in the growth and production efficiency of organisms under different temperature environments simply by editing key sites of the organism's own homologous genes without introducing foreign genes. It 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. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1The image shows the activity detection results of acCYTB protein under 30℃ treatment conditions; where wild type refers to the group transfected with empty plasmid pRS416; mutant genotype refers to the group transfected with recombinant plasmids carrying the acCYTB protein mutant encoding gene; and original genotype refers to the group transfected with recombinant plasmids carrying the acCYTB protein encoding gene.

[0032] Figure 2 The image shows the activity detection results of acCYTB protein under 4℃ treatment conditions; where wild type refers to the group transfected with empty plasmid pRS416; mutant genotype refers to the group transfected with recombinant plasmids carrying the acCYTB protein mutant encoding gene; and original genotype refers to the group transfected with recombinant plasmids carrying the acCYTB protein encoding gene. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0038] Example 1

[0039] This invention isolates and clones a cytochrome B gene with low-temperature activity from a species of the Actinoscyphiidae family, which is named acCYTB gene. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the acCYTB protein it encodes 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 to leucine (Leu) (L), amino acid Phe (F) at position 333 to leucine (Leu) (L), and amino acid His (H) at position 375 to arginine (Arg) (R). The optimal activity temperature of this mutant increased. 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] Selective medium (i.e., synthesis-deficient (SC) medium): prepared from 6.7 g / L amino acid-free yeast nitrogen (YNB), 20 g / L glucose, 0.1 g / L Lue, 0.1 g / L His, and 0.1 g / L Trp. Solid medium is prepared by adding 1.5% (w / v) agar.

[0051] pRS416 is a Saccharomyces cerevisiae expression vector, which can be routinely purchased from biotechnology companies (such as Shanghai Zeye Biotechnology Co., Ltd.).

[0052] 2. Construction of recombinant yeast strains

[0053] The coding genes of the acCYTB protein and its mutants 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. Then, using a lithium acetate (LiOAc)-mediated yeast transformation method, these two recombinant plasmids were transformed into *Saccharomyces cerevisiae*, with the empty plasmid pRS416 serving as a control. The specific steps are as follows:

[0054] A fresh single colony was incubated overnight at 30°C. The culture was then diluted to OD. 600 The concentration was 0.1, and the medium was placed in 5 mL of fresh YPD medium and cultured at 30°C until the OD value reached 0.1. 600 Approximately 0.6. Cells were collected, washed with 1 mL sterile ddH2O and 500 μL 0.1 M LiOAc, and then resuspended in 100 μL 0.1 M LiOAc. Next, 20 μL of cells were mixed with 80 μL transformation buffer (58.6 μL 50% PEG3350, 7.7 μL 1 M LiOAc, 9.0 μL DMSO, 4.7 μL ssDNA) and the recombinant plasmid. The mixture was gently aspirated and incubated at 30 °C for 35 min, followed by heat shock at 42 °C for 15 min. The supernatant was discarded by centrifugation, and the cells were resuspended in 200 μL sterile ddH2O and plated onto selective solid medium, incubated at 30 °C for 3 days.

[0055] 3. Yeast mitochondrial extraction

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

[0057] 4. Electron transport chain subunit activity assay

[0058] The mitochondria obtained in the previous step were sonicated (200W power, 5 seconds sonication, 10-second interval, repeated 15 times). The respiratory chain complex obtained from the mitochondrial lysis was quantified using the BCA protein assay kit (23225, Thermo Fisher Scientific). Subsequently, the activity of the electron transport chain subunit acCYTB protein was measured using the Mitochondrial Respiratory Chain Complex I Activity Assay Kit (BC0515, Solarbio), according to the manufacturer's instructions. Protein activity was measured at 30℃ and 4℃, respectively, according to the experimental groups. Finally, the enzyme activity of each mitochondrial electron transport chain complex was measured using a multi-mode microplate reader at the corresponding temperatures; three technical replicates were set for each group to ensure the reliability of the results. Finally, the complex activity values ​​were normalized, i.e., Δmeasured value = actual measured value / mean of the three technical replicates in the control group. A larger Δmeasured value indicates higher enzyme activity.

[0059] The results of the activity assay for the electron transport chain subunit acCYTB protein are shown in [the table below]. Figure 1 and Figure 2 The results showed that the activity signal of acCYTB protein under 4℃ treatment was significantly stronger than that of the mutant and the control. Under 30℃ treatment, 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 serve as a potential gene resource for cold-resistant breeding, for the genetic improvement of cold-resistant properties in organisms such as yeast. Furthermore, amino acids 304, 333, and 375 of the acCYTB protein are key temperature-determining sites for its activity. Mutating amino acid 304 to Leu(L), amino acid 333 to Leu(L), and amino acid 375 to Arg(R) can effectively increase the activity temperature of the acCYTB protein, which has broad applications in improving the tolerance of organisms to different temperatures.

[0060] The codon-optimized sequence of the gene encoding the 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 preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for increasing the optimal activity temperature of electron transport chain subunits, characterized in that, The amino acid sequence of the electron transport chain subunit is shown in SEQ ID NO.2; The method involves mutating the 304th amino acid of the electron transport chain subunit to leucine, the 333rd amino acid to leucine, and the 375th amino acid to arginine.

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

3.

3. The encoding gene of the mutant as described in claim 2.

4. A biomaterial for expressing the mutant of claim 2, characterized in that, Substances including any one of (b1)-(b3): (b1) A gene expression cassette containing the encoding gene of claim 3; (b2) A recombinant vector containing the gene expression cassette; (b3) A recombinant microbial strain containing the recombinant vector.

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