CN116875522A - Engineering bacteria containing alcohol dehydrogenase mutant gene and application thereof - Google Patents

Engineering bacteria containing alcohol dehydrogenase mutant gene and application thereof Download PDF

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CN116875522A
CN116875522A CN202310914824.9A CN202310914824A CN116875522A CN 116875522 A CN116875522 A CN 116875522A CN 202310914824 A CN202310914824 A CN 202310914824A CN 116875522 A CN116875522 A CN 116875522A
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pma5
alcohol dehydrogenase
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CN116875522B (en
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方诩
李宾
韩丽娟
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Shandong Henglu Biotechnology Co ltd
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Abstract

The invention provides engineering bacteria containing alcohol dehydrogenase mutant genes and application thereof. The invention relates to a genetic engineering bacterium containing an alcohol dehydrogenase mutant and application thereof in the aspect of synthesizing rare sugar. The genetically engineered bacterium is obtained by constructing an alcohol dehydrogenase mutant gene into bacillus or saccharomyces cerevisiae, wherein the alcohol dehydrogenase mutant is selected from one of the following mutations of PDH enzyme shown in SEQ ID NO. 1: D36A/I37R, D G/I37R, D R/I37R, D H/I37R, D K/I37R, D I/I37R, D F/I37R, A14T/D36A/I37R, or A14T/D36G/I37R, or GDH alcohol dehydrogenase as shown in SEQ ID NO:2 is mutated as follows: E42A. The genetically engineered bacterium provided by the invention has the advantage of improved activity of the synthetic tagatose.

Description

Engineering bacteria containing alcohol dehydrogenase mutant gene and application thereof
Technical Field
The invention relates to a genetic engineering bacterium and application thereof, in particular to application of the genetic engineering bacterium containing an alcohol dehydrogenase mutant gene in the aspect of synthesizing rare sugar, belonging to the technical field of synthesizing rare sugar in enzyme engineering and cell factories.
Background
Rare sugar is a kind of monosaccharide and derivative which exist in nature but have very small content, and generally plays an important role in the fields of diet, health care, medicine and the like. Tagatose (tagatose) is a rare natural ketose, belonging to the rare sugar category. The sweet taste is similar to sucrose, the sweetness can reach 92% of the sucrose, the generated heat is only 30% of the sucrose, no aftertaste can be generated, and no bad flavor can be generated, so that the low-calorie sweetener is a novel low-calorie sweetener which is used in the fields of foods and medicines, is widely focused in recent years, is one of the low-energy sweeteners approved by the FDA, and has the most similar taste, sweetness and sucrose, and is an ideal sucrose substitute.
The biological method for synthesizing tagatose has the advantages of environmental friendliness, high specificity, few byproducts and the like, most of the existing industrial tagatose is synthesized by adopting an enzyme method, and has the characteristics of high efficiency and high specificity, while the enzyme method for synthesizing tagatose mainly adopts isomerase, and the theoretical conversion rate is below 50% due to reversibility of isomerization reaction, so that a substrate (galactose) and a product (tagatose) are often mixed and are difficult to separate, and the industrial production cost is high.
In nature, tagatose synthesis first reduces galactose to produce galactitol using xylose reductase XR, which is NADPH cofactor dependent, and then oxidizes galactitol to produce tagatose using alcohol dehydrogenase (PDH), which is NAD + The cofactor dependence, different cofactor dependence of the two-step reaction limits the efficient progress of the reaction, and the cofactor preference of one of the enzymes is reversed through cofactor engineering to realize the unification of the cofactors, so that the production efficiency is improved, and the production cost is reduced.
Disclosure of Invention
The invention aims to: provides bacillus and saccharomyces cerevisiae genetically engineered bacteria and application thereof in the aspect of producing rare sugar. The rare sugar is tagatose.
In view of the above, the present invention provides a genetically engineered bacterium containing an NADPH cofactor-preferential polyol dehydrogenase mutant, which is capable of efficiently oxidizing an intermediate galactitol to tagatose, and forming an NADPH cofactor cycle in an enzyme reaction system.
The technical scheme of the invention is as follows: the genetically engineered bacterium is obtained by constructing an alcohol dehydrogenase mutant gene into bacillus or saccharomyces cerevisiae, wherein the alcohol dehydrogenase mutant is a mutant obtained by improving a PDH (polymerase chain reaction) with an amino acid sequence shown as SEQ ID NO.1 or a GDH alcohol dehydrogenase gene shown as SEQ ID NO. 2.
The genetically engineered bacterium has improved activity of synthesizing tagatose.
The alcohol dehydrogenase mutant has cofactor preference of NAD + 。
The alcohol dehydrogenase is derived from Paracoccus denitrificansParacoccus denitrificans) Polyol dehydrogenase (polyol dehydrogenase, PDH, genBank ID: WP_ 011751060), the amino acid sequence of which is shown as SEQ ID NO.1 and is named as PdPDH; or from rhizobium pisum of peaRhizobium leguminosarum) Alcohol dehydrogenases of (galactitol dehydrogenase, GDH, genBank ID: WP_ 011650422.1), the amino acid sequence of which is shown in SEQ ID NO.2, is named RlGDH. As shown in figure 1, the amino acid sequence identity of SEQ ID NO.1 and SEQ ID NO.2 is more than 30%.
In some embodiments, the alcohol dehydrogenase is described, further preferred, comprising an enzyme having at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity to SEQ ID No.1 or SEQ ID No. 2.
The alcohol dehydrogenase PDH or GDH mutant has its cofactor dependent on NADP + Dependency, the cofactor preference (NAD) of alcohol dehydrogenases PDH or GDH was reversed + Dependent), with dependent NADP + Cofactors oxidize galactitol to tagatose activity.
The alcohol dehydrogenase PDH mutant is one of the following mutations of D36A/I37R, D36G/I37R, D36R/I37R, D H/I37R, D K/I37R, D I/I37R, D F/I37R, A T/D36A/I37R or A14T/D36G/I37R at the N-terminal of the amino acid sequence shown in SEQ ID NO. 1.
The alcohol dehydrogenase GDH mutant is an E42A mutation at the N end of an amino acid sequence shown in SEQ ID NO. 2.
The application of the alcohol dehydrogenase PDH or GDH mutant in the aspect of synthesizing tagatose takes galactose from lactose as a substrate, takes Xylose Reductase (XR) and the alcohol dehydrogenase mutant as catalysts, and takes NADPH and NADP generated by the NADP reductase + Forming a cofactor recycle reaction for catalyzing cofactor; the Xylose Reductase (XR) is derived from pichia pastorisPichia stipitis) GenBank ID XP-001385181, the amino acid sequence of which is shown in SEQ ID NO. 3.
Figure 2 of the accompanying drawings depicts two different routes for the synthesis of tagatose using galactose as substrate. The first step of the two paths utilizes xylose reductase XR to reduce galactose to generate galactitol, wherein the xylose reductase XR is NADPH cofactor dependence; synthetic pathway I is a natural pathway, and in the second step, galactitol is oxidized to tagatose by alcohol dehydrogenase (PDH) which is NAD + Cofactor dependence the different cofactor dependencies of the two-step reaction limit the efficient progress of the reaction.
In summary, the cofactor for the enzymatic reaction of the synthesis pathway I aldose reductase XR is NADPH, while the coenzyme for the wild-type alcohol dehydrogenase is NAD + Thus, the addition of NADPH and NAD is required in the catalytic production of tagatose using both + Two cofactors, the amount of tagatose produced depends on the amount of cofactor added, and a large amount of cofactor is consumed, resulting in productionThe cost is too high.
Synthetic pathway II is a pathway of the mutant of alcohol dehydrogenase PDH or GDH of the present invention to produce tagatose, wherein the mutant of alcohol dehydrogenase PDH or GDH in the second reaction can directly utilize NADP generated in the first reaction + The cofactor oxidizes galactitol to produce tagatose.
The PDH or GDH mutant reverses the preference of alcohol dehydrogenase PDH/GDH for cofactors from NAD + Dependence to NADP + Depending on, cofactor recycling between the first and second step reactions is established when acting synergistically with xylose reductase XR. Compared with the natural path of the synthetic path I, the invention realizes the cyclic utilization of the cofactor by the enzyme-linked reaction to synthesize tagatose, obviously reduces the consumption of the cofactor and does not need to additionally add NAD + Cofactor, simplified post-treatment process, greatly reduced manufacturing cost, and possibility for the large-scale production of tagatose.
The expression of the alcohol dehydrogenase or the mutant thereof, and the host cell thereof comprises a wild strain separated in nature, a variant strain subjected to physical and chemical mutagenesis, or engineering bacteria subjected to genetic engineering. The host cells are cultured in a nutrient medium suitable for producing the alcohol dehydrogenase or mutant thereof using methods known in the art. For example, the cells may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid state fermentation) in a laboratory or industrial fermentor under conditions that allow expression and/or isolation of the alcohol dehydrogenase or mutant thereof. Cultivation occurs in a suitable nutrient medium comprising a carbon and/or nitrogen source and/or inorganic salts using procedures known in the art. Suitable media are commercially available or are prepared according to the disclosed compositions.
The alcohol dehydrogenase or mutant thereof may be recovered using methods known in the art. For example, the alcohol dehydrogenase or mutant thereof can be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
In some embodiments of the invention, bacillus genetically engineered bacteria are described that contain genes for alcohol dehydrogenase PDH or GDH mutants as previously described.
The bacillus genetically engineered strain can be a wild strain, a variant strain after physical and chemical mutagenesis or an engineering strain after genetic engineering modification.
The bacillus may be polymyxa or bacillus subtilisB.subtilis)Bacillus cereus, bacillus licheniformis, bacillus brevis, bacillus anthracis and bacillus alcalophilusB.alcalophilus) Bacillus thermophilus @B.thermophilus) Or salt-tolerant bacillusB.halodurans)。
The bacillus cereus family member is selected from bacillus anthracis, bacillus cereus, bacillus thuringiensis, bacillus mycoides, bacillus pseudomycoides, bacillus wei-gii and the like.
In a preferred embodiment, the bacillus is selected from the group consisting of bacillus subtilisBacillus subtilis 168。
Due to its GRAS (generally recognized safe) status, several species of Bacillus species, e.g., bacillus amyloliquefaciensB.amyloliquefaciens) Bacillus licheniformisB.licheniformis) And bacillus subtilis @Bacillus subtilis) Are used in biotechnological production of a variety of proteins and compounds used in the food and pharmaceutical industries. Carbohydrases and proteases from bacillus subtilis are recognized as safe (GRAS) by the united states food and safety administration (FDA). Bacillus subtilis is also entitled "safety qualification (Qualified Presumption of Safety)" by the european food safety agency.
In other embodiments, saccharomyces cerevisiae genetically engineered bacteria are described that contain genes for mutants of alcohol dehydrogenase PDH or GDH as previously described.
The saccharomyces cerevisiae is @ theSaccharomyces) Saccharomyces cerevisiae is an important microorganism in the fermentation industry, mainly used for alcohol, beer and breadIndustry. The traditional Saccharomyces cerevisiae breeding improvement methods mainly comprise natural screening, mutation breeding and hybridization breeding, but the methods have weak transferability. The genetic engineering technology improves the specificity of breeding and improving the saccharomyces cerevisiae. Saccharomyces cerevisiae has been recognized by the U.S. FDA as a safe organism, the expression product of which does not require a large number of host safety experiments, and the fermentation process is simple, and the applicant has found that it has excellent properties of synthesizing rare sugars after introducing the gene of the mutant of alcohol dehydrogenase PDH or GDH described in the present statement.
In a preferred embodiment, the Saccharomyces cerevisiae is selected from the group consisting of Saccharomyces cerevisiae @Saccharomyces cerevisiae）BY4741。
The strains of the above species can be easily purchased commercially in the domestic market, and can be obtained by public in many culture collections, such as China general microbiological culture Collection center (CGMCC), china Center for Type Culture Collection (CCTCC), guangdong province microbiological culture collection center (GDMCC), and the like.
It will be appreciated by those of ordinary skill in the art that genetic alterations may be described with reference to a suitable host organism and its corresponding metabolic response or a suitable source organism for the desired genetic material (e.g., genes of the desired metabolic pathway). However, one of ordinary skill in the art can apply the genetically engineered recombinant techniques of the present invention to other organisms, given the wide variety of organisms' whole genome sequencing and the high level of skill in the genomics arts.
In some embodiments, methods of constructing the spore or saccharomyces cerevisiae genetically engineered bacteria are also described. The construction method comprises the step of introducing genes of mutants of alcohol dehydrogenase PDH or GDH as described above BY using bacillus subtilis 168 and Saccharomyces cerevisiae BY4741 cells as starting strains.
The construction method of the genetically engineered bacterium preferably comprises the following steps: genes for introducing alcohol dehydrogenase PDH or mutants of GDH.
The construction method of the genetically engineered bacterium preferably comprises the following steps: firstly constructing PDH mutant or GDH mutant construct or recombinant expression vector, and then introducing the PDH mutant or GDH mutant construct or recombinant expression vector into host cells to construct the engineering host cells.
In a possible embodiment, it is preferred, for example, that the host cell is Bacillus subtilis, in which case the plasmid used to transform the recombinant expression vector of the host cell is pMA5-PDH and pMA5-PDH1 to pMA5-PDH9, or pMA5-GDH, pMA5-GDH1.
Specifically, the construction method of the genetically engineered bacterium comprises the following steps:
step 1, synthesizing genes of mutant PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH1 according to gene sequences of alcohol dehydrogenase and mutants thereof;
step 2, respectively connecting the genes synthesized in the step 1 to pMA5 plasmids containing different resistance genes and integration sites, and transforming escherichia coli competence to obtain recombinant expression plasmids pMA5-XR, pMA5-PDH1, pMA5-PDH2, pMA5-PDH3, pMA5-PDH4, pMA5-PDH5, pMA5-PDH6, pMA5-PDH7, pMA5-PDH8, pMA5-PDH9, pMA5-GDH and pMA5-GDH1;
and 3, transforming the recombinant plasmid obtained in the step 2 into bacillus subtilis.
In a possible embodiment, it is preferred that, for example, the host cell is Saccharomyces cerevisiae, in which case the plasmids used for transforming the recombinant expression vector of the host cell are BYg-iXiP, BYg-iXiP1 to BYg-iXiP19 and BYg-iXiG, BYg-iXiG1.
Specifically, the construction method of the genetically engineered bacterium comprises the following steps:
step 1, synthesizing genes of mutant PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH1 according to gene sequences of alcohol dehydrogenase and mutants thereof;
and 2, respectively connecting the genes synthesized in the step 1 to BYg plasmids containing different resistance genes and integration sites, and transforming escherichia coli competence to obtain recombinant expression plasmids BYg-ixiP, BYg-ixiP1, BYg-ixiP2, BYg-ixiP3, BYg-ixiP4, BYg-ixiP5, BYg-ixiP6, BYg-ixiP7, BYg-ixiP8, BYg-ixiP9, BYg-ixiG and BYg-ixiG1.
And 3, transforming the recombinant plasmid obtained in the step 2 into saccharomyces cerevisiae.
The method for constructing the genetically engineered bacteria comprises the steps of separating a wild strain from the natural world, or a variant strain after physical and chemical mutagenesis, or engineering bacteria after genetic engineering modification.
In some embodiments, the use of the genetically engineered bacterium in the synthesis of D-tagatose is also described.
The application is preferably to take galactose from lactose source as a substrate.
The genetically engineered bacteria may be directly used as an unpurified solution containing the genetically engineered bacteria, preferably purified genetically engineered bacteria.
The use, preferably in combination with xylose reductase, implements the cofactors NADPH and NADP by means of an enzyme-linked reaction + The tagatose is synthesized by recycling the catalysis, and NAD is not required to be additionally added + The use amount of cofactor is reduced.
Engineering strains (Bacillus subtilis BStgtP 8) BStgtP8 and (Saccharomyces cerevisiae SctgtP 8) SctgtP8 were deposited in China center for type culture Collection, address: wu Changou Lopa university of 16 Wuhan, university of mountain road, wuhan, hubei province, china, telephone number 027-68754052, storage date 2023, 6, 29, and storage numbers are respectively: CCTCC No. M20231126 and CCTCC No. M20231127.
Terminology: modification refers to any chemical modification of the amino acid sequence of the enzyme or its homologous sequence, as well as genetic manipulation of the DNA encoding the polypeptide. The modification may be a substitution, deletion and/or insertion of one or more amino acids, and a substitution of one or more amino acid side chains.
The beneficial effects are that: the invention successfully constructs bacillus or saccharomyces cerevisiae gene engineering bacteria containing the alcohol dehydrogenase mutant gene and synthesizes tagatose by using the bacillus or saccharomyces cerevisiae gene engineering bacteria. The alcohol dehydrogenase mutant reverses the preference of cofactor of the original enzyme, realizes the recycling of the cofactor in the two-step reaction of synthesizing tagatose, greatly reduces the consumption of the cofactor in the reaction process, simultaneously eliminates the generation of cofactor reaction by-product impurities, and is beneficial to the purification of later-stage products; the alcohol dehydrogenase mutant is introduced into engineering bacteria constructed by Saccharomyces cerevisiae or bacillus, so that the tagatose production capacity of the basic strain is improved. The technical scheme of the invention is simple and feasible, is suitable for industrial production and application, solves the problem of high cost in large-scale industrial production, and has important economic significance.
Drawings
FIG. 1 alignment of the amino acid sequences of SEQ ID NO.1 and SEQ ID NO. 2.
FIG. 2 shows the pathway of tagatose synthesis from galactose.
FIG. 3 SDS-PAGE analysis of XR enzyme and alcohol dehydrogenase mutants obtained in example 1. Lanes 1 through 13, respectively, from left to right. Lane 1 is XR enzyme, lanes 2-10 are PDH enzyme and its mutants PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9, lanes 11-12 are GDH enzyme and its mutant GDH1; lane 13 is a protein Marker.
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; all materials, reagents, etc., unless otherwise specified, are commercially available.
The present invention will be further described in detail by way of examples, and it should be understood that the embodiments described herein are merely illustrative of the present invention and are not intended to limit the invention, and that modifications and alterations may be made to the details and forms of the technical solution of the present invention without departing from the spirit and scope of the invention.
Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in the present specification and the experimental methods described below are those well known and commonly employed in the art.
In the following examples, lactose, galactose, galactitol, tagatose, and the like were analyzed by High Performance Liquid Chromatography (HPLC) under the following conditions: chromatographic column model: shodex SP-0810 column (300 mm X8mm,Showa Denko K.K., tokyo,japan), mobile phase ddH 2 O, column temperature 70 ℃, sample volume 10 μl, differential detector (RI detector L2490, HITACHI, japan HITACHI chromatoster), flow rate 1.0 mL/min, HPLC profile showing tagatose standard peak time rt=14.29 min.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. Furthermore, it will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components, and/or groups thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology in the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
Example 1 recombination and expression of alcohol dehydrogenase PDH or GDH and mutants thereof.
The alcohol dehydrogenase (PDH/GDH) gene was sent to the Gene company for synthesis. According to the amino acid sequence of alcohol dehydrogenase (PDH/GDH) and its amino acid, synthesizing alcohol dehydrogenase (PDH/GDH) gene and connecting it to PET32a plasmid vector so as to obtain the invented PET32a-PDH and PET32a-GDH recombinant expression plasmid.
The DNA synthesized by PDH/GDH genes is used as a template, the amino acid sequences of the DNA are shown as SEQ ID NO.1 and SEQ ID NO.2, upstream primers and downstream primers (shown in tables 1 and 2 in detail) of mutants are designed, and a point mutation kit is utilized to finally transform escherichia coli DH5 alpha so as to obtain recombinant expression plasmids.
The recombinant plasmid which was confirmed to be correct was transformed into E.coli BL21 as follows: BL21 competent bacteria were taken and placed on ice for 30 min to melt, 100 mu L of competent cells were taken and mixed with 10 mu L of the recombinant plasmid (concentration 50 ng/. Mu.L), placed in a 42 ℃ water bath for heat shock 45 s, immediately cooled in the ice bath for 2 min, 1 mL fresh LB medium (LB medium: peptone 1.0%, yeast extract 0.5%, naCl 1.0% and 1.5% agar powder) was added to the mixture, and the mixture was subjected to resuscitating culture at 37 ℃ and 100 rpm for 1 h, after which 100 mu L of bacterial liquid was taken and coated on LB plates containing ampicillin (100 mu g/ml), and after 12 h culture in a 37 ℃ constant temperature incubator, single colonies were picked up and sequenced to verify that the recombinant plasmid construction was successful.
Then, the positive transformant is selected and inoculated into LB liquid medium, and placed on a shaking table at 37 ℃ and 200 rpm for shaking culture for 12 h, so as to obtain seed liquid; then inoculating the seed solution into fresh LB culture medium with 1% (v/v) inoculum size, culturing at 37 ℃ under shaking until OD 600 is 0.8, inducing with isopropyl-beta-D-thiogalactopyranoside (IPTG) with 0.1 mmol/L final concentration, inducing with 12 h at 16 ℃ and rotating at 200 rpm. After induction of expression, the fermentation broth was centrifuged at 5000 r/min for 30 min at 4℃to collect the cells. After the cells were resuspended in 20 mM PBS buffer pH7.4, the cells were disrupted by sonication at plus on 5s/off 5s for 30 min; the disrupted liquid was centrifuged at 13000 Xg at 4℃for 30 min to remove cell debris, and the supernatant was collected.
Purifying by using a nickel column affinity chromatography to obtain the alcohol dehydrogenase mutant, wherein the process is as follows: adding deionized water to the top of the nickel column, naturally eluting, and eluting with Binding buffer with 5 times of volume; and then the crude enzyme solution filtered by a 0.45 mu m filter membrane is put on a column, the sample is fully combined with a nickel column at the flow rate of 1.5 ml/min, after the sample is drained, the Washing buffer with the volume of 5 times of the column is used for continuous gradient Elution to remove the impurity protein, and finally the target protein is eluted with the buffer with the volume of 5 times of the Washing buffer, and the eluent is collected. Then SDS-PAGE is used to analyze the expression condition of target protein (see figure 3 in detail), the gene engineering bacteria has obvious specific expression bands after induction, the obtained protein is mutant PDH1-PDH9 or GDH1 of the alcohol dehydrogenase, and the corresponding relation between the mutant PDH1-PDH9 or GDH1 and the mutation mode of the primordial enzyme PDH or GDH is shown in table 3.
TABLE 1 alcohol dehydrogenase PDH mutant and primers
。
TABLE 2 alcohol dehydrogenase GDH mutant and primers
。
TABLE 3 correspondence between alcohol dehydrogenase mutants and mutation patterns
。
Example 2 alcohol dehydrogenase mutant catalyzes the synthesis of tagatose (one-step reaction, coenzyme NADPH 2 mM).
20. 20 mM galactose, 0.02 mg Xylose Reductase (XR), 1.728 mg alcohol dehydrogenase mutant (PDH 1, or PDH2, or PDH3, or PDH4, or PDH5, or PDH6, or PDH7, or PDH8, or PDH9, or GDH 1), 2mM cofactor NADPH,100 mM Tri-HCl buffer (pH 8.0) were mixed, reacted at 30℃for 15 hours, and the reaction was terminated, followed by purification by gel column method. The concentrations of tagatose in the reaction solutions were measured by HPLC chromatography, respectively, and the results are shown in Table 4.
TABLE 4 study of alcohol dehydrogenase mutants and XR catalyzed Synthesis of tagatose
。
Example 3 preparation of recombinant Bacillus.
Genes were synthesized based on the gene sequences of xylose reductase XR and alcohol dehydrogenase and mutants thereof (PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH 1), respectively. The three synthesized genes are respectively connected to pMA5 plasmids containing different resistance genes and integration sites by a recombinant cloning kit method, and escherichia coli competence is transformed to obtain recombinant expression plasmids pMA5-XR, pMA5-PDH1, pMA5-PDH2, pMA5-PDH3, pMA5-PDH4, pMA5-PDH5, pMA5-PDH6, pMA5-PDH7, pMA5-PDH8, pMA5-PDH9, pMA5-GDH and pMA5-GDH1.
The recombinant plasmid, which was confirmed to be correct as described above, was transformed into bacillus subtilis (Bacillus subtilis) 168 as follows:
the fresh activated bacillus subtilis single colony is inoculated in 5 mL of GMI culture medium, cultured overnight at 30 ℃, transferred to the GMI culture medium according to 10 percent of inoculum size, cultured at 220 rpm for 3.5 h, transferred to GMII culture medium according to 10 percent of inoculum size, cultured at 37 ℃ for 90 min and 5000 g at 125 rpm, and centrifuged for 10 min to collect the thalli to obtain competent cells. To 0.5. 0.5 mL competence, 5. Mu.g of the recombinant expression plasmid was added, and after shaking culture at 37℃and 220 rpm for 30 min, the resultant was plated on a corresponding resistance plate, and cultured overnight at 37℃to obtain transformants.
And respectively picking single colonies of each transformant, inoculating the single colonies into LB culture medium, culturing at 37 ℃ and 220 rpm for 48 hours to respectively obtain strains BStgtP, BStgtP, BStP 2, BStP 3, BStP 4, BStP 5, BStP 6, BStP 7, BStP 8, BStP 9 and BStgtG, BStgtG1, and screening to obtain synthetic strains of high-yield tagatose. Wherein, the strain BStgtP8 is preserved in China center for type culture collection, address: university of martial arts, storage date 2023, 6, 29, deposit number: cctccc No. M20231126.
TABLE 5 Bacillus engineering bacteria and genotypes thereof
。
Example 4. Preparation of recombinant Saccharomyces cerevisiae.
Genes were synthesized based on the gene sequences of Xylose Reductase (XR) and alcohol dehydrogenase mutants (PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH 1), respectively. The three genes synthesized are respectively connected to BYg expression plasmids containing resistance genes and integration sites by using a recombinant cloning kit method, and escherichia coli competence is transformed to obtain recombinant expression plasmids BYg-iXiP, BYg-iXiP1, BYg-iXiP2, BYg-iXiP3, BYg-iXiP4, BYg-iXiP5, BYg-iXiP6, BYg-iXiP7, BYg-iXiP8, BYg-iXiP9, BYg-iXiP g and BYg-iXiP 1.
The recombinant plasmid, which was verified to be correct as described above, was transformed into Saccharomyces cerevisiae BY4741 as follows:
and (3) carrying out quick ice bath cooling for 3min after treating the Carrier DNA at the temperature of 95-100 ℃ for 3min, and repeating the operation once to obtain the single-stranded DNA.
Taking 100 uL purchased Saccharomyces cerevisiae BY4741 competent cells, melting on ice, sequentially adding precooled target plasmid BYg-iXiG M Or BYg-iXiP M 10 uL of single-stranded Carrier DNA, PEG/LiAc 500 uL are blown by a gun for a few times and fully mixed, the mixture is placed in a water bath at 42 ℃ for 15 min after ice bath at 30 ℃ for 40s at 5000 rpm, the supernatant is discarded, 400uL of ddH2O is added for heavy suspension precipitation, the supernatant is removed by centrifugation again, 50 uL of ddH2O is added for heavy suspension, the mixture is coated on YPD plates containing antibiotics, and the mixture is cultured for 3 days at 30 ℃.
Single colonies of each transformant were picked up and inoculated into YPD medium, and cultured at 30℃and 220 rpm for 72℃ 72 h, to obtain recombinant strains SctgtP, sctgtP, sctgtP2, sctgtP3, sctgtP4, sctgtP5, sctgtP6, sctgtP7, sctgtP8, sctgtP9 and SctgtG, sctgtG, respectively, to obtain synthetic strains of tagatose. Wherein, the strain SctgtP8 is preserved in China center for type culture Collection, address: university of martial arts, storage date 2023, 6, 29, deposit number: cctccc No. M20231127.
TABLE 6 Saccharomyces cerevisiae Gene engineering bacteria and genotypes thereof
。
Example 5. Study of the Bacillus subtilis genetically engineered strain obtained in example 3 and the Saccharomyces cerevisiae genetically engineered strain obtained in example 4 to synthesize tagatose.
Shake flask fermentation is as follows:
1) Preparation of first-stage: the bacillus subtilis genetic engineering strain obtained in the example 3 and the saccharomyces cerevisiae genetic engineering strain obtained in the example 4 are respectively taken to be single-colony in 10 mL LB/YPD liquid culture medium 37 ℃/30 o C. Culturing overnight at 200 rpm, and obtaining the strain as the first-class strain.
2) Preparation of the second-stage seed: LB/YPD liquid culture inoculated in 1L of primary seedBased on the addition of 1% glucose and 1% galactose at a high concentration at the same time, at 30 o C,200 rpm culture for 3-5 days, sampling, HPLC method detection of the generated tagatose yield, results are recorded in Table 7-8.
TABLE 7 study of Synthesis of tagatose by genetically engineered Bacillus subtilis obtained in EXAMPLE 3
。
TABLE 8 study of the Synthesis of tagatose by Saccharomyces cerevisiae Gene engineering bacteria obtained in example 4
。
Table 7 and table 8 data illustrate: the bacillus subtilis genetically engineered bacteria and saccharomyces cerevisiae genetically engineered bacteria containing the alcohol dehydrogenase mutant genes have improved activity of synthesizing tagatose.
It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the scope of the technical solutions of the present invention.

Claims (7)

1. The genetically engineered bacterium containing the alcohol dehydrogenase mutant gene is characterized in that the genetically engineered bacterium is obtained by constructing an alcohol dehydrogenase mutant gene into a host cell, wherein the host cell is selected from bacillus or saccharomyces cerevisiae, the alcohol dehydrogenase mutant is selected from PDH enzyme with an amino acid sequence shown as SEQ ID NO.1, and the PDH alcohol dehydrogenase with an amino acid sequence shown as SEQ ID NO.2 has one of the following mutations, namely D36A/I37R, D G/I37R, D36R/I37R, D H/I37R, D K/I37R, D I/I37R, D F/I37R, A14T/D36A/I37R, or A14T/D36G/I37R: E42A.
2. The method for constructing genetically engineered bacteria of claim 1, comprising the steps of:
step 1, constructing a PDH mutant or a construction body or a recombinant expression vector of the GDH mutant;
and 2, introducing the construct or the recombinant expression vector obtained in the step 1 into a host cell to construct an engineering host cell.
3. The method for constructing genetically engineered bacteria of claim 2, wherein the host cell is bacillus subtilis, comprising the steps of:
step 1, synthesizing genes of mutant PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH1 according to gene sequences of alcohol dehydrogenase and mutants thereof;
step 2, respectively connecting the genes synthesized in the step 1 on pMA5 plasmid, and transforming escherichia coli competence to obtain recombinant expression plasmids pMA5-XR, pMA5-PDH1, pMA5-PDH2, pMA5-PDH3, pMA5-PDH4, pMA5-PDH5, pMA5-PDH6, pMA5-PDH7, pMA5-PDH8, pMA5-PDH9, pMA5-GDH and pMA5-GDH1;
and 3, transforming the recombinant plasmid obtained in the step 2 into bacillus subtilis.
4. The method for constructing genetically engineered bacteria of claim 2, wherein when the host cell is saccharomyces cerevisiae, the method comprises the following steps:
step 1, synthesizing genes of mutant PDH, PDH1, PDH2, PDH3, PDH4, PDH5, PDH6, PDH7, PDH8, PDH9 or GDH, GDH1 according to gene sequences of alcohol dehydrogenase and mutants thereof;
step 2, respectively connecting the genes synthesized in the step 1 on BYg plasmids, and transforming escherichia coli competence to obtain recombinant expression plasmids BYg-ixiP, BYg-ixiP1, BYg-ixiP2, BYg-ixiP3, BYg-ixiP4, BYg-ixiP5, BYg-ixiP6, BYg-ixiP7, BYg-ixiP8, BYg-ixiP9 and BYg-ixiG and BYg-ixiG1;
and 3, transforming the recombinant plasmid obtained in the step 2 into saccharomyces cerevisiae.
5. The use of the genetically engineered bacterium of claim 1 in the synthesis of tagatose.
6. The use according to claim 5, wherein NADP dependent + Cofactors oxidize galactitol to tagatose.
7. The use according to claim 5, wherein the substrate is selected from galactose of lactose origin.
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