CN117625564A - Erythrose reductase mutant and application thereof - Google Patents
Erythrose reductase mutant and application thereof Download PDFInfo
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- CN117625564A CN117625564A CN202311537001.5A CN202311537001A CN117625564A CN 117625564 A CN117625564 A CN 117625564A CN 202311537001 A CN202311537001 A CN 202311537001A CN 117625564 A CN117625564 A CN 117625564A
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- erythrose reductase
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- yarrowia lipolytica
- erythritol
- erythrose
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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
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to an erythrose reductase mutant and application thereof.
Background
Erythritol (1, 2,3, 4-tetrol) is white, odorless, non-hygroscopic, non-optical active, good in heat stability and easy to dissolve in water, has sweetness of 60% -70% of sucrose, has calorie of 0.2kcal/g, is only 5% of the calorie of sucrose, and is a low-calorie sweetener. The food has the advantages of cool taste, no decayed tooth, good crystallization and the like, and has wider application in the field of food industry. Erythritol can be synthesized by a chemical method and a microbial fermentation method, however, the chemical synthesis method has the defects of low production efficiency, high cost, operation danger and the like; the microbial fermentation method has mild and easily controlled production process, and becomes a main way for producing erythritol nowadays. Yarrowia lipolytica Yarrowia lipolytica is taken as a recognized food-safe microorganism, and is a main utilization strain of erythritol produced at home and abroad at present due to the advantages of strong stress resistance, unique gene structure, wide available substrates and the like.
In yarrowia lipolytica, erythritol is produced by the Pentose Phosphate Pathway (PPP) as an osmoprotectant. The last step in the metabolic synthesis pathway of erythritol is the reduction of D-erythrose to erythritol by catalysis of the enzyme Erythrose Reductase (ER), which in the catalytic process takes NADPH as cofactor. Erythrose reductase is the only enzyme in the last step of the erythritol biosynthetic pathway and is considered to be a key enzyme for the overall reaction.
As a rate-limiting enzyme for producing erythritol, the expression and activity of the erythritol reductase are critical to the efficiency of erythritol synthesis. However, the protein engineering work for the enzyme is little at present, so that the obtained erythrose reductase mutant with improved catalytic capability has important significance for further improving the yield of erythritol.
Disclosure of Invention
In order to solve the problem of low erythritol yield in the prior art, the invention provides an erythritol reductase mutant derived from yarrowia lipolytica, a coding gene thereof and application thereof in producing erythritol by microbial fermentation. Compared with 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%; and the provided high-yield erythritol genetic engineering bacteria containing the coding gene of the erythritol reductase mutant has obviously improved erythritol yield compared with a single strain over-expressing the wild type of the erythritol reductase.
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.
The invention models the amino acid sequence of the erythrose reductase from yarrowia lipolytica by using alpha fold, combines substrates D-erythrose and NADPH model, uses Autodock for molecular docking, selects 16 key sites influencing enzyme activity, and further uses FoldX for stability analysis after mutation of the 16 sites. After molecular docking and thermal stability screening, mutation sites with better stability are selected for constructing the erythrose reductase mutant. The constructed single mutant of the erythrose reductase is transformed into Escherichia coli BL (DE 3) through a vector for expression, crude enzyme liquid is collected, and the reactive enzyme activity of each single mutant of the erythrose reductase is measured by detecting the consumption of NADPH in the process of catalyzing D-erythrose to generate erythritol, so that 6 single mutants of the erythrose reductase with the enzyme activity positively improved are roughly screened. And carrying out pairwise combination on the 6 single mutants of the erythrose reductase obtained by screening to obtain 15 double mutants of the erythrose reductase, and measuring the reactive enzyme activity of the double mutants to finally determine a mutant K26N, K N/V295M, K N/G215N, K26N/F216Y with obviously improved enzyme activity. The coding genes of the four erythrose reductase mutants are further constructed into double expression vectors together with glucose dehydrogenase GDH coding genes, and transformed into Escherichia coli BL (DE 3) to construct the genetically engineered bacterium, the wet thalli of the genetically engineered bacterium are used as catalysts for carrying out biocatalysis reaction for generating erythritol by using D-erythrose as a substrate and glucose as an auxiliary substrate, so that the influence of the erythrose reductase mutants on the enzyme activity expression of the erythrose reductase is explored. The results show that the enzyme activities of the four erythrose reductase mutants are improved by 1.26-1.44 times compared with the wild type erythrose reductase, wherein the enzyme activities of the erythrose reductase mutants K26N/V295M are improved by 44 percent, and the four erythrose reductase mutants have the highest enzyme activities. In order to further obtain the genetic engineering bacteria for producing the erythritol, the CRISPR-Cas9 gene editing technology is applied, yarrowia lipolytica Po g (ATCC 20460) of yarrowia lipolytica is taken as a chassis strain, the complete gene expression frame of the erythrose reductase mutant K26N/V295M is inserted into the genome of the chassis strain, and a combined promoter hp4d is selected as a promoter of the expression frame, so that the overexpression of the erythrose reductase mutant gene is realized, and the biosynthesis yield of the erythritol is further improved.
Preferably, the erythrose reductase mutant is subjected to a combination mutation as compared to the erythrose reductase encoded by the YALI0F18590g gene from yarrowia lipolytica Yarrowia lipolytica:
(1) Lysine at position 26 is mutated to asparagine and glycine at position 215 is mutated to asparagine;
(2) Lysine at position 26 is mutated to asparagine and phenylalanine at position 216 is mutated to tyrosine;
(3) Lysine at position 26 is mutated to asparagine and valine at position 295 is mutated to methionine.
As a further preference, the erythrose reductase mutant is subjected to the following combination mutations compared to the erythrose reductase encoded by the YALI0F18590g gene derived from yarrowia lipolytica Yarrowia lipolytica: lysine at position 26 is mutated to asparagine and valine at position 295 is mutated to methionine.
Specifically, the invention mutates lysine at position 26 of the erythrose reductase with the amino acid sequence shown as SEQ ID NO.1 into asparagine to obtain an erythrose reductase mutant with the amino acid sequence shown as SEQ ID NO. 2; or, mutating the 26 th lysine of the erythrose reductase with the amino acid sequence shown as SEQ ID NO.1 into asparagine, and mutating the 215 th glycine into asparagine to obtain an erythrose reductase mutant with the amino acid sequence shown as SEQ ID NO. 3; or, mutating the 26 th lysine of the erythrose reductase with the amino acid sequence shown as SEQ ID NO.1 into asparagine, and mutating the 216 th phenylalanine into tyrosine to obtain an erythrose reductase mutant with the amino acid sequence shown as SEQ ID NO. 4; or, mutating the 26 th lysine of the erythrose reductase with the amino acid sequence shown as SEQ ID NO.1 into asparagine, and mutating the 295 th valine into methionine to obtain the erythrose reductase mutant with the amino acid sequence shown as SEQ ID NO. 5.
Because of the specificity of the amino acid sequences, any fragment of a peptide protein or variant thereof, such as a conservative variant, biologically active fragment or derivative thereof, comprising an amino acid sequence of the present invention is within the scope of the present invention, as long as the fragment of the peptide protein or peptide protein variant has a homology of 90% or more to the amino acid sequence described above. In particular, the alteration comprises a deletion, insertion or substitution of an amino acid in the amino acid sequence; wherein, for conservative changes of the variant, the substituted amino acid has similar structure or chemical properties as the original amino acid, such as replacement of isoleucine with leucine, the variant may also have non-conservative changes, such as replacement of glycine with tryptophan.
The invention also provides genes for encoding the erythrose reductase mutants, and the nucleotide sequences of the genes for encoding the erythrose reductase mutants are preferably shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9. Because of the specificity of the nucleotide sequence, any variant of the polynucleotides of the present invention, as long as it has more than 90% homology with the aforementioned polynucleotides, falls within the scope of the present invention. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide may be variants that are either naturally occurring or non-naturally occurring, including substitution, deletion 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, without substantially altering the function of the peptide protein it encodes.
The invention also provides a recombinant vector containing the coding gene of the erythrose reductase mutant. The recombinant vector comprises a polynucleotide operably linked to control sequences suitable for directing expression in a host cell. Various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., are linked to the nucleotide sequence of the erythrose reductase mutant of the present invention, and are intended to fall within the scope of the present invention. The recombinant vector preferably uses a plasmid pET-28a (+) as an expression vector, and clones the coding gene of the erythrose reductase mutant to the plasmid pET-28a (+). In addition, when the gene encoding the erythrose reductase mutant is further constructed together with the gene encoding glucose dehydrogenase GDH into a double expression vector, it is generally possible to select pCDF-Duet (+) as the double expression vector.
The invention also provides a genetic engineering bacterium containing the coding gene of the erythrose reductase mutant. Introducing exogenous erythrose reductase mutant coding genes into host cells through a genetic engineering technology to construct genetically engineered bacteria and expressing the genetically engineered bacteria so as to obtain the erythrose reductase mutant; wherein the host cell can be bacteria, fungi, plant cells or animal cells, and preferably Escherichia coli Escherichia coli BL (DE 3) and yarrowia lipolytica Yarrowia lipolytica are used as expression hosts.
The invention also provides a construction method of the high-yield erythritol genetic engineering bacteria, which comprises the following steps: and (3) taking a yarrowia lipolytica Yarrowia lipolytica Po g strain as a chassis strain, inserting a gene expression frame of the erythrose reductase mutant into Yarrowia lipolytica Po g genome at fixed points and performing overexpression, so as to construct the high-yield erythritol genetic engineering strain. Preferably, the combined promoter hp4d is selected as the promoter of the gene expression cassette to enhance expression of the erythrose reductase.
The invention also provides the erythrose reductase mutant and the coding gene, the recombinant vector and the genetic engineering bacteria thereof, or the application of the high-yield erythrose alcohol genetic engineering bacteria constructed by the method in the microbial fermentation production of the erythrose alcohol.
Preferably, the application includes: the coding gene of the erythrose reductase mutant and the coding gene of the glucose dehydrogenase GDH are constructed together into a double expression vector pCDF-Duet (+) and transformed into Escherichia coli BL (DE 3) to construct the genetically engineered bacterium, and the wet thalli or crude enzyme liquid prepared by crushing the wet thalli of the genetically engineered bacterium is used as a catalyst to carry out biocatalysis reaction for generating erythritol by using D-erythrose as a substrate and glucose as an auxiliary substrate.
Preferably, the application includes: inoculating the high-yield erythritol genetic engineering bacteria into a fermentation medium, and carrying out shake flask fermentation to produce erythritol by taking glycerol as a carbon source.
The invention has the beneficial effects that: according to the invention, the site selection is carried out through molecular docking and thermal stability screening, site-directed directional mutation is carried out to change protein amino acid residues, and the amino acid near the catalytic center of the enzyme activity is mutated to obtain the erythrose reductase mutant with improved activity; compared with the wild type erythrose reductase from yarrowia lipolytica, the mutant enzyme activity is improved by 26-44%. After the erythrose reductase mutant gene is inserted into the genome of the yarrowia lipolytica Yarrowia lipolytica Po g of the chassis strain at fixed points, the yield of the erythritol of the obtained high-yield erythritol genetic engineering strain is obviously improved compared with that of a strain which singly overexpresses the wild type erythrose reductase, and the erythrose reductase mutant gene has great significance for industrial production of the erythritol.
Drawings
FIG. 1 is a nucleic acid gel electrophoresis diagram of the constructed dual expression vector of erythrose reductase and glucose dehydrogenase; lanes: 1: a 250kb marker;2: amplification products of ER fragments; 3: amplification products of ER K26N fragments; 4: amplification products of ER K26N/G215N fragments; 5: amplification products of ER K26N/F216Y fragment; 6: amplification products of ERK26N/V295M fragments; 7: amplification product of pCDF-Dute-GDH expression cassette.
FIG. 2 is a gel electrophoresis diagram of a mutant strain erythrose reductase and glucose dehydrogenase double-expressed protein; lanes: 1: a marker;2: original strain E.coli BL21-pCDF-Dute-ER-GDH;3: e.coli BL21-pCDF-Dute-ER K26N-GDH;4: e.coli BL21-pCDF-Dute-ER K26N/G215N-GDH;5: e.coli BL21-pCDF-Dute-ER K26N/F216Y-GDH;6: e.coli BL21-pCDF-Dute-ER K26N/V295M-GDH.
FIG. 3 is a graph showing the time course of the reaction of the substrate catalyzed by erythrose reductase.
FIG. 4 shows the relative enzyme activities of the erythrose reductase wild type and mutant K26N, K N/V295M, K N/G215N, K26N/F216Y.
FIG. 5 shows the yield and OD of erythritol 144h by shake flask fermentation after overexpression of the wild-type coding gene of erythrose reductase and the mutant K26N/V295M coding gene in yarrowia lipolytica Yarrowia lipolytica Po g chassis strain 600 。
Detailed Description
The following specific examples are presented to illustrate the present invention, and those skilled in the art will readily appreciate the additional advantages and capabilities of the present invention as disclosed herein. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. The methods used in the examples of the present invention are conventional methods, and the reagents used are commercially available.
[ method for preparing Medium and solution ]
LB liquid medium: 10g/L peptone, 5g/L yeast powder, 5g/L NaCl and sterilizing at 121 ℃ for 20min.
LB solid medium: 15g/L agar powder is added on the basis of LB liquid medium, and sterilization is carried out for 20min at 121 ℃.
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, the pH of the concentrated HCl is adjusted to 6.0.
YPD liquid medium: glucose 20g/L, yeast powder 10g/L, peptone 20g/L,115 ℃ and sterilization for 20min.
YPD solid medium: 20g/L agar powder is added on the basis of YPD liquid culture medium, and sterilization is carried out for 20min at 115 ℃.
SD solid medium: glucose 20g/L, YNB 6.7g/L, agar powder 20g/L,115 ℃ and sterilization for 20min.
ESM fermentation medium: glycerin 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, concentrated H 2 SO 4 Adjusting pH to 3.0, sterilizing at 121deg.C for 20min, adding sterilized CaCO before inoculation 3 3 g/L。
Example 1: erythrose reductase mutant screening
(1) Acquisition of erythrose reductase mutants
The amino acid sequence of the erythrose reductase from yarrowia lipolytica Yarrowia lipolytica was modeled using alpha Fold, the PubChem website downloaded two substrate D-erythrose and NADPH models, molecular docking was performed using Autodock, 16 key sites affecting enzyme activity were selected, the 16 sites were further subjected to post-mutation stability analysis using Fold X, and the mutation with the best stability was selected for the construction of erythrose reductase mutants. Constructing each erythrose reductase mutant expression vector by taking the constructed vector pET-28a-ER as a template and introducing mutation sites by a primer PCR with reasonable design; wherein, the primers are shown in Table 1.
Table 1: primer sequences
(2) Obtaining of wet cell
Transforming each of the erythrose reductase mutant expression vectors into host competence Escherichia coli BL (DE 3); the successful transformant bacterial liquid is streaked on a kanamycin solid LB plate with the final concentration of 100 mu g/ml, is subjected to static culture for 12 hours at 37 ℃, single colony is selected and inoculated on an LB liquid culture medium with the final concentration of 100 mu g/ml, and is subjected to shaking culture at 220rpm for about 7 hours at 37 ℃ until reaching OD 600 0.8 to 1.0 to obtain seed liquid. The seed solution was inoculated in an inoculum size of 1% by volume into LB liquid medium containing kanamycin at a final concentration of 100. Mu.g/ml, cultured at 37℃for 2 hours, and then subjected to induction at 220rpm for 12 hours with the addition of IPTG at a final concentration of 0.1 mM. Centrifuging the obtained bacterial liquid in an ultralow temperature high-speed centrifuge at 8000g and 4 ℃ for 10min, and storing the obtained wet bacterial liquid in a refrigerator at-20 ℃.
(3) Preparation of crude enzyme solution
2g of resting cells were weighed, resuspended in 10mL of 50mM PBS (pH=6.0) buffer, and broken by sonication to release intracellular proteins, the broken solution was always placed in an ice bath, and the breaking procedure was: the power is 250W, the crushing time is 1s, the interval time is 2s, and the total crushing time is 10min. After the crushing, the mixed solution is placed at 10000g of rotating speed, and is centrifuged for 10min at 4 ℃ to remove solid matters such as cell fragments and the like, and the supernatant is collected to be crude enzyme liquid. It was diluted 40-fold with 50mM PBS (ph=6.0) buffer, which was available for subsequent enzymatic assay reactions.
(4) Coarse screening of mutants
The erythrose reductase requires consumption of NADPH as a cofactor in catalyzing the production of erythritol from D-erythrose, and the coarse screen reacts the enzymatic activity of the erythrose reductase by detecting the consumption of NADPH. And (3) taking the obtained crude enzyme solution as a catalytic solution, detecting the change value of absorbance at 340nm by using an enzyme-labeling instrument, calculating the consumption of NADPH, and then reacting the enzyme activity, thereby realizing the rough screening of the erythrose reductase mutant.
The reaction system of the coarse screening enzyme activity is as follows: 150. Mu.L of 50mM PBS (pH=6.0), 10. Mu. L D-erythrose (400 mM), 20. Mu.LNADPH (2 mM), 20. Mu.L of crude enzyme solution.
The method for measuring the enzyme activity of the coarse screen comprises the following steps: the reactions were performed in 96-well cell culture plates in a total volume of 200. Mu.L, 150. Mu.L of 50mM PBS (pH=6.0) was added first, then 10. Mu.L of 400mM D-erythrose was added, then 20. Mu.L of 2mM NADPH was added, and finally 20. Mu.L of crude enzyme solutions of the wild-type erythrose reductase and the mutant diluted to an appropriate multiple were added, respectively, using eight-row chromatography, and the reaction was performed at 30℃and the change in absorbance at 340nm was detected by an enzyme-labeling instrument for 1min before the reaction to calculate the consumption of NADPH. The amount of enzyme required to consume 1mM NADPH 1min before the start of the reaction was taken as one enzyme activity unit (U), and the relative enzyme activity= (mutant enzyme activity unit/wild-type enzyme activity unit) ×100%. Thus, 6 single mutants of the erythrose reductase with forward lifting of the enzyme activity are determined, 15 double mutants are constructed by combining the 6 mutation points in pairs, and the finally obtained mutants K26N, K N/G215N, K N/F216Y, K26N/V295M respectively lift 32.2%, 35.6%, 27.7% and 48.0% compared with the wild type enzyme activity, and further catalytic reactions are carried out on the mutants.
Example 2: influence of the erythrose reductase mutant on the expression of the enzyme Activity of the erythrose reductase
PCR was performed using pET-28a-ER as a template and primer K26N-F, K N-R (as in Table 1), the glycine at position 26 was mutated to asparagine, and the band size was verified to be correct and then transferred to Escherichia coli BL (DE 3) for competence, giving rise to the site-directed mutagenesis strain designated Escherichia coli BL (DE 3) pET-28a-ERK26N. Primers G215N-F/G215N-R, F216Y-F/F216Y-R, V295M-F/V295M-R (as in Table 1) were designed and PCR was performed using Escherichia coli BL (DE 3) pET-28a-ER K26N as template to construct double point mutant erythrose mutant strains Escherichia coli BL (DE 3) pET-28a-ERK26N/G215N, escherichia coli BL (DE 3) respectively
pET-28a-ER K26N/F216Y，Escherichia coli BL21(DE3)pET-28a-ER K26N/V295M。
Using the erythrose reductase wild type strain pET28a-ER and the constructed individual erythrose reductase mutant strains with increased enzyme activities (pET-28 a-ER K26N, pET-28a-ER K26N/G215N, pET-28a-ER K26N/F216Y, pET-28a-ER K26N/V295M) as templates, the primers shown in Table 1 were used to amplify gene fragments of the erythrose reductase wild type ER and mutants (ER K26N, ER K26N/G215N, ER K26N/F216Y, ER K26N/V295M), respectively. The glucose dehydrogenase GDH expression cassette was amplified using the existing laboratory strain containing pCDF-Dute-GDH as a template with the pCDF-Dute-GDH-F/pCDF-Dute-GDH-R primer pair. The length of the band is verified by nucleic acid gel electrophoresis for the amplified fragments, the amplified fragments are normal, and the nucleic acid gel diagram is shown in figure 1. The two fragments containing the coding gene of the erythrose reductase wild type or mutant and the coding gene of glucose dehydrogenase are cloned in one step to obtain a double expression vector pCDF-Dute-ER-GDH, and transferred into a host Escherichia coli BL (DE 3). Escherichia coli BL21 (DE 3)/pCDF-Dute-ER-GDH wet cells containing the erythrose reductase mutant and glucose dehydrogenase coexpression gene were prepared in the same manner as in example 1 (2), resuspended in 50mM PBS (pH=6.0) buffer, and the protein expression was confirmed by SDS-PAGE gel electrophoresis, and the recombinant bacteria correctly expressed the protein, as shown in FIG. 2.
In order to explore the influence of the erythrose reductase mutant on the enzyme activity expression of the erythrose reductase, the collected wet thalli are used as a catalyst to carry out a biocatalytic reaction for generating erythritol by using D-erythrose as a substrate, and the catalytic activity of the erythrose reductase wild type or the mutant thereof is detected.
The catalytic reaction system contains D-erythrose with the final concentration of 50mM, glucose with the final concentration of 100mM and wet thalli with the final concentration of 20 g/L; the reaction was carried out at 30℃and 800 rpm.
The method for measuring the enzyme activity of the erythrose reductase in the catalytic reaction comprises the following steps: the reaction was carried out in a 1.5mL EP tube with a single reaction total system of 500. Mu.L, 362. Mu.L of 50mM PBS (pH=6.0) was added, 25. Mu.L of 2M glucose was then added, 50. Mu.L of wet cells co-expressed with the erythrose reductase wild type/mutant and glucose dehydrogenase diluted to an appropriate multiple were then added, the above reaction mixture was reacted for 2 minutes in advance to mobilize the coenzyme circulation system, and 63. Mu.L of 400mM D-erythrose as a substrate was finally added. The reaction was terminated thermally after 30min from the start of the addition of the substrate, i.e. 10min for a boiling water bath of 1.5mL EP tube with the reaction mixture. Centrifuging the treated reaction liquid, sucking the supernatant, diluting the supernatant by 2 times, and performing high performance liquid phase detection. Erythritol solutions of different concentrations were prepared and peak areas were detected with a differential liquid phase, and yields were calculated from a standard curve, relative enzyme activity= (mutant erythritol yield/starting strain erythritol yield) ×100%. The results are shown in FIG. 4. Wherein the enzyme activity of the mutant K26N is improved by 25.9%, the enzyme activity of the K26N/G215N is improved by 25.7%, the enzyme activity of the K26N/F216Y is improved by 38.5%, and the enzyme activity of the K26N/V295M is improved by 43.9%.
In order to explore proper reaction time, the catalytic reaction firstly explores the reaction time course in recombinant fungus wet cells co-expressed by the wild type of the erythrose reductase and the glucose dehydrogenase, samples are taken at 1min, 3min, 5min, 10min, 30min, 60min and 90min of the reaction, and the contents of D-erythrose and erythritol are detected by high performance liquid chromatography respectively, and the reaction course is shown in a graph in figure 3. The reaction is carried out for 90min to reach equilibrium, the difference of the enzyme activities of the reaction mutant and the wild type is taken as the reaction time of the comparison between the erythrose reductase mutant and the wild type, and the total time of the subsequent reaction is determined to be 30min.
Example 3: construction and application of high-yield erythritol genetically engineered bacteria
Site-directed insertion of the erythrose reductase wild type and mutant ER K26N/V295M coding genes is realized by using CRISPR-Cas9 gene editing technology. First, the gene fragments of the wild type ER and mutant ER K26N/V295M of the erythrose reductase are integrated into the middle of the hp4d combined promoter and XPR2 TER terminator on the pINA1312 vector, and the complete expression frame of the ER and ER K26N/V295M genes is obtained. Then constructing vectors pCRISPRyl-URA-MHY (ER) and pCRISPRyl-URA-MHY (ERK 26N/V295M), and placing the complete erythrose reductase wild type or mutant gene expression frame between upstream homology arm and downstream homology arm of MHY site. Plasmids of pCRISPRyl-URA-MHY (ER) and pCRISPRyl-URA-MHY (ERK 26N/V295M) strains were extracted and transferred into a competent yarrowia lipolytica of Yarrowia lipolytica Po g, and Yarrowia lipolytica Po g of a strain Yarrowia lipolytica Po g of which the site-specific insertion of an erythrose reductase expression frame was successful, yarrowia lipolytica Po g of which were ER K26N/V295M, were obtained by verification.
Wherein, the preparation method of the competence of the yarrowia lipolytica Yarrowia lipolytica Po g chassis strain comprises the following steps: mu.L of Yarrowia lipolytica Po g of the bacterial liquid was taken out of the stored glycerol tube and applied to YPD solid plates, and the culture was allowed to stand in an incubator at 30℃for about 24 hours. The appropriate amount of cultured yarrowia lipolytica cells were scraped with a sterile gun head, resuspended in a pre-chilled sterile EP tube containing 1ml TE Buffer (1 m, ph=7), centrifuged at 5000rpm for 5min and the supernatant was decanted, the cells resuspended in 600 μl of lithium acetate (0.1 m, ph=6.0) solution and incubated in a 30 ℃ incubator for 1h with a flip-flop every 10min. And centrifuging at 5000rpm for 5min, pouring out the supernatant, and adding 100 mu L of 0.1M lithium acetate to resuspend thalli to obtain competent cells. Competent split-up into 1.5mL sterile EP tubes, 40 μl per tube, was used for the subsequent transformation experiments.
The method for transforming plasmids into the Yarrowia lipolytica Po g chassis strain competence comprises the following steps: 40. Mu.L of competent cells were taken, 2. Mu.L of 10mg/ml salmon sperm DNA and 10. Mu.L of plasmid were sequentially added, and after being mixed by blowing, they were allowed to stand in a constant temperature incubator at 30℃for 15 minutes. 350. Mu.L of PEG4000-LiAc (0.1M, pH=6.0) and 16.7. Mu.L of 1M DTT were added in this order, and incubated in a constant temperature incubator at 30℃for 1 hour. 46.7 mu LDMSO was added and heat-shocked in a 39℃water bath for 10min. 600. Mu.L of 0.1M lithium acetate was added and mixed by slow blowing, and the mixture was allowed to stand in a incubator at 30℃for 30 minutes, followed by application to SD solid medium.
Respectively carrying out stationary culture on Yarrowia lipolytica Po g of the successfully transformed genetic engineering strain (ER, yarrowia lipolytica Po g of ER K26N/V295M) and Yarrowia lipolytica Po g of chassis strain (Yarrowia lipolytica Po g) in a YPD solid plate at 30 ℃ for 24 hours, picking single bacterial colony, inoculating the single bacterial colony to a YPD liquid culture medium, and placing the single bacterial colony in a shaking table at 200rpm for culturing at 30 ℃ for 24 hours to obtain seed liquid; the seed solution was inoculated in an ESM fermentation medium at an inoculum size of 2% by volume and cultured at 220rpm for 144 hours. Centrifuging the fermented culture solution, diluting the supernatant to an appropriate multiple, and carrying out Agilent differential liquid phase detection on the erythritol yield by using a Bio-Radamine HPX-87H chromatographic column. As shown in FIG. 5, the yield of erythritol of the high-yield erythritol genetic engineering bacterium Yarrowia lipolytica Po g is 47g/L, and the yield of erythritol of ER K26N/V295M is improved by 14.59% and 8.4% respectively compared with the yield of the erythritol of the chassis strain Yarrowia lipolytica Po g and the yield of the genetic engineering strain Yarrowia lipolytica Po g of the over-expressed wild-type erythritol reductase encoding gene.
The above examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. An erythrose reductase mutant characterized by having undergone a single point mutation or a combination of mutations at the following sites compared to the erythrose reductase encoded by the 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.
2. The erythrose reductase mutant of claim 1, which has undergone a combination mutation as compared to the erythrose reductase encoded by the YALI0F18590g gene derived from yarrowia lipolytica Yarrowia lipolytica:
(1) Lysine at position 26 is mutated to asparagine and glycine at position 215 is mutated to asparagine;
(2) Lysine at position 26 is mutated to asparagine and phenylalanine at position 216 is mutated to tyrosine;
(3) Lysine at position 26 is mutated to asparagine and valine at position 295 is mutated to methionine.
3. The erythrose reductase mutant of claim 1, which has undergone the following combination mutations compared to the erythrose reductase encoded by the YALI0F18590g gene derived from yarrowia lipolytica Yarrowia lipolytica:
lysine at position 26 is mutated to asparagine and valine at position 295 is mutated to methionine.
4. A gene encoding the erythrose reductase mutant according to any one of claims 1 to 3.
5. A recombinant vector comprising the gene encoding the erythrose reductase mutant of claim 4.
6. A genetically engineered bacterium comprising a gene encoding the erythrose reductase mutant of claim 4.
7. The construction method of the high-yield erythritol genetically engineered bacterium is characterized by comprising the following steps of: taking yarrowia lipolytica Yarrowia lipolytica Po g strain as a chassis strain, inserting the gene expression frame of the erythrose reductase mutant of any one of claims 1-3 into the genome at fixed points and performing over-expression, so as to construct the high-yield erythritol genetic engineering strain.
8. The method according to claim 7, wherein a combined promoter hp4d is selected as the promoter of the gene expression cassette.
9. The use of the erythritol reductase mutant according to any one of claims 1 to 3, or the genetically engineered bacterium according to claim 6, or the high-yield erythritol genetically engineered bacterium constructed by the method according to claim 8 in the production of erythritol by microbial fermentation.
10. The application of claim 9, wherein the application comprises: inoculating high-yield erythritol genetic engineering bacteria into a fermentation culture medium, and carrying out shake flask fermentation to produce erythritol by taking glycerol as a carbon source.
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