Erythriose reductase mutant and application thereof

Abstract

The invention discloses an erythritol reductase mutant and application thereof, and belongs to the technical field of variation or genetic engineering. In particular discloses an erythritol reductase mutant with at least one of the following mutations relative to an amino acid sequence shown as SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A, and application of the erythritol reductase mutant in increasing the yield of erythritol. The mutant capable of promoting synthesis of erythritol is obtained through three-dimensional modeling and key catalytic pocket searching, and the yield of the erythritol of the yarrowia lipolytica is remarkably increased by using the genetic engineering strain constructed by the mutant and can be increased by 77% to the maximum. The erythritol synthesis capability of the erythritol reductase mutant is greatly improved, the erythritol reductase mutant is more suitable for industrial application, the production cost can be remarkably reduced, and the production efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of mutation or genetic engineering, and specifically relates to an erythrose reductase mutant and an application thereof. Background Art

[0002] Erythritol (Erythritol) scientific name 1,2,3,4-butanetetrol, molecular formula C 4 H 10 O 4 , is a four-carbon polyol, a natural substance widely found in plants and organisms in nature. As a natural sweetener, erythritol has the characteristics of low calories, strong stability, and little effect on blood sugar. Its sweetness is about 60%-80% of sucrose, and its calories are 0.2 kcal / g, which is only 5% of sucrose calories. It is a sugar alcohol with low calories and energy. Erythritol has limited absorption in the human body, and most of it will be excreted from the body, which makes its glycemic index (GI value) approach zero. Therefore, it is considered to be safe for human health and will not cause dental caries. It is 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. Because erythritol has the above-mentioned many advantages, it has been widely used in food, medicine, chemical industry and other fields. In recent years, the output of the erythritol industry has continued to grow, mainly driven by the improvement of health awareness and the increase in demand for low-sugar foods. As consumers' demand for low-calorie, low-sugar alternatives continues to grow, especially in the context of increasingly prominent health problems such as diabetes and obesity, erythritol, as a natural, low-calorie, low-glycemic sweetener, has become one of the core raw materials for the upgrading of the food industry. The industrial production of erythritol mainly relies on microbial fermentation, and the key to producing erythritol based on fermentation is to obtain high-yield and excellent production bacteria. At present, the industrial strains for producing erythritol by fermentation mainly include Candida spp., Pichia spp. and Yarrowia spp.

[0003] Compared with the fermentation production of other sugar alcohols, the production of erythritol by microbial fermentation has low yield and conversion rate. In order to increase the synthesis of erythritol by bacteria, the characteristics of bacteria need to be modified. At present, 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 genetic engineering means to transform production bacteria at the molecular level and continuously develop high-yield strains, it can not only improve production efficiency, but also significantly reduce production costs, win competitive advantages for enterprises, and provide 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 increase the production of erythritol in microorganisms by genetic modification of genes. The technical problem to be solved is not limited to the described technical subject matter. Those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] In order to solve the above technical problems, the present invention first provides an erythrose reductase mutant, wherein the erythrose reductase mutant is at least selected from any one of the following: A1) a mutant, wherein the amino acid sequence of the mutant comprises a sequence obtained by mutation of an amino acid residue site in the sequence shown in SEQ ID NO.1, wherein the mutation of the amino acid residue site is selected from at least one of the following: The 42nd position K mutated to R; I at position 49 mutated to A or V; The 75th position F mutated to S; L mutated to Y at position 107; The Y at position 125 mutated to A or L; The 296th position I mutated to A; A2) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1).

[0006] The connection in A2) can be directly connected via a peptide bond or connected via a linker.

[0007] The label protein described in A2) includes but is not limited to: GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), Strep label protein, MBP (maltose binding protein) label protein, Flag label protein, SUMO (small molecule ubiquitin-like modifier) ​​label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein, or a combination of the above label proteins. Those skilled in the art know how to select a suitable label protein according to the desired purpose. The use of the label does not change the function of the target protein, and its purpose is to separate, purify, detect or trace, so the label protein suitable for this application is not limited to a specific type. 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 to remove the tag by cleavage with TEV protease).

[0008] The amino acid sequence shown in SEQ ID NO: 1 may be the amino acid sequence of wild-type erythrose reductase ER25.

[0009] 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. The name of the erythrose reductase mutant with 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, and the nucleotide sequence of its encoding gene can be shown as SEQ ID NO:3.

[0010] The name of the erythrose reductase mutant with 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 shown as SEQ ID NO:4.

[0011] The name of the erythrose reductase mutant with 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, and the nucleotide sequence of its encoding gene can be shown as SEQ ID NO:5.

[0012] The erythrose reductase mutant with F75S mutation may be named Phe75Ser, and 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 may be shown as SEQ ID NO:6.

[0013] The name of the erythrose reductase mutant with L107Y mutation can be Leu107Tyr, and its amino acid sequence is obtained by mutating Leu at position 107 of SEQ ID NO:1 to Tyr. The nucleotide sequence of its encoding gene can be shown as SEQ ID NO:7.

[0014] The erythrose reductase mutant with Y125A mutation may be named Tyr125Ala, and its amino acid sequence is obtained by mutating Tyr at position 125 of SEQ ID NO:1 to Ala. The nucleotide sequence of its encoding gene may be shown as SEQ ID NO:8.

[0015] The erythrose reductase mutant with Y125L mutation may be named Tyr125Leu, and 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 shown in SEQ ID NO:9.

[0016] The name of the erythrose reductase mutant with I296A mutation can be Ile296Ala, and its amino acid sequence is obtained by mutating Ile at position 296 of SEQ ID NO:1 to Ala. The nucleotide sequence of its encoding gene is shown in SEQ ID NO:10.

[0017] The present invention also provides a biomaterial, wherein the biomaterial comprises any one of the following: B1) a nucleic acid molecule encoding the erythrose reductase mutant; 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 microorganism containing the nucleic acid molecule described in B1); B5) A recombinant host cell containing the nucleic acid molecule described in B1).

[0018] Furthermore, the biological materials can all express the nucleic acid molecules described in B1).

[0019] In the above biological material, the nucleic acid molecule B1) includes a DNA molecule whose coding sequence comprises any one of SEQ ID NOs: 3-10.

[0020] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by modifying the codon preference based on the nucleotide sequence shown in any one of SEQ ID NOs: 3-10.

[0021] The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0022] The recombinant vector described herein can be constructed using an expression vector. The structure of the expression vector is well known to those skilled in the art. The expression vector usually contains elements required for the expression of the target gene, such as a promoter, a multiple cloning site, a terminator, a ribosome binding site, etc., and may also contain a selection marker gene (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.). The expression vector can be constructed using any method known in the art (such as recombinant technology, synthetic technology, etc.), or it can be purchased commercially. For example, in one or more embodiments of the present invention, the expression vector is pYLEX1.

[0023] The recombinant vector can 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 embodiment provided by the present invention is a pYLEX1 vector, the present invention is not limited to this specific vector. Those skilled in the art can use other suitable vectors (such as yeast expression vectors pYES2, pPICZaA, pUG6, etc.), as long as the vector can express the erythrose reductase mutant.

[0024] The microorganisms described herein may include Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis, and Escherichia coli.

[0025] In the above biological materials, the recombinant microorganism described in B4) may be a recombinant Yarrowia lipolytica.

[0026] Furthermore, the recombinant Yarrowia lipolytica may be a recombinant Yarrowia lipolytica obtained by mutating a protein in Yarrowia lipolytica whose amino acid sequence includes SEQ ID NO: 1; the mutation includes at least one of the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A.

[0027] Further, the mutation may be the following mutation relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L or I296A.

[0028] The present invention also provides the use of the erythritol reductase mutant or the biological material in preparing erythritol or products containing erythritol.

[0029] The products include but are not limited to food, cosmetics, medicines, feed and daily chemical products.

[0030] The present invention also provides a method for increasing the yield of erythritol in a target microorganism or preparing erythritol, the method comprising mutating a protein containing an amino acid sequence of SEQ ID NO: 1 in the target microorganism to obtain a recombinant microorganism having a higher yield of erythritol than the target microorganism, wherein the mutation comprises at least one of the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A.

[0031] Further, the mutation may be the following mutation relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L or I296A.

[0032] Furthermore, the method may further include: culturing the recombinant microorganism in a culture medium, and collecting the erythritol from the culture.

[0033] The culture 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 batch feeding culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0034] The mutation method is 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.

[0035] Furthermore, the method for preparing erythritol can be a fermentation method for preparing erythritol.

[0036] The present invention also provides a method for producing erythritol, the method comprising the following steps: Step 1, constructing a recombinant cell or recombinant microorganism capable of expressing any of the erythrose reductase mutants described herein; Step 2: culturing the recombinant cell or recombinant microorganism to obtain erythritol.

[0037] In the above application or method, the microorganism comprises Yarrowia lipolytica.

[0038] The present invention also provides a whole-cell catalyst, which comprises any one of the erythrose reductase mutants or the biological material described herein.

[0039] In this document, when referring to an erythrose reductase mutant, its amino acid sequence is determined by reference to the amino acid sequence of wild-type erythrose reductase ER25 (SEQ ID NO: 1), and when referring to a mutation, the mutation is described in the following manner: "single-letter abbreviation of amino acid before mutation, mutation position, single-letter abbreviation of 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 mutates from lysine to arginine. The first methionine (Met) of the amino acid sequence shown in SEQ ID NO: 1 is at position 1.

[0040] The present invention obtains the three-dimensional structural model of erythrose reductase (ER) ER25 by structural prediction, and obtains the structural model of the complex of ER25 and its substrates erythrose and DADP by molecular docking. Further, the structural pocket of erythrose binding to ER25 and the key amino acid residues (K42, I49, F75, L107, Y215, I296) that may affect the catalytic activity of ER25 are determined according to the complex model, and mutants that may improve the enzyme activity are proposed through rational analysis, and these amino acid residues are changed by site-directed mutagenesis to improve the catalytic performance of ER25. The wild type of ER25 and the designed mutants of ER25 are expressed in engineering bacteria, and the ability of the mutants to catalyze erythrose to produce erythritol in Yarrowia lipolytica is compared. The results show that the erythrose reductase mutants designed by the present invention can significantly increase the yield of erythritol in Yarrowia lipolytica. Among them, the K42R mutant has the best ability to catalyze the production of erythritol. After expressing the K42R mutant in Yarrowia lipolytica, the erythritol production is 1.45 times that of expressing the wild-type ER25. Compared with the starting strain of Yarrowia lipolytica YP060-3ΔKU70, the production is increased by 72%; compared with the starting strain of Yarrowia lipolytica Po1gΔKU70, the production is increased by 77%. It has better performance and can significantly increase the production of biosynthesized erythritol.

[0041] Bacteria are the core of the fermentation industry, and there has long been an urgent need for high-yield bacteria in the industry. At present, there are still bottlenecks in the industrial production of erythritol by Yarrowia lipolytica, such as weak anabolism, which need to be broken through. The present invention uses three-dimensional modeling to find key catalytic pockets, and performs site-directed mutagenesis on erythritol reductase ER25 to improve its catalytic efficiency and achieve efficient synthesis of erythritol. Finally, erythritol reductase mutants are obtained. The ability of genetically engineered strains constructed using these erythritol reductase mutants to synthesize erythritol is greatly improved, which is more suitable for industrial applications, can significantly reduce production costs, and improve production efficiency.

[0042] Definition of terms 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. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0043] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a target gene. A typical expression cassette contains a promoter, an MCS (multiple cloning site) and / or a terminator. An expression cassette may also include a target gene, a marker gene (such as TK gene, DHFR gene, CAT gene and NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected through a linker.

[0044] The term "vector" generally refers to a vector that can carry exogenous DNA or target gene into host cells 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, so that the genetic material elements it carries are 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 properties of the recipient cell. The vector includes, but is not limited to, plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e., Cosmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (such as SV40), herpes virus (such as herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including but not limited to promoter sequence, transcription start sequence, enhancer sequence, selection element and reporter gene. In addition, the vector may also contain a replication start site.

[0045] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from the genus Corynebacterium ( Corynebacterium sp. ) (such as Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium blunt-toothed, etc.), Brevibacterium ( Brevibacterium sp. ) (such as Brevibacterium lactis, Brevibacterium flavum, Brevibacterium ammoniae, etc.), Escherichia coli ( Escherichia sp. ) (such as Escherichia coli), Erwinia spp. ( Erwinia sp. ), Agrobacterium ( Agrobacterium sp. ) (such as Agrobacterium tumefaciens), Flavobacterium ( Flavobacteriumsp. )、Alcaligenes spp. Alcaligenes sp. ), Pseudomonas ( Pseudomonas sp. ) and Bacillus ( Bacillus sp. ) (such as Bacillus), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from the genus Saccharomyces ( Saccharomyces sp. ) (such as Saccharomyces cerevisiae, Candida, Methanol yeast, Pichia pastoris), Fusarium ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. ), Verticillium ( Verticillium sp. )、Penicillium( Penicillium sp. )、Aspergillus( Aspergillus sp. ) and Cephalosporium ( Cephalosporium sp. ) etc. The actinomycetes may be from the genus Streptomyces ( Streptomyces sp. ) (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus ( Fucus sp. ), the genus A. Achnanthes sp. ), Cocoon algae ( Amphiprora sp. ), Diplocoriaceae ( Amphora sp. ), Fibrocystis spp. Ankistrodesmus sp. ), Astrophytum ( Asteromonas sp. ) and Chromophytes ( Boekelovia sp. )wait.

[0046] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, among which: the plant cell can be Arabidopsis thaliana ( Arabidopsis thaliana ),tobacco( Nicotiana tabacum ),corn( Zea mays )、Rice( Oryza sativa ),wheat( Triticum aestivum) and other plant cells, but not limited thereto; the animal cells may be mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (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 (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frogs ( Xenopus laevis ) cells or giant salamander ( Andrias davidianus ) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc. but are not limited thereto.

[0047] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0048] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0049] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0050] The term "link" generally refers to the association of two or more molecules. The link can be covalent or non-covalent. The link described herein can be directly connected by a peptide bond, or connected by a linker (joint).

[0051] The term "mutation" generally refers to changes in amino acid sequences or nucleotide sequences, which may include changes in the base pair composition or arrangement order of the gene structure, such as point mutations caused by changes in a single base, or deletions, duplications and insertions of multiple bases, etc. It may also include replacement, deletion and insertion (addition) of one or more amino acid residues of a protein.

[0052] The term "site-directed mutagenesis" generally refers to the process of changing one or several bases in a gene through site-directed mutagenesis, including base addition, deletion, point mutation, etc., which results 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.

[0053] The term "gene editing" generally refers to the technology that can change a specific gene sequence in any cell, and can cause base deletion, duplication, insertion, frameshift mutation, replacement and knockout of target genes, replacement, deletion, shearing and single base change of genome sequences, etc., that is, the technology of "editing" the genome or the sequence of a specific gene at will. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology and CRISPR gene editing technology.

[0054] The term "homologous recombination" generally refers to a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. Exemplarily, the site-directed mutagenesis strategy based on homologous recombination can be achieved by the following steps: connecting the two ends of the nucleic acid molecule encoding the erythrose reductase mutant of the present invention (e.g., the DNA molecule shown in any one of SEQ ID NOs: 3-10) to the homologous arms of the wild-type erythrose reductase gene and then introducing it into the recipient bacteria to replace the wild-type erythrose reductase gene, thereby achieving the purpose of site-directed mutagenesis.

[0055] The term "culture" generally refers to a liquid or solid product (all materials in the culture container) that has a microbial community after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or contain a certain amount of culture medium, metabolites or other components produced during the culture process.

[0056] The term "fermentation" generally refers to a biological reaction process, including microbial fermentation, that produces and accumulates desired products through the growth, reproduction and metabolic activities of organisms.

[0057] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a schematic diagram of the molecular simulation of the binding of ER25 and NADP (yellow) and erythrose (blue) in Example 1. The key amino acid residues to be mutated are marked in green.

[0059] Figure 2The figure is a nucleic acid gel electrophoresis verification diagram of the erythrose reductase mutant plasmid in Example 2. M, marker; 1 to 9 are: pYLEX1-ER25, pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, pYLER296A.

[0060] Figure 3 The nucleic acid gel electrophoresis verification diagram of the positive transformant containing erythrose reductase before and after mutation of YP060-3△KU70 in Example 2. M, marker; 1 to 9 are: 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.

[0061] Figure 4 The nucleic acid gel electrophoresis verification diagram of the positive transformant containing erythrose reductase before and after mutation of Po1g△KU70 in Example 2. M, marker; 1-9 are: Po1g ER25, Po1g ER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, Po1g ER296A. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0063] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0064] The following examples were processed using Graphpad Pism9.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test method was used, and P < 0.05 (*) indicated statistical difference, P < 0.01 (**) indicated statistically significant difference, and P < 0.001 (***) indicated extremely significant statistical difference. The quantitative experiments in the following examples were set up for three biological replicates unless otherwise specified, and the results were averaged.

[0065]

[0066] Example 1: Structural simulation of the endogenous gene erythrose reductase of Yarrowia lipolytica and molecular simulation of the erythrose binding pocket Random mutagenesis screening of mutants lacks precise design. In this example, the three-dimensional structural model of erythrose reductase (ER) ER25 was obtained by structural prediction, and the structural model of ER25 and its substrate erythrose and DADP complex was obtained by molecular docking ( Figure 1 ). The details are as follows: 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. Erythrose small molecules and NADP were imported and converted into .pdbqt files respectively. The possible binding pockets were analyzed, and the binding pockets were determined based on this model. The amino acids near the pocket and the amino acid residues that may affect the catalytic performance of the pocket were selected for site-directed mutagenesis.

[0067] The results showed that key amino acid residues (K42, I49, F75, L107, Y215, I296) may affect the ability of ER protein to bind erythritol and the swing of erythritol during the binding process, thereby affecting its catalytic activity. According to the properties of their amino acids, a variety of mutants were designed and screened: Lys42Arg, Ile49Ala, Ile49Val, Phe75Ser, Leu107Tyr, Tyr125Ala, Tyr125Leu, Ile296Ala.

[0068] Example 2. Obtaining erythrose reductase strains and site-directed mutation strains This example constructs a strain containing the following erythrose reductase mutant coding genes: 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 encoding gene is shown in SEQ ID NO:3.

[0069] 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 encoding gene is shown in SEQ ID NO:4.

[0070] 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 encoding gene is shown in SEQ ID NO:5.

[0071] 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 shown in SEQ ID NO:6.

[0072] 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 shown in SEQ ID NO:7.

[0073] 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 shown in SEQ ID NO:8.

[0074] 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 shown in SEQ ID NO:9.

[0075] 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 shown in SEQ ID NO:10.

[0076] The specific construction steps are as follows: 1) According to the known erythrose reductase ER25 gene (SEQ ID NO: 2), it was introduced into the pYLEX1 plasmid to construct a recombinant plasmid pYLEX1-ER25 containing the erythrose reductase ER25 gene. The erythrose reductase ER25 gene was obtained by polymerase chain reaction (PCR) method, and the construction of the recombinant plasmid can be carried out by the common methods in the art.

[0077] 2) Design primers: Introduce mutation sites by reverse PCR to construct erythrose reductase mutant expression plasmid. Use pYLEX1-ER25 plasmid as template and Primer Premier 5.0 for primer design. The primer sequences are shown in Table 1:

[0078] 3) Inverse PCR: The reaction system and reaction procedure of inverse PCR are shown in Tables 2 and 3, respectively.

[0079]

[0080]

[0081] The PCR product was digested with 0.25 μL of DpnⅠ to remove methylation, and then transformed E.coli DH5α strain, culture for 12 to 14 hours to pick a single colony, extract the plasmid after overnight culture, and verify by DNA sequencing whether the 42nd lysine of erythrose reductase ER25 (SEQ ID NO: 1) is successfully replaced by arginine, the 49th isoleucine is replaced by alanine, the 49th isoleucine is replaced by valine, the 75th phenylalanine is replaced by serine, the 107th leucine is replaced by tyrosine, the 125th tyrosine is replaced by alanine, the 125th tyrosine is replaced by leucine, and the 296th isoleucine is replaced by alanine. The mutant plasmids pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, and pYLER296A are successfully obtained by PCR and sequencing verification (as shown in the verification figure). Figure 2 ).

[0082] Among them, the mutant plasmid pYLER42R contains a Lys42Arg mutant encoding gene (SEQ ID NO: 3), pYLER49A contains a Ile49Ala mutant encoding gene (SEQ ID NO: 4), pYLER49V contains a Ile49Val mutant encoding gene (SEQ ID NO: 5), pYLER75S contains a Phe75Ser mutant encoding gene (SEQ ID NO: 6), pYLER107Y contains a Leu107Tyr mutant encoding gene (SEQ ID NO: 7), pYLER125A contains a Tyr125Ala mutant encoding gene (SEQ ID NO: 8), pYLER125L contains a Tyr125Leu mutant encoding gene (SEQ ID NO: 9), and pYLER296A contains a Ile296Ala mutant encoding gene (SEQ ID NO: 10).

[0083] 4) The original plasmid pYLEX1-ER25 and the correctly sequenced mutant plasmids pYLER42R, pYLER49A, pYLER49V, pYLER75S, pYLER107Y, pYLER125A, pYLER125L, and pYLER296A were transformed into competent cells of YP060-3ΔKU70 and Po1gΔKU70 strains, and after visible colonies grew on the hygromycin-resistant plates, single colonies were randomly picked, and the genome was extracted after overnight culture for PCR verification. After verification, Figure 3 As shown, the YP060-3 ER25 strain and mutant strains YP060-3 ER42R, YP060-3 ER49A, YP060-3 ER49V, YP060-3 ER75S, YP060-3ER107Y, YP060-3 ER125A, YP060-3 ER125L, and YP060-3 ER296A were successfully obtained; Figure 4 As shown, the Po1gER25 strain and mutant strains Po1g ER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, and Po1g ER296A were successfully obtained.

[0084] Among them, mutant strains YP060-3 ER42R and Po1g ER42R contained mutant plasmid pYLER42R; mutant strains YP060-3 ER49A and Po1g ER49A contained mutant plasmid pYLER49A; mutant strains YP060-3 ER49V and Po1gER49V contained mutant plasmid pYLER49V; mutant strains YP060-3 ER75S and Po1g ER75S contained mutant plasmid pYLER75S; mutant strains YP060-3 ER107Y and Po1g ER107Y contained mutant plasmid pYLER107Y; mutant strains YP060-3 ER125A and Po1g ER125A contained mutant plasmid pYLER125A; mutant strains YP060-3 ER125L and Po1gER125L contain the mutant plasmid pYLER125L; mutant strains YP060-3 ER296A and Po1g ER296A contain the mutant plasmid pYLER296A.

[0085] The pYLEX1 plasmid was purchased from Yeastern Biotech Co., Ltd. The pYLEX1 plasmid carries an auxotrophic screening gene leucine expression cassette, a marker gene Amp, a promoter hp4d, and a terminator XPR2 term.

[0086] Yarrowia lipolytica ( Yarrowia lipolytica )YP060-3 is known and is described in the patent application with publication number CN115584327A.

[0087] The Yarrowia lipolytica Po1g strain was purchased from Yeastarn Biotech Co., Ltd.

[0088] Yarrowia lipolytica YP060-3ΔKU70 was constructed by knocking out the KU70 encoding gene responsible for non-homologous recombination from Yarrowia lipolytica YP060-3; Yarrowia lipolytica Po1gΔKU70 was constructed by knocking out the KU70 encoding gene responsible for non-homologous recombination from Yarrowia lipolytica Po1g; the KU70 gene knockout method is routinely performed and known to those skilled in the art, and reference can be made to the following literature: Yu AQ, Pratomo N, Ng TK, Ling H, Cho HS, Leong SS, ChangMW. Genetic engineering of an unconventional yeast for renewable biofuel andbiochemical production. Journal of Visualized Experiments, 2016, 115, e54371.

[0089] Example 3: Effect of erythrose reductase mutants on erythritol production 1) The strains constructed in Example 2: YP060-3 ER25 strain and mutant strains YP060-3 ER42R, YP060-3ER49A, YP060-3 ER49V, YP060-3 ER75S, YP060-3 ER107Y, YP060-3 ER125A, YP060-3ER125L, YP060-3 ER296A and original strain YP060-3ΔKU70; Po1g ER25 strain and mutant strains Po1gER42R, Po1g ER49A, Po1g ER49V, Po1g ER75S, Po1g ER107Y, Po1g ER125A, Po1g ER125L, Po1g ER296A and the original strain Po1gΔKU70 were activated twice and inoculated into the fermentation medium at an inoculum rate of 1%, and fermented in shake flasks at 30°C and 200 r / min for 6 days.

[0090] 2) The activation step of the genetically engineered bacteria is as follows: pick a single colony on the YPD solid plate, inoculate it into a test tube containing 5 mL of seed culture medium, and culture it at 30°C, 220 r / min, and shake it for 24 h (complete the first activation). Then, inoculate it into 25 mL of seed culture medium at a 1% inoculum amount, and continue to shake it at 30°C, 220 r / min for 16 h (complete the second activation).

[0091] The seed culture medium was YPD medium, the specific composition of which was: peptone 20 g / L, yeast extract powder 10 g / L, glucose 20 g / L, 115°C, sterilized for 20 min.

[0092] The fermentation medium composition was: glucose 300 g / L, yeast powder 8 g / L, KH 2 PO 4 0.35 g / L, NaCl 0.5 g / L, 115℃, sterilization for 20 min.

[0093] 3) After fermentation, pour the fermentation broth into a 50 mL centrifuge tube and centrifuge at 4000 r / min for 30 min. Take the supernatant and dilute the sample 25 times with ultrapure water. Centrifuge the sample dilution at 10000 rpm for 5 min, filter it with a 0.22 μm microporous filter membrane into a brown vial, and determine the erythritol content in the sample by HPLC.

[0094] Wherein, the above-mentioned HPLC detection conditions are: Chromatographic column HPX-87H (300×7.8mm, Bio-Rad), mobile phase 5 mmol / L dilute sulfuric acid, flow rate: 0.6 mL / min, column temperature: 35℃, RID: 40℃, injection volume 10 μL.

[0095] 4) After HPLC detection, the changes in the production of erythritol synthesized by the recombinant bacteria with erythrose reductases having different mutation sites are shown in Table 4.

[0096]

[0097] It was determined that the mutated erythrose reductase catalyzed the production of erythritol to varying degrees, indicating that the enzyme activity and substrate binding ability were improved to a certain extent after the mutation. Among them, YP060-3ΔKU70 was used as the starting strain, and the mutant YP060-3 ER42R strain had the greatest effect on increasing erythritol production, with a yield increase of 72% compared with the starting strain YP060-3ΔKU70 of Yarrowia lipolytica; compared with the unmutated strain YP060-3 ER25, the yield was increased by 46%. Taking Po1gΔKU70 as the starting strain, the mutant Po1g ER42R strain had the most outstanding effect on increasing erythritol production, with a yield increase of 77% compared with the starting strain Po1gΔKU70, and a yield increase of 47% compared with the unmutated strain Po1gΔKU70 ER25. It can be seen that after modification, the erythrose reductase mutants have an improved effect on the production of erythritol in Yarrowia lipolytica, among which the mutant Lys42Arg (K42R) has a significant effect.

[0098] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes 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, wherein the amino acid sequence of the mutant comprises a sequence obtained by mutation of an amino acid residue site in the sequence shown in SEQ ID NO.1, wherein the mutation of the amino acid residue site is selected from at least one of the following; The 42nd position K mutated to R; I at position 49 mutated to A or V; The 75th position F mutated to S; L mutated to Y at position 107; The Y at position 125 mutated to A or L; The 296th position I mutated to A; A2) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant described in A1).

2. Biomaterial, characterized in that The biological material includes any of the following: B1) a nucleic acid molecule encoding the erythrose reductase mutant according to 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 microorganism containing the nucleic acid molecule described in B1); B5) A recombinant host cell containing the nucleic acid molecule described in B1).

3. The biomaterial according to claim 2, characterized in that B1) The nucleic acid molecule comprises a DNA molecule whose coding sequence comprises any one of SEQ ID NOs: 3-10.

4. The biomaterial according to claim 2, characterized in that B4) The recombinant microorganism is a recombinant Yarrowia lipolytica.

5. Use of the erythrose reductase mutant according to claim 1 or the biomaterial according to any one of claims 2 to 4 in the preparation of erythritol or a product containing erythritol.

6. A method for increasing the yield of erythritol of a target microorganism or preparing erythritol, characterized in that: The method comprises mutating a protein having an amino acid sequence of SEQ ID NO: 1 in a target microorganism to obtain a recombinant microorganism having a higher yield of erythritol than the target microorganism, wherein the mutation comprises at least one of the following mutations relative to SEQ ID NO: 1: K42R, I49A, I49V, F75S, L107Y, Y125A, Y125L and I296A.

7. The method according to claim 6, characterized in that The microorganism includes Yarrowia lipolytica.

8. A method for producing erythritol, characterized in that: The method comprises the following steps: Step 1, constructing a recombinant cell or recombinant microorganism capable of expressing the erythrose reductase mutant according to claim 1; Step 2: culturing the recombinant cell or recombinant microorganism to obtain erythritol.

9. A whole-cell catalyst, characterized in that The whole cell catalyst comprises the erythrose reductase mutant according to claim 1 or the biological material according to any one of claims 2-4.

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