Erythrose reductase mutant and its application

Abstract

The present invention discloses an erythrose reductase mutant and its application, belonging to the field of mutation or genetic engineering technology. Specifically, it discloses an erythrose reductase mutant having at least one of the following mutations relative to the amino acid sequence shown in SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L, and I296A, and its application in increasing the yield of erythritol. Through three-dimensional modeling, the present invention finds the key catalytic pocket and obtains mutants that can promote the synthesis of erythritol. The genetically engineered strains constructed using these mutants significantly increase the yield of erythritol in Yarrowia lipolytica, and the maximum increase in yield can reach 77%. The ability of the erythrose reductase mutant of the present invention to synthesize erythritol is greatly improved, making it more suitable for industrial applications, significantly reducing production costs, and improving production efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of mutation or genetic engineering, and particularly relates to an erythritol reductase mutant and its application. Background Art

[0002] Erythritol, scientifically named 1,2,3,4-butanetetraol, has the molecular formula C4H 10 O4, is a four-carbon polyol and a natural substance widely present in plants and organisms in nature. As a natural sweetener, erythritol has characteristics such as low calories, strong stability, and little impact on blood sugar. Its sweetness is about 60%-80% of that of sucrose, and the calorie content is 0.2 kcal / g, only 5% of the calorie content of sucrose, making it a sugar alcohol with relatively low calories and energy. The absorption of erythritol in the human body is limited, and the vast majority will be excreted from the body, which makes its glycemic index (GI value) approach zero. Therefore, it is considered relatively safe for human health and does not cause dental caries, being a sugar alcohol with low pathogenicity. Compared with other sugar alcohols, the maximum no-effect dose of erythritol causing diarrhea is the highest among polyols. Due to the above-mentioned numerous advantages of erythritol, it has been widely used in fields such as food, medicine, and chemical industry. In recent years, the output of the erythritol industry has been continuously increasing, mainly driven by the improvement of health awareness and the increasing demand for low-sugar foods. With the continuous growth of consumers' demand for low-calorie and low-sugar alternatives, especially against the backdrop of the increasing prominence of health problems such as diabetes and obesity, erythritol, as a natural, low-calorie, and low-blood sugar response sweetener, has become one of the core raw materials for the upgrading of the food industry. The industrial production of erythritol mainly relies on the microbial fermentation method, and the key to producing erythritol based on the fermentation method is to obtain high-yield and excellent production strains. Currently, the industrial strains for producing erythritol by fermentation mainly include Candida spp., Pichia spp., and Yarrowia spp., etc.

[0003] When producing erythritol by microbial fermentation, compared with the fermentation production of other sugar alcohols, there is a phenomenon of low yield and conversion rate. In order to increase the synthesis amount of erythritol in the strain, it is necessary to modify the traits of the strain. Currently, the domestic and foreign market demand for erythritol is increasing year by year, which poses new challenges to the industrial production of erythritol. Therefore, by means of genetic engineering to modify the production strain at the molecular level and continuously develop high-yield strains can not only improve production efficiency but also significantly reduce production costs, winning a competitive advantage for enterprises and providing strong support for the sustainable development of the erythritol industry. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the yield of microbial erythritol through genetic modification of genes. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention first provides erythrose reductase mutants, and the erythrose reductase mutants are at least selected from any one of the following:

[0006] A1) Mutants, the amino acid sequence of which contains a sequence obtained by mutating the amino acid residue sites in the sequence shown in SEQ ID NO.1, and the amino acid residue site mutations are selected from at least one of the following:

[0007] The K at position 42 is mutated to R;

[0008] The I at position 49 is mutated to A or V;

[0009] The F at position 75 is mutated to S;

[0010] The L at position 107 is mutated to Y;

[0011] The Y at position 125 is mutated to A or L;

[0012] The I at position 296 is mutated to A;

[0013] A2) A fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1).

[0014] The connection in A2) can be directly connected through a peptide bond or connected through a linker.

[0015] The tag proteins described in A2) include but are not limited to: GST (glutathione S-transferase) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substance A (NusA) tag protein, His tag protein (His-tag), Strep tag protein, MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO (small ubiquitin-like modifier) tag protein, HA (hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, bacteriophage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein, or a combination of the above tag proteins. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of the tag does not change the function of the target protein, and its purpose is for separation, purification, detection or tracing. Therefore, the tag proteins applicable to this application are not limited to specific types. The tag can be separated from the target protein by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0016] The amino acid sequence shown in SEQ ID NO:1 can be the amino acid sequence of wild-type erythrose reductase ER25.

[0017] Furthermore, the amino acid sequence of the erythrose reductase mutant may have the following mutations relative to the amino acid sequence shown in SEQ ID NO:1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L or I296A. Among them:

[0018] The name of the erythrose reductase mutant with the K42R mutation can be Lys42Arg, and its amino acid sequence is obtained by mutating Lys at position 42 of SEQ ID NO:1 to Arg. The nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:3.

[0019] The name of the erythrose reductase mutant with the I49A mutation can be Ile49Ala, and its amino acid sequence is obtained by mutating Ile at position 49 of SEQ ID NO:1 to Ala. The nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:4.

[0020] The name of the erythrose reductase mutant with the I49V mutation can be Ile49Val, and its amino acid sequence is obtained by mutating Ile at position 49 of SEQ ID NO:1 to Val. The nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:5.

[0021] The name of the erythrose reductase mutant with the F75S mutation can be Phe75Ser. Its amino acid sequence is obtained by mutating Phe at position 75 of SEQ ID NO:1 to Ser, and the nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:6.

[0022] The name of the erythrose reductase mutant with the L107Y mutation can be Leu107Tyr. Its amino acid sequence is obtained by mutating Leu at position 107 of SEQ ID NO:1 to Tyr, and the nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:7.

[0023] The name of the erythrose reductase mutant with the Y125A mutation can be Tyr125Ala. Its amino acid sequence is obtained by mutating Tyr at position 125 of SEQ ID NO:1 to Ala, and the nucleotide sequence of its encoding gene can be as shown in SEQ ID NO:8.

[0024] The name of the erythrose reductase mutant with the Y125L mutation can be Tyr125Leu. Its amino acid sequence is obtained by mutating Tyr at position 125 of SEQ ID NO:1 to Leu, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:9.

[0025] The name of the erythrose reductase mutant with the I296A mutation can be Ile296Ala. Its amino acid sequence is obtained by mutating Ile at position 296 of SEQ ID NO:1 to Ala, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:10.

[0026] The present invention also provides biological materials, including any one of the following:

[0027] B1) A nucleic acid molecule encoding the erythrose reductase mutant;

[0028] B2) An expression cassette containing the nucleic acid molecule of B1);

[0029] B3) A recombinant vector containing the nucleic acid molecule of B1);

[0030] B4) A recombinant microorganism containing the nucleic acid molecule of B1);

[0031] B5) A recombinant host cell containing the nucleic acid molecule of B1).

[0032] Furthermore, the above biological materials can all express the nucleic acid molecule of B1).

[0033] Among the above biological materials, the nucleic acid molecule of B1) includes a DNA molecule whose coding sequence contains any one shown in SEQ ID NO:3 - 10.

[0034] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequences shown in any of SEQ ID NOs: 3-10.

[0035] The nucleic acid molecules described herein may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecules may also be RNA, such as mRNA or hnRNA, etc.

[0036] The recombinant vectors described herein can be constructed using expression vectors. The structure of expression vectors is well-known to those skilled in the art. Expression vectors usually contain elements required for the expression of the target gene, such as promoters, multiple cloning sites, terminators, ribosome binding sites, etc., and may also contain selection marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). Expression vectors can be constructed using any methods known in the art (such as recombinant technology, synthetic technology, etc.), or can be commercially purchased. For example, in one or more embodiments of the present invention, the expression vector is pYLEX1.

[0037] The recombinant vector may be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding the erythrose reductase mutant (Lys42Arg, Ile49Ala, Ile49Val, Phe75Ser, Leu107Tyr, Tyr125Ala, Tyr125Leu or Ile296Ala) into an expression vector (such as a prokaryotic expression vector or a eukaryotic expression vector). Although the expression vector used in the examples provided by the present invention is the pYLEX1 vector, the present invention is not limited to this specific vector. Those skilled in the art can adopt other suitable vectors (such as yeast expression vectors pYES2, pPICZaA, pUG6, etc.), as long as the vector can express the erythrose reductase mutant.

[0038] The microorganisms described herein may include Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis and Escherichia coli.

[0039] Among the above biological materials, the recombinant microorganism in B4) may be a recombinant Yarrowia lipolytica yeast.

[0040] Furthermore, the recombinant Yarrowia lipolytica yeast may be a recombinant Yarrowia lipolytica yeast obtained by mutating the protein with an amino acid sequence comprising SEQ ID NO: 1 in Yarrowia lipolytica; the mutations include at least one of the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A.

[0041] Further, the mutation may be the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L or I296A.

[0042] The present invention also provides the use of the erythrose reductase mutant or the biomaterial in the preparation of erythritol or a product containing erythritol.

[0043] The products include but are not limited to foods, cosmetics, pharmaceuticals, feeds and daily chemical products.

[0044] The present invention also provides a method for increasing the yield of erythritol of a target microorganism or preparing erythritol, the method comprising mutating a protein comprising the amino acid sequence of SEQ ID NO: 1 in the target microorganism to obtain a recombinant microorganism with a higher erythritol yield than the target microorganism, and the mutation comprising at least one of the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A.

[0045] Further, the mutation may be the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L or I296A.

[0046] Further, the method may further comprise: culturing the recombinant microorganism in a culture medium and collecting the erythritol from the culture.

[0047] The culturing can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to the actual situation.

[0048] The methods for mutation are well known to those skilled in the art, such as PCR-mediated site-directed mutagenesis, oligonucleotide primer-mediated site-directed mutagenesis, cassette mutagenesis, gene editing technology or homologous recombination technology.

[0049] Further, the method for preparing erythritol may be a fermentation method for preparing erythritol.

[0050] The present invention also provides a method for producing erythritol, the method comprising the following steps:

[0051] Step 1, constructing a recombinant cell or recombinant microorganism capable of expressing any of the erythrose reductase mutants described herein;

[0052] Step 2: Cultivate the recombinant cell or recombinant microorganism to obtain erythritol.

[0053] In the above application or method, the microorganism includes Yarrowia lipolytica.

[0054] The present invention also provides a whole-cell catalyst, which includes any erythrose reductase mutant or the biomaterial described herein.

[0055] In this article, when referring to an erythrose reductase mutant, its amino acid sequence is determined by referring to the amino acid sequence of the wild-type erythrose reductase ER25 (SEQ ID NO: 1). When referring to a mutation, the mutation is described in the following way: "single-letter abbreviation of the amino acid before mutation, mutation position, single-letter abbreviation of the amino acid after mutation". Exemplarily, "K42R" means that at the 42nd position of the amino acid sequence shown in SEQ ID NO: 1, the amino acid is mutated from lysine to arginine. The first methionine (Met) of the amino acid sequence shown in SEQ ID NO: 1 is the 1st position.

[0056] The present invention obtained a three-dimensional structure model of erythrose reductase (ER) ER25 through structure prediction, and obtained a structure model of the complex of ER25 with its substrates erythrose and DADP by molecular docking. Further, according to the complex model, the structural pocket where erythrose binds to ER25 and the key amino acid residues (K42, I49, F75, L107, Y215, I296) that may affect the catalytic activity of ER25 were determined. Mutants that may improve enzyme activity were proposed through rational analysis, and these amino acid residues were changed by site-directed mutagenesis to improve the catalytic performance of ER25. The wild-type ER25 and each mutant of designed ER25 were respectively expressed in engineering bacteria, and the ability of the mutants to catalyze erythrose to produce erythritol in Yarrowia lipolytica was compared. The results showed that all the erythrose reductase mutants designed by the present invention could significantly increase the yield of erythritol in Yarrowia lipolytica. Among them, the K42R mutant had the best ability to catalyze the production of erythritol. After expressing the K42R mutant in Yarrowia lipolytica, the yield of erythritol was 1.45 times that of expressing the wild-type ER25. Compared with the starting strain YP060-3ΔKU70 of Yarrowia lipolytica, the yield increase could reach 72%; compared with the starting strain Po1gΔKU70 of Yarrowia lipolytica, the yield increase could reach 77%, with better performance and could significantly improve the yield of biosynthetic erythritol.

[0057] Strain is the core of the fermentation industry, and there has long been an urgent need for high-yield strains in the industry. At present, there are still bottleneck problems that need to be broken through, such as weak anabolism, in the process of industrial production of erythritol by Yarrowia lipolytica. In the present invention, through three-dimensional modeling, the key catalytic pocket was searched, and site-directed mutagenesis was carried out on the erythrose reductase ER25 to improve its catalytic efficiency and achieve the efficient synthesis of erythritol. Finally, erythrose reductase mutants were obtained. The ability of the genetically engineered strains constructed using these erythrose reductase mutants to synthesize erythritol was greatly improved, making them more suitable for industrial applications, significantly reducing production costs, and improving production efficiency.

[0058] Term Definition

[0059] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0060] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a gene of interest. A typical expression cassette contains a promoter, MCS (multiple cloning site), and / or terminator. The expression cassette may also include the gene of interest, marker genes (such as TK gene, DHFR gene, CAT gene, and NEO gene), ribosome recognition and binding site (SD), transcription factor binding site (TFBS), enhancer, silencer, repressor, intron, poly(A) signal sequence, and / or mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0061] The term "vector" generally refers to a vector that can carry exogenous DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into the host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to be amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vectors include, but are not limited to: plasmids, phages (such as phage λ or M13 phage), cosmids (i.e., cosmid plasmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), P1 artificial chromosomes (PACs), or Ti plasmid artificial chromosomes (TACs)), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, polyomaviruses (such as SV40), herpesviruses (such as herpes simplex virus)). A vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector can also contain an origin of replication.

[0062] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria can be from the genus Corynebacterium ( Corynebacterium sp. ), such as Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium crenatum, etc.), the genus Brevibacterium ( Brevibacterium sp. ), such as Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniagenes, etc.), the genus Escherichia ( Escherichia sp. ), such as Escherichia coli), the genus Erwinia ( Erwinia sp. ), the genus Agrobacterium ( Agrobacterium sp. ), such as Agrobacterium tumefaciens), the genus Flavobacterium ( Flavobacterium sp. ), the genus Alcaligenes ( Alcaligenes sp. ), the genus Pseudomonas ( Pseudomonas sp. ), and the genus Bacillus ( Bacillus sp. ), such as Bacillus subtilis, etc.). The viruses can include rotaviruses, baculoviruses, retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, polyomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus), etc. The fungi can be from the genus Saccharomyces ( Saccharomyces sp. ), such as Saccharomyces cerevisiae, Candida spp., Pichia methanolica, Pichia pastoris), the genus Fusarium ( Fusarium sp. ), the genus Rhizoctonia (Rhizoctonia sp. ), Verticillium Verticillium sp. ), Penicillium Penicillium sp. ), Aspergillus Aspergillus sp. ), and Cephalosporium Cephalosporium sp. ), etc. The actinomycetes can be from the genus Streptomyces ( Streptomyces sp. )(such as Streptomyces). The algae can be from the phylum Cyanophyta (such as cyanobacteria), Fucus ( Fucus sp. ), Achnanthes ( Achnanthes sp. ), Amphiprora ( Amphiprora sp. ), Amphora ( Amphora sp. ), Ankistrodesmus ( Ankistrodesmus sp. ), Asterocystis ( Asteromonas sp. ), and Chrysosphaera ( Boekelovia sp. ), etc.

[0063] The term "host cell" is also referred to as recipient cell and generally refers to any type of cell that can be used to introduce a vector, such as plant cells and animal cells. The host cell can be understood to refer not only to a specific recipient cell but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or alterations, the progeny may not have to be exactly the same as the original parental cell but is still included within the scope of the host cell. Suitable host cells are known in the art, where: the plant cells can be Arabidopsis thaliana ( Arabidopsis thaliana ), Nicotiana tabacum ( Nicotiana tabacum ), Zea mays ( Zea mays ), Oryza sativa ( Oryza sativa ), Triticum aestivum ( Triticum aestivum ), etc. plant cells but not limited to these; the animal cells can be mammalian cells (such as Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell sub-line (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (such as chicken or duck cells), amphibian cells (such as Xenopus laevis ( Xenopus laevis ) cells or Andrias davidianus ( Andrias davidianus ) cells), fish cells (such as grass carp, common carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc. but not limited to these.

[0064] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating an exogenous target gene and a vector in vitro, which can be constructed in any suitable manner as long as the constructed recombinant vector can carry the exogenous target gene into a recipient cell and provide the ability for the exogenous target gene to replicate, integrate, amplify, and / or express in the recipient cell.

[0065] The term "recombinant microorganism" generally refers to a microorganism whose genes are manipulated and modified to obtain a recombinant microorganism with changed functions. For example, an exogenous target gene or a recombinant vector is introduced into the target microorganism, or the endogenous genes of the target microorganism are directly gene-edited.

[0066] The term "recombinant host cell" generally refers to a host cell whose genes are manipulated and modified to obtain a recombinant host cell with changed functions. For example, an exogenous target gene or a recombinant vector is introduced into the host cell, or the endogenous genes of the host cell are directly gene-edited.

[0067] The term "ligation" generally refers to the association of two or more molecules. Ligation can be covalent or non-covalent. The ligation described herein can be directly through a peptide bond or through a linker (adapter).

[0068] The term "mutation" generally can refer to a change in an amino acid sequence or a nucleotide sequence, which can include a change in the base pair composition or arrangement order in the structure of a gene, such as a point mutation caused by a single base change, or deletions, duplications, and insertions of multiple bases, etc., and can also include substitutions, deletions, and insertions (additions) of one or more amino acid residues of a protein.

[0069] The term "site-directed mutagenesis" generally can refer to changing one or several bases in a certain gene by site-directed mutagenesis methods, including base addition, deletion, point mutation, etc., resulting in a change in the amino acid composition of the corresponding protein. Site-directed mutagenesis methods include oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis, etc.

[0070] The term "gene editing" generally refers to a technology that can complete changes in specific gene sequences in any cell, and can cause base deletions, duplications, insertions, frameshift mutations, and replacement and knockout of target genes in a gene, realizing replacement, deletion, cleavage, and single-base changes in genomic sequences, that is, the technology of arbitrarily "editing" the sequence of the genome or a specific gene. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology, and CRISPR gene editing technology.

[0071] The term "homologous recombination" generally refers to a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Exemplarily, a site-directed mutagenesis strategy based on homologous recombination can be achieved through the following steps: The nucleic acid molecule encoding the erythrose reductase mutant of the present invention (for example, the DNA molecule shown in any of SEQ ID NO: 3-10) is ligated with homologous arms of the wild-type erythrose reductase gene at both ends and then introduced into the recipient bacterium to replace the wild-type erythrose reductase gene, thereby achieving the purpose of site-directed mutagenesis.

[0072] The term "culture" generally refers to the collective term for liquid or solid products (all substances in the culture container) with a microbial population after artificial inoculation and cultivation. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms or can contain a certain amount of culture medium, metabolites or other components generated during the cultivation process.

[0073] The term "fermentation" generally refers to a biological reaction process in which the desired product is produced and accumulated through the growth, reproduction and metabolic activities of organisms, including microbial fermentation.

[0074] The term "comprising" is not intended to be restrictive, is intended to be inclusive and means that there may be other elements in addition to the listed elements, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". In this article, the terms "comprising" and "including" can be used interchangeably. Brief Description of the Drawings

[0075] Figure 1 It is a schematic diagram showing the binding of molecularly simulated ER25 and NADP (yellow), erythrose (blue) in Example 1. The key amino acid residues to be mutated are marked in green.

[0076] Figure 2 It is a verification diagram of nucleic acid gel electrophoresis of the erythrose reductase mutant plasmid in Example 2. M, marker; 1 to 9 are respectively: pYLEX1-ER25, pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, pYLER296A.

[0077] Figure 3This is the nucleic acid gel electrophoresis verification diagram of the positive transformants containing erythrose reductase before and after mutation in YP060-3△KU70 in Example 2. M, marker; 1-9 are respectively: YP060-3 ER25, YP060-3 ER42R, YP060-3ER49A, YP060-3 ER49V, YP060-3 ER75S, YP060-3 ER107Y, YP060-3 ER125A, YP060-3ER125L, YP060-3 ER296A.

[0078] Figure 4 This is the nucleic acid gel electrophoresis verification diagram of the positive transformants containing erythrose reductase before and after mutation in Po1g△KU70 in Example 2. M, marker; 1-9 are respectively: Po1g ER25, Po1g ER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, Po1g ER296A. Specific implementation manners

[0079] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0080] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0081] The following examples use Graphpad Pism9.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The one-way analysis of variance test method is used. P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a significant statistical difference, and P < 0.001 (***) indicates an extremely significant statistical difference. In the following examples, quantitative experiments are set with three biological replicate experiments and the results are averaged unless otherwise specified.

[0082]

[0083] Example 1: Structure simulation of the endogenous gene erythrose reductase and molecular simulation of the erythrose binding pocket in Yarrowia lipolytica

[0084] Random mutation screening for mutants lacks precise design. In this example, a three-dimensional structural model of erythrose reductase (ER) ER25 was obtained through structure prediction, and a structural model of the complex of ER25 with its substrate erythrose and DADP was obtained by molecular docking ( Figure 1 ). The specific steps are as follows:

[0085] Using erythrose reductase ER25 (YALI0C13508p, GenBank accession number: CAG82106.1, SEQ ID NO:1) as a template, the predicted ER25 protein model was imported using Autodock Tools software and converted into a.pdbqt file. The erythrose small molecule and NADP were imported and converted into.pdbqt files respectively. The possible binding pockets were analyzed, and based on this model, the amino acids near the binding pocket and the amino acid residues that may affect the catalytic performance of the pocket were selected for site-directed mutagenesis.

[0086] The results showed that the key amino acid residues (K42, I49, F75, L107, Y215, I296) may affect the ability of the ER protein to bind erythritol and the swing of erythritol during the binding process, thereby affecting its catalytic activity. According to the properties of the amino acids, a variety of mutants were designed and screened: Lys42Arg, Ile49Ala, Ile49Val, Phe75Ser, Leu107Tyr, Tyr125Ala, Tyr125Leu, Ile296Ala.

[0087] Example 2: Obtaining of erythrose reductase strains and site-directed mutagenesis strains

[0088] In this example, strains containing the coding genes of the following erythrose reductase mutants were constructed:

[0089] Erythrose reductase mutant Lys42Arg: Its amino acid sequence is obtained by mutating Lys at position 42 of SEQ ID NO:1 to Arg, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO:3.

[0090] Erythrose reductase mutant Ile49Ala: Its amino acid sequence is obtained by mutating Ile at position 49 of SEQ ID NO:1 to Ala, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO:4.

[0091] Erythrose reductase mutant Ile49Val: Its amino acid sequence is obtained by mutating Ile at position 49 of SEQ ID NO:1 to Val, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO:5.

[0092] Erythrose reductase mutant Phe75Ser: Its amino acid sequence is obtained by mutating Phe at position 75 of SEQ ID NO:1 to Ser, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:6.

[0093] Erythrose reductase mutant Leu107Tyr: Its amino acid sequence is obtained by mutating Leu at position 107 of SEQ ID NO:1 to Tyr, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:7.

[0094] Erythrose reductase mutant Tyr125Ala: Its amino acid sequence is obtained by mutating Tyr at position 125 of SEQ ID NO:1 to Ala, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:8.

[0095] Erythrose reductase mutant Tyr125Leu: Its amino acid sequence is obtained by mutating Tyr at position 125 of SEQ ID NO:1 to Leu, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:9.

[0096] Erythrose reductase mutant Ile296Ala: Its amino acid sequence is obtained by mutating Ile at position 296 of SEQ ID NO:1 to Ala, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO:10.

[0097] The specific construction steps are as follows:

[0098] 1) According to the known erythrose reductase ER25 gene (SEQ ID NO:2), it is introduced into the pYLEX1 plasmid to construct a recombinant plasmid pYLEX1-ER25 containing the erythrose reductase ER25 gene. The erythrose reductase ER25 gene is obtained by the method of polymerase chain reaction (PCR), and the construction of the recombinant plasmid can be carried out by the common methods in the art.

[0099] 2) Design primers: Introduce mutation sites by the method of inverse PCR to construct an erythrose reductase mutant expression plasmid. Using the pYLEX1-ER25 plasmid as a template, primers are designed using Primer Premier 5.0, and the primer sequences are shown in Table 1:

[0100]

[0101] 3) Inverse PCR: The reaction system and reaction program of inverse PCR are shown in Table 2 and Table 3 respectively.

[0102]

[0103]

[0104] The PCR products were added with 0.25 μL of DpnⅠ to digest and remove methylation, and then transformed E.coli into DH5α strains. After culturing for 12 to 14 hours, single colonies were picked, and plasmids were extracted after overnight culture. DNA sequencing was used to verify whether lysine at position 42 of erythrose reductase ER25 (SEQ ID NO:1) was successfully replaced with arginine, isoleucine at position 49 was replaced with alanine, isoleucine at position 49 was replaced with valine, phenylalanine at position 75 was replaced with serine, leucine at position 107 was replaced with tyrosine, tyrosine at position 125 was replaced with alanine, tyrosine at position 125 was replaced with leucine, and isoleucine at position 296 was replaced with alanine. After verification by PCR and sequencing, mutant plasmids pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, and pYLER296A were successfully obtained (verification figures are as shown in Figure 2 ).

[0105] Among them, the mutant plasmid pYLER42R contains the Lys42Arg mutant coding gene (SEQ ID NO:3), pYLER49A contains the Ile49Ala mutant coding gene (SEQ ID NO:4), pYLER49V contains the Ile49Val mutant coding gene (SEQ ID NO:5), pYLER75S contains the Phe75Ser mutant coding gene (SEQ ID NO:6), pYLER107Y contains the Leu107Tyr mutant coding gene (SEQ ID NO:7), pYLER125A contains the Tyr125Ala mutant coding gene (SEQ ID NO:8), pYLER125L contains the Tyr125Leu mutant coding gene (SEQ ID NO:9), and pYLER296A contains the Ile296Ala mutant coding gene (SEQ ID NO:10).

[0106] 4) The original plasmid pYLEX1-ER25 and the correctly sequenced mutant plasmids pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, and pYLER296A were respectively transformed into the competent cells of YP060-3ΔKU70 and Po1gΔKU70 strains. After visible colonies grew on the hygromycin-resistant plates, single colonies were randomly picked, and genomic DNA was extracted after overnight culture for PCR verification. After verification, as shown in Figure 3As shown, the YP060-3 ER25 strain and mutant strains YP060-3 ER42R, YP060-3 ER49A, YP060-3 ER49V, YP060-3 ER75S, YP060-3 ER107Y, YP060-3 ER125A, YP060-3 ER125L, YP060-3 ER296A were successfully obtained; as Figure 4 shown, the Po1gER25 strain and mutant strains Po1g ER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, Po1g ER296A were successfully obtained.

[0107] Among them, the mutant strains YP060-3 ER42R and Po1g ER42R contain the mutant plasmid pYLER42R; the mutant strains YP060-3 ER49A and Po1g ER49A contain the mutant plasmid pYLER49A; the mutant strains YP060-3 ER49V and Po1g ER49V contain the mutant plasmid pYLER49V; the mutant strains YP060-3 ER75S and Po1g ER75S contain the mutant plasmid pYLER75S; the mutant strains YP060-3 ER107Y and Po1g ER107Y contain the mutant plasmid pYLER107Y; the mutant strains YP060-3 ER125A and Po1g ER125A contain the mutant plasmid pYLER125A; the mutant strains YP060-3 ER125L and Po1g ER125L contain the mutant plasmid pYLER125L; the mutant strains YP060-3 ER296A and Po1g ER296A contain the mutant plasmid pYLER296A.

[0108] The pYLEX1 plasmid was purchased from Yeastern Biotech Co., Ltd., and the pYLEX1 plasmid carries a nutritional defect screening gene leucine expression cassette, a marker gene Amp, a promoter hp4d, and a terminator XPR2 term.

[0109] Yarrowia lipolytica ( Yarrowia lipolytica ) YP060-3 is known and is described in the patent application with the publication number CN115584327A.

[0110] The Yarrowia lipolytica Po1g strain was purchased from Yeastern Biotech Co., Ltd.

[0111] Yarrowia lipolytica YP060-3ΔKU70 was constructed by knocking out the coding gene KU70 responsible for non-homologous recombination from Yarrowia lipolytica YP060-3; Yarrowia lipolytica Po1gΔKU70 was constructed by knocking out the coding gene KU70 responsible for non-homologous recombination from Yarrowia lipolytica Po1g; The method for knocking out the KU70 gene is a routine operation and is known to those skilled in the art. For reference, see the following literature: Yu AQ, Pratomo N, Ng TK, Ling H, Cho HS, Leong SS, Chang MW. Genetic engineering of an unconventional yeast for renewable biofuel and biochemical production. Journal of Visualized Experiments, 2016, 115, e54371.

[0112] Example 3. Effect of Erythrose Reductase Mutants on Erythritol Yield

[0113] 1) The strains constructed in Example 2: YP060-3 ER25 strain and mutant strains YP060-3 ER42R, YP060-3 ER49A, YP060-3 ER49V, YP060-3 ER75S, YP060-3 ER107Y, YP060-3 ER125A, YP060-3 ER125L, YP060-3 ER296A and the original strain YP060-3ΔKU70; Po1g ER25 strain and mutant strains Po1g ER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, Po1g ER296A and the original strain Po1gΔKU70 were each activated twice and then inoculated into the fermentation medium at an inoculation amount of 1%, and shake-flask fermented at 30 °C and 200 r / min for 6 days.

[0114] 2) The activation step of the genetically engineered bacteria is as follows: Pick a single colony on the YPD solid plate and inoculate it into a test tube containing 5 mL of seed medium. After shaking culture at 30 °C and 220 r / min for 24 h (completing the first activation), inoculate it into 25 mL of seed medium at an inoculation amount of 1% and continue shaking culture at 30 °C and 220 r / min for 16 h (completing the second activation).

[0115] The seed culture medium is YPD medium, and its specific composition is: peptone 20 g / L, yeast extract powder 10 g / L, glucose 20 g / L, sterilized at 115 °C for 20 min.

[0116] The fermentation medium composition is: glucose 300 g / L, yeast powder 8 g / L, KH2PO4 0.35 g / L, Nacl 0.5 g / L, sterilized at 115 °C for 20 min.

[0117] 3) After the fermentation is completed, pour the fermentation broth into a 50 mL centrifuge tube, centrifuge at 4000 r / min for 30 min, take the supernatant, dilute the sample 25 times with ultrapure water, centrifuge the sample dilution at 10000 rpm for 5 min, filter it through a 0.22 μm microporous filter membrane into a brown vial, and determine the erythritol content in the sample by HPLC.

[0118] Among them, the above HPLC detection conditions are as follows:

[0119] Chromatographic column: HPX-87H (300×7.8 mm, Bio-Rad), mobile phase: 5 mmol / L dilute sulfuric acid, flow rate: 0.6 mL / min, column temperature: 35 °C, RID: 40 °C, injection volume: 10 μL.

[0120] 4) After HPLC detection, the yield changes of erythritol catalyzed by recombinant bacteria with different mutant sites of erythrose reductase are shown in Table 4.

[0121]

[0122] After determination, the yield of erythritol catalyzed by the mutated erythrose reductase has increased to varying degrees, indicating that the enzyme activity and the binding ability with the substrate have been improved to a certain extent after mutation. Among them, using YP060-3ΔKU70 as the starting strain, after mutation, the YP060-3 ER42R strain has the greatest effect on improving the yield of erythritol. Compared with the starting strain YP060-3ΔKU70 of Yarrowia lipolytica, the yield has increased by 72%; compared with the strain YP060-3 ER25 before mutation, the yield has increased by 46%. And using Po1gΔKU70 as the starting strain, after mutation, the Po1g ER42R strain has the most prominent effect on increasing the production of erythritol. Compared with the starting strain Po1gΔKU70, the yield has increased by 77%, and compared with the strain Po1gΔKU70 ER25 before mutation, the yield has increased by 47%. It can be seen that after modification, the erythrose reductase mutant has an effect on improving the yield of erythritol in Yarrowia lipolytica, and among them, the mutant Lys42Arg (K42R) has a significant effect.

[0123] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. An erythrose reductase mutant, characterized in that, The erythrose reductase mutant is at least selected from any one of the following: A1) A mutant, the amino acid sequence of which is a sequence obtained by mutating the amino acid residue site in the sequence shown in SEQ ID NO.1, and the amino acid residue site mutation is that K at the 42nd position is mutated to R; A2) A fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1).

2. A biological material, characterized in that, The biological material includes any one of the following: B1) A nucleic acid molecule encoding the erythrose reductase mutant described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1); B4) A recombinant Yarrowia lipolytica yeast containing the nucleic acid molecule described in B1).

3. The biomaterial according to claim 2, wherein The nucleic acid molecule described in B1) is the DNA molecule shown in SEQ ID NO:

3.

4. Use of the erythrose reductase mutant described in claim 1 or the biological material described in claim 2 or 3 in the preparation of erythritol or a product containing erythritol.

5. A method for increasing the yield of erythritol by a target microorganism or for preparing erythritol, characterized in that, The method includes mutating the protein with the amino acid sequence of SEQ ID NO:1 in the target microorganism to obtain a recombinant microorganism with a higher erythritol yield than the target microorganism, and the mutation is the following mutation relative to SEQ ID NO:1: K42R; The microorganism is Yarrowia lipolytica yeast.

6. A method for producing erythritol, characterized in that, The method includes the following steps: Step 1, constructing a recombinant cell or recombinant Yarrowia lipolytica yeast capable of expressing the erythrose reductase mutant described in claim 1; Step 2, culturing the recombinant cell or recombinant Yarrowia lipolytica yeast to obtain erythritol.

7. Whole cell catalyst, characterized in that, The whole-cell catalyst includes the erythrose reductase mutant described in claim 1 or the biological material described in claim 2 or 3.

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