CN117625564A - Erythrose reductase mutant and application thereof - Google Patents

Abstract

The invention provides an erythrose reductase mutant, which is subjected to single-point mutation or combined mutation of the following sites compared with the erythrose reductase coded by YALI0F18590g gene derived from yarrowia lipolytica Yarrowia lipolytica: (1) lysine at position 26 is mutated to asparagine; (2) glycine at position 215 is mutated to asparagine; (3) phenylalanine at position 216 is mutated to tyrosine; (4) valine at position 295 is mutated to methionine. Compared with the wild type erythrose reductase from yarrowia lipolytica, the erythrose reductase mutant provided by the invention has the advantage that the enzyme activity is improved by 26% -44%. The erythritol yield of the high-yield erythritol genetic engineering strain obtained by inserting the erythrose reductase mutant gene into the genome of the yarrowia lipolytica strain Yarrowia lipolytica Po g at fixed points is obviously improved compared with that of a strain which singly overexpresses the erythrose reductase wild type, and the high-yield erythritol genetic engineering strain has great significance for industrial production of erythritol.

Description

An erythrose reductase mutant and its application

Technical field

The invention belongs to the field of genetic engineering, and specifically relates to an erythrose reductase mutant and its application.

Background technique

Erythritol (1,2,3,4-butanetetraol) is a four-carbon alcohol that is white, odorless, non-hygroscopic, non-optically active, has good thermal stability and is easily soluble in water. Its sweetness is that of sucrose. 60% to 70% of sugar, with a caloric value of 0.2kcal/g, only 5% of the caloric content of sucrose. It is a low-calorie sweetener. Because of its cool taste, non-caries, good crystallization and other advantages, it has been widely used in the food industry. Erythritol can be synthesized through chemical methods and microbial fermentation methods. However, chemical synthesis methods have shortcomings such as low production efficiency, high costs, and dangerous operations. The production process of microbial fermentation methods is gentle and easy to control, and it has become the main method for erythritol production today. way. Yarrowia lipolytica, as a recognized food safety microorganism, has become the main strain used to produce erythritol at home and abroad due to its strong resistance to stress, unique genetic structure, and wide range of available substrates.

In Yarrowia lipolytica, erythritol is produced as an osmoprotectant via the pentose phosphate pathway (PPP). The last step of the erythritol metabolic synthesis pathway is the reduction of D-erythrose to erythritol through the catalysis of erythrose reductase (ER). At the same time, erythrose reductase uses NADPH as a supplement during the catalytic process. factor. Erythrose reductase is the only enzyme in the last step of the erythritol biosynthetic pathway and is considered the key enzyme for the entire reaction.

As the rate-limiting enzyme for the production of erythritol, the expression and activity of erythritol reductase are crucial to the synthesis efficiency of erythritol. However, there is currently very little protein engineering work on this enzyme. Therefore, obtaining an erythrose reductase mutant with improved catalytic ability is of great significance to further increase the production of erythritol.

Contents of the invention

In order to solve the problem of low erythritol production in the prior art, the present invention provides an erythrose reductase mutant derived from Yarrowia lipolytica and its encoding gene, as well as in the production of erythritol by microbial fermentation. Applications. Compared with wild-type erythrose reductase derived from Yarrowia lipolytica, the enzyme activity of the erythrose reductase mutant provided by the invention is increased by 26% to 44%; and the erythrose reductase mutant provided by the invention contains an erythrose reductase mutation. The erythritol production of the high-yield erythritol genetically engineered strain encoding the somatic gene is significantly improved compared to the single-overexpressing wild-type strain of erythrose reductase.

The present invention provides an erythrose reductase mutant, which undergoes a single point mutation or a combination of mutations at the following sites compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica:

(1) Lysine at position 26 is mutated into asparagine;

(2) Glycine at position 215 is mutated into asparagine;

(3) Phenylalanine at position 216 is mutated into tyrosine;

(4) The valine at position 295 was mutated into methionine.

In this invention, the amino acid sequence of erythrose reductase derived from Yarrowia lipolytica was modeled using AlphaFold, combined with the substrate D-erythrose and NADPH models, and Autodock was used for molecular docking, and 16 influencing enzymes were selected. For the active key sites, FoldX was further used to conduct post-mutation stability analysis on these 16 sites. After molecular docking combined with thermal stability screening, mutation sites with better stability were selected to construct erythrose reductase mutants. The constructed single mutants of erythrose reductase were transformed into Escherichia coli BL21 (DE3) through vectors for expression and the crude enzyme solution was collected. The performance of each single mutant of erythrose reductase in catalyzing the production of D-erythrose was detected. The consumption of NADPH during the process of erythritol was used to measure the reaction enzyme activity. From this, 6 erythrose reductase single mutants with positive increase in enzyme activity were identified through rough screening. The 6 erythrose reductase single mutants obtained through screening were combined in pairs to construct 15 erythrose reductase double mutants, and their reaction enzyme activities were measured, and finally the mutants with significantly improved enzyme activity were determined. K26N, K26N/V295M, K26N/G215N, K26N/F216Y. The coding genes of these four erythrose reductase mutants were further co-constructed with the glucose dehydrogenase GDH coding gene into a dual expression vector and transformed into Escherichia coli BL21 (DE3) to construct a genetically engineered strain. The wet cells of the bacteria were used as catalysts to carry out biocatalytic reactions using D-erythrose as the substrate and glucose as the co-substrate to produce erythritol, in order to explore the effects of erythrose reductase mutants on erythrose reductase. Effects on enzyme activity expression. The results showed that compared with wild-type erythrose reductase, the enzyme activity of the above four erythrose reductase mutants increased by 1.26 to 1.44 times, among which the enzyme activity of the erythrose reductase mutant K26N/V295M increased by 44%. Relatively has the highest enzyme activity. In order to further obtain genetically engineered bacteria with high erythritol production, the present invention applied CRISPR-Cas9 gene editing technology, using Yarrowia lipolytica Po1g (ATCC20460) as the chassis strain, and transformed the erythrose reductase mutant K26N/V295M The complete gene expression cassette was inserted into the genome of the chassis strain, and the combined promoter hp4d was selected as the promoter of the expression cassette to achieve overexpression of the erythrose reductase mutant gene, further improving the biological properties of erythritol. Synthetic Yield.

Preferably, the erythrose reductase mutant has undergone one of the following combined mutations compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica:

(1) The lysine at position 26 is mutated into asparagine and the glycine at position 215 is mutated into asparagine;

(2) The lysine at position 26 is mutated into asparagine and the phenylalanine at position 216 is mutated into tyrosine;

(3) The lysine at position 26 was mutated into asparagine and the valine at position 295 was mutated into methionine.

As a further preference, the erythrose reductase mutant has undergone the following combined mutations compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica: lysine at position 26 The acid was mutated to asparagine and the valine at position 295 was mutated to methionine.

Specifically, the present invention mutates lysine at position 26 of erythrose reductase as shown in SEQ ID NO.1 to asparagine to obtain erythrose with an amino acid sequence as shown in SEQ ID NO.2 Reductase mutant; alternatively, mutate lysine at position 26 to asparagine and glycine at position 215 to asparagine of erythrose reductase whose amino acid sequence is as shown in SEQ ID NO.1 to obtain amino acids An erythrose reductase mutant with a sequence as shown in SEQ ID NO.3; or a mutation of lysine at position 26 of an erythrose reductase with an amino acid sequence as shown in SEQ ID NO.1 to asparagine, At the same time, phenylalanine at position 216 was mutated to tyrosine to obtain an erythrose reductase mutant with an amino acid sequence as shown in SEQ ID NO.4; or, an erythrose reductase mutant with an amino acid sequence as shown in SEQ ID NO.1 was obtained. Lysine at position 26 of erythrose reductase was mutated to asparagine, and valine at position 295 was mutated to methionine, thereby obtaining an erythrose reductase mutant with the amino acid sequence shown in SEQ ID NO.5.

Due to the particularity of the amino acid sequence, any fragment of the peptide protein or its variant containing the amino acid sequence shown in the present invention, such as its conservative variant, biologically active fragment or derivative, as long as the fragment of the peptide protein or the variant of the peptide protein is consistent with The above-mentioned amino acid sequence homology is more than 90%, and all fall within the protection scope of the present invention. Specifically, the changes include the deletion, insertion or substitution of amino acids in the amino acid sequence; wherein, for conservative changes in variants, the replaced amino acids have similar structures or chemical properties to the original amino acids, such as replacing different amino acids with leucine. Leucine, variants can also have non-conservative changes, such as the substitution of tryptophan for glycine.

The present invention also provides a gene encoding the above-mentioned erythrose reductase mutant. The nucleotide sequence of the encoding gene of the erythrose reductase mutant is preferably as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 are shown. Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in the present invention, as long as it has more than 90% homology with the aforementioned polynucleotide, falls within the protection scope of the present invention. The polynucleotide variant refers to a polynucleotide sequence that has one or more nucleotide changes. Variants of this polynucleotide may be biological substitution variants or non-genetic variants, including substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide, but does not substantially change the function of the peptide protein it encodes.

The present invention also provides a recombinant vector containing the gene encoding the erythrose reductase mutant. The recombinant vector comprises a polynucleotide operably linked to control sequences suitable for directing expression in a host cell. Various conventional vectors in this field, such as various plasmids, phages or viral vectors, etc., connected to the erythrose reductase mutant nucleotide sequence of the present invention, should all fall within the protection scope of the present invention. The recombinant vector preferably uses plasmid pET-28a(+) as an expression vector, and the coding gene of the erythrose reductase mutant is cloned into the plasmid pET-28a(+). In addition, when the gene encoding the erythrose reductase mutant is further co-constructed with the gene encoding glucose dehydrogenase GDH into a dual expression vector, pCDF-Duet(+) can generally be selected as the dual expression vector.

The invention also provides genetically engineered bacteria containing the gene encoding the erythrose reductase mutant. The exogenous erythrose reductase mutant encoding gene is introduced into the host cell through genetic engineering technology to construct the genetically engineered bacteria and expressed to obtain the erythrose reductase mutant of the present invention; wherein, the host The cells can be bacteria, fungi, plant cells or animal cells, and Escherichia coli BL21 (DE3) and Yarrowia lipolytica are preferred as expression hosts.

The invention also provides a method for constructing a genetically engineered strain with high yield of erythritol. The method includes: using Yarrowia lipolytica Polg strain as the chassis strain, and converting the erythrose reductase mutant into The gene expression frame was inserted into the genome of Yarrowia lipolytica Po1g and overexpressed to construct a genetically engineered strain with high yield of erythritol. Preferably, the combinatorial promoter hp4d is selected as the promoter of the gene expression box to enhance the expression of erythrose reductase.

The invention also provides the erythrose reductase mutant and its encoding gene, recombinant vector, genetically engineered bacteria, or the high-yield erythritol genetically engineered bacteria constructed by the above method in the production of erythritol through microbial fermentation. Applications.

Preferably, the application includes: co-constructing the erythrose reductase mutant encoding gene and the glucose dehydrogenase GDH encoding gene into the dual expression vector pCDF-Duet (+) and transforming it into Escherichia coli BL21 (DE3) for construction Genetically engineered bacteria are obtained, and the wet cells of the genetically engineered bacteria or the crude enzyme liquid obtained after crushing the wet cells are used as catalysts to generate erythrose with D-erythrose as the substrate and glucose as the auxiliary substrate. Biocatalytic reactions of alcohols.

Preferably, the application includes: inoculating the high-yield erythritol genetically engineered bacteria into a fermentation medium, using glycerol as a carbon source, and performing shake flask fermentation to produce erythritol.

Beneficial effects of the present invention: The present invention selects sites through molecular docking combined with thermal stability screening and performs site-directed mutations to change protein amino acid residues, mutates amino acids near the enzyme active catalytic center, and obtains erythrose reductase with improved activity. Mutant; compared with wild-type erythrose reductase derived from Yarrowia lipolytica, the enzyme activity of the mutant increased by 26% to 44%. After the erythrose reductase mutant gene was inserted into the genome of the chassis strain Yarrowia lipolytica Po1g, the erythritol production of the high-producing erythritol genetically engineered strain was higher than that of the wild type overexpressing erythrose reductase alone. The strain has been significantly improved, which is of great significance to the industrial production of erythritol.

Description of drawings

Figure 1 is a nucleic acid gel electrophoresis diagram of the constructed dual expression vector of erythrose reductase and glucose dehydrogenase; lanes: 1: 250kb marker; 2: amplification product of ER fragment; 3: amplification product of ER K26N fragment ; 4: Amplification product of ER K26N/G215N fragment; 5: Amplification product of ER K26N/F216Y fragment; 6: Amplification product of ERK26N/V295M fragment; 7: Amplification product of pCDF-Dute-GDH expression framework.

Figure 2 is a gel electrophoresis diagram of the mutant strain erythrose reductase and glucose dehydrogenase dual-expression protein gel electrophoresis; lanes: 1: marker; 2: original strain E.coli BL21-pCDF-Dute-ER-GDH; 3: E. coli BL21-pCDF-Dute-ERK26N-GDH; 4: E.coli BL21-pCDF-Dute-ER K26N/G215N-GDH; 5: E.coli BL21-pCDF-Dute-ERK26N/F216Y-GDH; 6: E. coli BL21-pCDF-Dute-ER K26N/V295M-GDH.

Figure 3 shows the time course of the substrate reaction catalyzed by erythrose reductase.

Figure 4 shows the relative enzyme activities of erythrose reductase wild type and mutants K26N, K26N/V295M, K26N/G215N, and K26N/F216Y.

Figure 5 shows the erythritol production and OD ₆₀₀ of the wild-type encoding gene of erythrose reductase and the mutant K26N/V295M encoding gene after overexpression in Yarrowia lipolytica Po1g chassis strain after shake flask fermentation for 144 hours.

Detailed ways

The following describes the implementation of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, as long as there is no conflict, the following embodiments and the features in the embodiments can be combined with each other. Unless otherwise specified, the methods used in the examples of the present invention are conventional methods, and all reagents used can be obtained from commercial sources.

[How to prepare culture media and solutions]

LB liquid culture medium: peptone 10g/L, yeast powder 5g/L, NaCl 5g/L, sterilized at 121°C for 20 minutes.

LB solid medium: Add 15g/L agar powder to LB liquid medium, sterilize at 121°C for 20 minutes.

50mM PBS buffer: NaCl 4g/L, KCl 0.1g/L, Na ₂ HPO ₄ 0.72 g/L, KH ₂ PO ₄ 0.12 g/L, and concentrated HCl to adjust pH to 6.0.

YPD liquid medium: glucose 20g/L, yeast powder 10g/L, peptone 20g/L, 115°C, sterilized for 20 minutes.

YPD solid medium: Add 20g/L agar powder to YPD liquid medium, sterilize at 115°C for 20 minutes.

SD solid medium: glucose 20g/L, YNB 6.7g/L, agar powder 20g/L, 115°C, sterilized for 20 minutes.

ESM fermentation medium: glycerol 100g/L, (NH ₄ ) ₂ SO ₄ 2.3 g/L, KH ₂ PO ₄ 0.22 g/L, MgSO ₄ ·7H ₂ O1g/L, yeast powder 1g/L, NaCl 25g/L , adjust the pH to 3.0 with concentrated H ₂ SO ₄ , sterilize at 121°C for 20 minutes, and add sterilized CaCO ₃ 3 g/L before inoculation.

Example 1: Screening of erythrose reductase mutants

(1) Obtaining erythrose reductase mutants

The amino acid sequence of erythrose reductase derived from Yarrowia lipolytica was modeled using AlphaFold. The two-substrate D-erythrose and NADPH model was downloaded from the PubChem website, and Autodock was used for molecular docking. 16 were selected. For the key sites that affect enzyme activity, Fold Using the constructed vector pET-28a-ER as a template, reasonably designed primers were used to introduce mutation sites through PCR to construct expression vectors for each erythrose reductase mutant; the primers are shown in Table 1.

Table 1: Primer sequences

(2) Obtaining wet bacteria

Each erythrose reductase mutant expression vector was transformed into the competent host Escherichia coli BL21 (DE3); the successfully verified transformant strain was streaked on a kanamycin solid LB plate with a final concentration of 100 μg/ml, 37 Cultivation for 12 hours at ℃, pick a single colony and inoculate it into LB liquid medium with kanamycin at a final concentration of 100 μg/ml, and culture it on a shaking table at 37 ℃ and 220 rpm for about 7 hours until the OD ₆₀₀ is 0.8 to 1.0 to obtain the seed liquid. The seed solution was inoculated into LB liquid medium with a final concentration of kanamycin of 100 μg/ml at an inoculation volume of 1%. After culturing for 2 hours at 37°C, IPTG was added at a final concentration of 0.1mM and induced at 28°C and 220 rpm for 12 hours. The obtained bacterial liquid was centrifuged in an ultra-low temperature high-speed centrifuge at 8000g and 4°C for 10 minutes, and the wet bacterial cells obtained were stored in a -20°C refrigerator.

(3) Preparation of crude enzyme solution

Weigh 2g of resting cells and resuspend them in 10mL of 50mM PBS (pH=6.0) buffer. Use ultrasonic disruption to release intracellular proteins. The disruption solution should always be placed in an ice bath. The disruption procedure is: power 250W, disruption time 1s, interval time 2s, total crushing time 10min. After the crushing is completed, place the mixed solution at 10,000 g and centrifuge at 4°C for 10 min to remove solid matter such as cell debris and collect the supernatant as crude enzyme solution. Dilute it 40 times with 50mM PBS (pH=6.0) buffer, and the diluted solution can be used for subsequent enzyme activity measurement reactions.

(4) Coarse screening of mutants

Erythrose reductase needs to consume NADPH as a cofactor in the process of catalyzing D-erythrose to produce erythritol. The coarse screen reflects the enzyme activity of erythrose reductase by detecting NADPH consumption. The crude enzyme solution obtained was used as the catalytic solution, and the change in absorbance at 340 nm was detected by a microplate reader to calculate the consumption of NADPH and then react the enzyme activity to achieve a rough screening of erythrose reductase mutants.

The crude screening enzyme activity reaction system is: 150 μL 50mM PBS (pH=6.0), 10μL D-erythrose (400mM), 20μL NADPH (2mM), 20μL crude enzyme solution.

Crude screening enzyme activity assay method: The reaction is carried out in a 96-well cell culture plate. The total volume of a single reaction is 200 μL. First add 150 μL 50mM PBS (pH=6.0), then add 10 μL 400 mM D-erythrose, and then add 20 μL 2mMNADPH. , and finally use eight consecutive rows to add 20 μL of crude enzyme solution of erythrose reductase wild type and mutant diluted to appropriate multiples. The reaction is carried out at 30°C, and the absorbance at 340nm is detected by a microplate reader 1 minute before the reaction. Change the value to calculate NADPH consumption. The amount of enzyme required to consume 1mM NADPH 1 minute before the reaction is started is one enzyme activity unit (U), and the relative enzyme activity = (mutant enzyme activity unit/wild-type enzyme activity unit)*100%. In this way, 6 single mutants of erythrose reductase with positive increase in enzyme activity were determined. These 6 mutation points were combined in pairs to construct 15 double mutants. The final mutants K26N, K26N/G215N, and K26N were obtained. The enzyme activities of /F216Y and K26N/V295M were increased by 32.2%, 35.6%, 27.7%, and 48.0% respectively compared with the wild type. Further catalytic reactions were carried out on these mutants.

Example 2: Effect of erythrose reductase mutants on the expression of erythrose reductase enzyme activity

Use pET-28a-ER as the template, use primers K26N-F and K26N-R (as shown in Table 1) to perform PCR, mutate the glycine at position 26 to asparagine, and transfer the band size to Escherichia coli BL21 ( DE3) competent state, the site-directed mutant strain was obtained and named Escherichia coli BL21(DE3)pET-28a-ERK26N. Design primers G215N-F/G215N-R, F216Y-F/F216Y-R, V295M-F/V295M-R (as shown in Table 1), and use Escherichia coli BL21(DE3)pET-28a-ER K26N as the template for PCR, respectively. Construction of double-point mutation erythrose reductase mutant strain Escherichia coli BL21(DE3)pET-28a-ERK26N/G215N, Escherichia coli BL21(DE3)

pET-28a-ER K26N/F216Y, Escherichia coli BL21(DE3)pET-28a-ER K26N/V295M.

The erythrose reductase wild-type strain pET28a-ER and the constructed erythrose reductase mutant strains with improved enzyme activity (pET-28a-ER K26N, pET-28a-ER K26N/G215N, pET-28a- ER K26N/F216Y, pET-28a-ER K26N/V295M) as template, use the primers shown in Table 1 to amplify erythrose reductase wild-type ER and mutants (ER K26N, ER K26N/G215N, ER K26N) respectively. /F216Y, ER K26N/V295M) gene fragment. Using the strain containing pCDF-Dute-GDH existing in the laboratory as a template, the pCDF-Dute-GDH-F/pCDF-Dute-GDH-R primer pair was used to amplify the glucose dehydrogenase GDH expression framework. The amplified fragment was verified by nucleic acid gel electrophoresis to verify the band length. The amplified fragment was normal, and the nucleic acid gel diagram is shown in Figure 1. The two fragments containing the coding gene for erythrose reductase wild type or mutant and the coding gene for glucose dehydrogenase were cloned in one step to obtain the dual expression vector pCDF-Dute-ER-GDH, and transformed into the host Escherichia coli BL21 ( DE3). Escherichia coli BL21(DE3)/pCDF-Dute-ER-GDH wet cells containing erythrose reductase mutant and glucose dehydrogenase co-expression genes were prepared in the same manner as in Example 1(2), and Resuspend in 50mM PBS (pH=6.0) buffer, and verify the protein expression through SDS-PAGE gel electrophoresis. The recombinant bacteria correctly express the protein. The expression is shown in Figure 2.

In order to explore the effect of erythrose reductase mutants on the expression of erythrose reductase enzyme activity, the wet bacterial cells collected above were used as catalysts to perform biocatalysis to generate erythritol using D-erythrose as the substrate. Reaction to detect the catalytic activity of erythrose reductase wild type or its mutants.

The catalytic reaction system contains D-erythrose with a final concentration of 50mM, 100mM glucose, and 20g/L wet cells; the reaction is carried out at 30°C and 800rpm.

Determination method of erythrose reductase enzyme activity in catalytic reaction: The reaction is carried out in a 1.5mL EP tube. The total system of a single reaction is 500μL. First add 362μL 50mM PBS (pH=6.0), then add 25μL 2M glucose, and then add 50μL Dilute the erythrose reductase wild type/mutant and glucose dehydrogenase to wet bacterial cells co-expressed to appropriate multiples. Pre-react the above reaction mixture for 2 minutes to mobilize the coenzyme circulation system. Finally, add 63 μL of 400 mM D-erythrose substrate as the substrate. Moss sugar. Start timing from the addition of substrate, and thermally terminate the reaction after 30 minutes of reaction, that is, place the 1.5 mL EP tube containing the reaction mixture in a boiling water bath for 10 minutes. The processed reaction solution was centrifuged, and the supernatant was aspirated and diluted 2 times for high-performance liquid phase detection. Prepare erythritol solutions of different concentrations, and use differential liquid phase to detect the peak area. Calculate the yield according to the standard curve. Relative enzyme activity = (mutant erythritol yield/starting strain erythritol yield)*100%. The results are shown in Figure 4. Among them, the enzyme activity of mutant K26N increased by 25.9%, the enzyme activity of K26N/G215N increased by 25.7%, the enzyme activity of K26N/F216Y increased by 38.5%, and the enzyme activity of K26N/V295M increased by 43.9%.

Among them, in order to explore the appropriate reaction time, the catalytic reaction was first carried out in wet cells of recombinant bacteria co-expressing wild-type erythrose reductase and glucose dehydrogenase. During the 1st, 3rd and 3rd minutes of the reaction, Samples were taken at 5 min, 10 min, 30 min, 60 min, and 90 min, and the contents of D-erythrose and erythritol were detected by high-performance liquid chromatography. The reaction process results are shown in Figure 3. The reaction reaches equilibrium after 90 minutes, which is the difference in enzyme activity between the reaction mutant and the wild type. The reaction time at 30% before the reaction reaches equilibrium is used as the reaction time for comparison between the erythrose reductase mutant and the wild type to determine the subsequent The total reaction time is 30min.

Example 3: Construction and application of genetically engineered bacteria with high yield of erythritol

CRISPR-Cas9 gene editing technology was used to achieve site-specific insertion of genes encoding erythrose reductase wild-type and mutant ER K26N/V295M. First, the gene fragments of erythrose reductase wild-type ER and mutant ER K26N/V295M were integrated into the hp4d combined promoter and XPR2 TER terminator on the pINA1312 vector to obtain the complete expression cassettes of the ER and ERK26N/V295M genes. Then the vectors pCRISPRyl-URA-MHY(ER) and pCRISPRyl-URA-MHY(ERK26N/V295M) were constructed respectively, and the complete erythrose reductase wild-type or mutant gene expression cassette was placed on the upstream homology arm of the MHY site. and between downstream homology arms. Extract the pCRISPRyl-URA-MHY(ER) and pCRISPRyl-URA-MHY(ERK26N/V295M) strain plasmids and transfer them into Yarrowia lipolytica Po1g competent cells. After verification, the site-directed insertion of the erythrose reductase expression framework is obtained. Successful strains Yarrowia lipolytica Po1g::ER, Yarrowialipolytica Po1g::ER K26N/V295M.

Among them, the method for preparing the competent base strain of Yarrowia lipolytica Po1g includes: taking 100 μL of Yarrowia lipolytica Po1g bacterial liquid from the preserved glycerol tube, spreading it on a YPD solid plate, and cultivating it statically in a 30°C incubator for about 24 hours. . Use a sterile pipette tip to scrape an appropriate amount of cultured Yarrowia lipolytica cells, resuspend them in a pre-cooled sterile EP tube containing 1ml TE Buffer (1M, pH=7), centrifuge at 5000rpm for 5 minutes and then pour it out. supernatant, resuspend the cells in 600 μL of lithium acetate (0.1M, pH=6.0) solution, incubate in a 30°C constant-temperature incubator for 1 hour, and turn over and resuspend every 10 minutes. Then centrifuge at 5000 rpm for 5 min, discard the supernatant, and add 100 μL of 0.1 M lithium acetate to resuspend the cells to obtain competent cells. Dispense the competent cells into 1.5 mL sterile EP tubes, 40 μL per tube, for subsequent transformation experiments.

The method of transforming the plasmid into the competent form of the above-mentioned Yarrowia lipolytica Po1g chassis strain includes: taking 40 μL of competent cells, adding 2 μL of 10 mg/ml salmon sperm DNA and 10 μL of plasmid in sequence, pipetting to mix, and then culturing statically in a 30°C constant-temperature incubator. 15 minutes. Add 350 μL PEG4000-LiAc (0.1M, pH=6.0) and 16.7 μL 1M DTT in sequence, and incubate in a 30°C constant-temperature incubator for 1 hour. Add 46.7 μL DMSO and heat shock in a water bath at 39°C for 10 min. Add 600 μL of 0.1M lithium acetate and mix slowly by pipetting, let stand in a 30°C constant temperature incubator for 30 minutes, and then spread on SD solid medium.

The above-mentioned successfully transformed genetically engineered strains Yarrowia lipolytica Po1g::ER, Yarrowialipolytica Po1g::ER K26N/V295M and chassis strain Yarrowia lipolytica Po1g were streaked on YPD solid plates and cultured statically at 30°C for 24 hours, and single colonies were picked and inoculated YPD liquid medium was placed in a shaker with a rotation speed of 200 rpm and cultured at 30°C for 24 hours to obtain a seed liquid; the seed liquid was inoculated into the ESM fermentation medium with an inoculation volume of 2% and cultured at 220 rpm for 144 hours. Centrifuge the post-fermentation culture fluid, take the supernatant and dilute it to an appropriate multiple, and use a Bio-RadAminex HPX-87H chromatography column to perform Agilent differential liquid phase detection of erythritol production. The results are shown in Figure 5. The erythritol production of the high-yield genetically engineered strain Yarrowia lipolytica Po1g::ER K26N/V295M reached 47g/L, which was lower than that of the bottom strain Yarrowia lipolytica Po1g and the overexpressed wild-type erythritol. The genetically engineered strain Yarrowia lipolytica Po1g::ER with enzyme encoding genes increased the production by 14.59% and 8.4% respectively.

The above-described embodiments are only descriptions of preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art may make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope of the present invention.

Claims (10)

1. An erythrose reductase mutant, characterized in that, compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica, it has undergone a single point mutation or combination of the following sites mutation: (1) Lysine at position 26 is mutated into asparagine; (2) Glycine at position 215 is mutated into asparagine; (3) Phenylalanine at position 216 is mutated into tyrosine; (4) The valine at position 295 was mutated into methionine. 2. The erythrose reductase mutant according to claim 1, characterized in that, compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica, it has undergone one of the following Combined mutations: (1) The lysine at position 26 is mutated into asparagine and the glycine at position 215 is mutated into asparagine; (2) The lysine at position 26 is mutated into asparagine and the phenylalanine at position 216 is mutated into tyrosine; (3) The lysine at position 26 was mutated into asparagine and the valine at position 295 was mutated into methionine. 3. The erythrose reductase mutant according to claim 1, characterized in that, compared with the erythrose reductase encoded by the YALIOF18590g gene derived from Yarrowia lipolytica, it has undergone the following combined mutations : The lysine at position 26 was mutated to asparagine and the valine at position 295 was mutated to methionine. 4. A gene encoding the erythrose reductase mutant according to any one of claims 1 to 3. 5. A recombinant vector containing the gene encoding the erythrose reductase mutant according to claim 4. 6. Genetically engineered bacteria containing the gene encoding the erythrose reductase mutant according to claim 4. 7. A method for constructing a high-yield erythritol genetically engineered bacterium, characterized in that the method includes: using the Yarrowia lipolytica Polg strain as the chassis strain, adding any one of claims 1 to 3 The gene expression frame of the erythrose reductase mutant was inserted into its genome at a specific point and overexpressed to construct a genetically engineered strain with high yield of erythritol. 8. The construction method according to claim 7, characterized in that the combinatorial promoter hp4d is selected as the promoter of the gene expression box. 9. The erythrose reductase mutant as claimed in any one of claims 1 to 3, or the genetically engineered bacterium as claimed in claim 6, or the high-yield erythritol genetically engineered bacterium constructed by the method as claimed in claim 8 Application in microbial fermentation to produce erythritol. 10. Application as claimed in claim 9, characterized in that the application includes: inoculating high-yield erythritol genetically engineered bacteria into a fermentation medium, using glycerol as a carbon source, and performing shake flask fermentation to produce erythritol. alcohol.