CN112175891B - Genetically engineered bacterium for producing trehalose and construction method and application thereof - Google Patents

Abstract

A genetic engineering bacterium for producing trehalose and a construction method and application thereof belong to the fields of genetic engineering and fermentation engineering. In order to solve the problem that organic carbon sources are needed to be provided for artificially as raw materials for producing trehalose at present and the production cost is increased, the invention provides a genetic engineering bacterium for producing trehalose and a construction method and application thereof. The genetically engineered bacterium is obtained by over-expressing maltooligosaccharide-based trehalose synthase MTse and maltooligosaccharide-based trehalose hydrolase MTHase by cyanobacteria and trehalose transporter TRET 1. The invention realizes the synthesis of trehalose by directly fixing carbon dioxide by solar energy in photosynthetic autotrophic microorganism cyanobacteria. The source of trehalose synthesis energy is solar energy and the source of carbon source is carbon dioxide. Compared with the traditional production mode, the trehalose synthesized by the method is not limited by insufficient raw materials and energy, and carbon emission is not increased, so that the trehalose is a real zero-emission trehalose product.

Description

Genetically engineered bacterium for producing trehalose and construction method and application thereof

Technical Field

The invention belongs to the field of genetic engineering and fermentation engineering. In particular to a genetic engineering bacterium for producing trehalose and a construction method and application thereof.

Background

Cyanobacteria (cyanobacteria) is the only prokaryote capable of performing plant-type oxygen-releasing photosynthesis, has great potential in terms of biocatalysts for directly converting carbon dioxide into fuels, chemicals and other high-added-value products, and is currently used as chassis cells for realizing synthesis of various biofuels, bio-based chemicals and sugar substances, such as sucrose, glycogen, fatty alcohols, aliphatic hydrocarbons, fatty acids, isoprene, ethanol, butanol, isobutyraldehyde, lactic acid, hydrogen and the like along with rapid development in the fields of metabolic engineering and synthetic biology.

Trehalose (Trehalose) is a non-reducing disaccharide formed by joining two glucose molecules with an alpha-1, 1-glycosidic linkage, and there is a synthetic pathway for Trehalose in bacteria, yeasts, fungi, insects, invertebrates and plants. The trehalose synthesized in organisms can be used as a carbon source and energy reserve of the organisms and help cells to cope with various environmental stresses (high temperature, high salt, low temperature, drought and the like), and the trehalose product obtained by industrial production can be used as an excellent activity protective agent of biological products, a unique ingredient for improving the quality of foods and an important component of moisturizing cosmetics, and is an important functional disaccharide.

Trehalose has multiple functions (carbon source and stress resistance) and wide application prospects in multiple fields (cosmetics, medicines and health products), and in order to realize industrialized production of trehalose, the yield of the trehalose is improved, the market price is reduced, so far scientific researchers have researched and developed various methods for preparing the trehalose: (1) direct extraction from organisms: in 2012, anning et al screen yeast SC2 (initial strain has trehalose content of 48mg/g dry weight) to obtain mutant SA12 (trehalose content of 64mg/g dry weight, 33% higher than initial strain), increase intracellular trehalose content to 149mg/g dry weight (yield: 3.1 g/L) by optimizing strain medium (seed medium and fermentation medium), increase intracellular trehalose content to 227mg/g dry weight (yield: 4.82 g/L) after 40g/L NaCl stress, and finally perform fermentation in a fed-batch mode by optimizing fermentation mode while salt stress treatment of mutant SA12 to obtain biomass of 23.3g/L and intracellular trehalose content to 229mg/g dry weight (yield: 5.4 g/L) by preliminary fermentation in a 7L fermenter; (2) extracting trehalose synthase by microbial fermentation, and synthesizing trehalose by an enzymatic method: the Japanese forest protogenesis research institute utilizes maltooligosaccharide-based trehalose synthase (Malrooligosyl-trehalose synthase, MTase) and maltooligosaccharide-based trehalose hydrolase (Malrooligosyl-trehalose trehalodehydrolase, MTHase) to form a mixed enzyme system, and degrades starch to generate trehalose; (3) trehalose production by microbial fermentation: for example, in 2015, the advanced et al realize the successful expression of the enzyme by introducing a trehalose bifunctional synthase gene (tpsp) of Haw fibrous phage (Cytophaga hutchinsonii) into escherichia coli, the yield of trehalose is increased to 1.2g/L, the relative conversion rate is 21%, the fermentation condition is optimized by a fermentation tank, the final yield of trehalose reaches 13.3g/L, and the relative conversion rate of glucose reaches 48.6%. In summary, no matter microbial fermentation (yeast or escherichia coli) or enzymatic synthesis is adopted, saccharide compounds such as starch, glucose and the like are required to be used as fermentation substrates, so that the production cost is greatly increased.

Disclosure of Invention

In order to solve the problem that organic carbon sources are needed to be provided for artificially as raw materials for producing trehalose at present and the production cost is increased, the invention provides a genetic engineering bacterium for producing trehalose and a construction method and application thereof.

The genetically engineered bacterium is a cyanobacterium over-expressed maltooligosaccharide-based trehalose synthase (MTSAE) gene or a homologous gene with the MTSAE gene, and a maltooligosaccharide-based trehalose hydrolase (MTHase) gene or a homologous gene with the MTHase gene;

the nucleotide sequence of the MTse gene is shown as SEQ ID NO. 1;

the homologous gene of the MTSAE gene refers to a gene with the homology of the coded amino acid sequence and the coded amino acid sequence of the MTSAE gene reaching more than 84 percent;

the nucleotide sequence of the MTHase gene is shown as SEQ ID NO. 2;

the homologous gene of the MTHase gene refers to a gene with the homology of the coded amino acid sequence and the coded amino acid sequence of the MTHase gene reaching more than 82%;

preferably, the genetically engineered bacterium also overexpresses the TRET 1gene, or a homologous gene to the TRET1 gene;

the TRET1 nucleotide sequence is shown as SEQ ID NO. 3;

the homologous gene of the TRET 1gene refers to a gene with the homology of the coded amino acid sequence and the amino acid sequence coded by the TRET 1gene reaching more than 58 percent.

Preferably, the cyanobacteria is Synechococcus.

Preferably, the Synechococcus is Synechococcus PCC7942 or Synechococcus Sye7942-C252Y.

The construction method of the genetically engineered bacterium comprises the following steps:

(1) Constructing a recombinant plasmid carrying MTase and MTHase genes;

(2) And (3) introducing the recombinant plasmid obtained in the step (1) into host bacteria to obtain genetically engineered bacteria, wherein the host bacteria are cyanobacteria.

Preferably, the method further comprises the steps of constructing a recombinant plasmid carrying a TRET 1gene, and introducing the recombinant plasmid carrying the TRET 1gene and the recombinant plasmid carrying MTase and MTHase genes into host bacteria to obtain genetically engineered bacteria.

Preferably, the MTase and MTHase genes in the recombinant plasmid in the step (1) are connected through a P3 connecting peptide gene, and the P3 connecting peptide gene is shown as SEQ ID NO. 16.

The genetically engineered bacterium disclosed by the invention is applied to the production of trehalose.

Preferably, the specific steps for producing trehalose are as follows:

(1) Culturing genetically engineered bacteria: culturing genetically engineered bacteria in column type photoreactor, inoculating concentration OD ₇₃₀ 0.1, the culture temperature is 28-30deg.C, and the light intensity is 100-200. Mu. Mol photons m ^-2 s ^- The gas used for culturing is 3% CO ₂ A mixture with 97% air;

(2) Transporter TRET1 induced expression: culturing the engineering bacteria in the step (1) until the 4 th day, adding isopropyl thiogalactoside (IPTG) to induce the expression of a transport protein TRET1, and adding the IPTG to induce for 24 hours to separate and purify the trehalose.

Preferably, the culture in step (1) is carried out in BG11 medium without antibiotics at 30℃and with a light intensity of 150. Mu. Mol photons m ^-2 s ^-1 。

Advantageous effects

The invention realizes the synthesis of trehalose by directly fixing carbon dioxide by solar energy in photosynthetic autotrophic microorganism cyanobacteria. The source of trehalose synthesis energy is solar energy, while the source of carbon source is carbon dioxide. Compared with the traditional production mode, the trehalose product synthesized by the method of the invention is not limited by insufficient raw materials and energy, and simultaneously carbon emission is not increased, thus being a real zero-emission trehalose product.

Drawings

FIG. 1 is a vector map of plasmid pQY 160;

FIG. 2 is a vector map of plasmid pQY165;

FIG. 3 shows screening and identifying electrophoretogram of genetically engineered strains cyanoQY038 and cyanoQY053, wherein SPS site series bands are obtained by PCR amplification by using the engineering strains and Wild Type (WT) genomes as templates and using primers 0808-T-Flong and 0808-T-Rlong, wherein the size of the engineering strains is 8085bp, and the size of the wild type bands is 2997bp; the NS3 site series band is obtained by PCR amplification by using engineering strains and Wild Type (WT) genomes as templates and using primers NS3-T1-F and NS3-T1-R, wherein the size of the engineering strains band is 4024bp, and the size of the wild type band is 289bp;

FIG. 4 shows the use of ion chromatography ICS5000 after induction of the genetically engineered strain cyanoQY038 IPTG ⁺ The peak diagram is measured, A is the peak diagram of a trehalose standard (50 mg/L), and the trehalose peak-out time is 2.819min; b is ICS5000 Using ion chromatography ⁺ Measuring peak diagram of culture supernatant (diluted 100 times) of CyanoQY038, wherein trehalose peak time is 2.821min, and the culture supernatant is matched with a standard substance;

FIG. 5A is a graph showing strain growth of genetically engineered strains CyanoQY038 and CyanoQY053 provided by the examples of the present invention under no salt stress; b is the accumulation of intracellular and extracellular trehalose; c is the accumulation of intracellular and extracellular trehalose of the unit OD strain; d is the percentage of extracellular trehalose in the total amount;

FIG. 6A is a strain growth curve of the genetically engineered strain CyanoQY031 provided by the embodiment of the invention under no salt stress; b is the accumulation condition of intracellular trehalose of the strain.

Detailed Description

The cyanobacteria Synechococcus PCC7942 (Syn 7942) used in the examples below, which was often used as a model strain, was subjected to experimental studies and detailed information is described in Microbiology,2005.151 (Pt 8): p.2605-13.doi:10.1099/mic.0.28030-0.Stability of the Synechococcus elongatus PCC 7942circadian clock under directed anti-phase expression of the kai genes.ditty, J.L., canales, S.R., anderson, B.E., williams, S.B., golden, S.S.

Another Synechococcus used in the examples below was Synechococcus PCC7942 mutant Sye7942-C252Y [ serine (C) 252 mutation in the ATP synthase FOF1 alpha subunit (AtpA) to tyrosine (Y) ], detailed information being described in Appl Environ Microbiol.2018.A Specific Single Nucleotide Polymorphism in the ATP Synthase Gene Significantly Improves Environmental Stress Tolerance of Synechococcus elongatus PCC 7942.Lou, W., X.Tan, K.Song, S.Zhang, G.Luan, C.Li and X.Lu.

The anabaena PCC7120 used in the examples below is a commonly used model alga, details are described in microbiology.2006.the role of a gene cluster for trehalose metabolism in dehydration tolerance of the filamentous cyanobacterium Anabaena sp.PCC 7120.Higo, A.Katoh, H.ohmori, K.Ikeuchi, M.ohmori, M.example 1, step (2) 3) pNS3-3B with cscB, a plasmid from Pamela A.silver laboratory, harvard medical college, U.S.A., details are recorded in Appl Environ Microbiol,2012.78 (8): p.2660-8.doi:10.1128/AEM.07901-11.Rerouting carbon flux to enhance photosynthetic productivity.Ducat,D.C, avelar-Rivas, J.A., way, J.C., silver, P.A.

The public of the materials can be obtained from the Qingdao biological energy and process institute, the microbiological metabolism engineering research group of the national academy of sciences of China from Laoshan mountain region Songling No. 189 of Qingdao, shandong.

The relevant terms related to the present invention are explained as follows:

cyanobacteria (cyanobacteria) is a class of prokaryotic microorganisms capable of photoautotrophic, which are able to utilize solar energy and carbon dioxide as energy and carbon sources for their own growth.

Maltooligosyl trehalose synthase (mtase) and Maltooligosyl trehalose hydrolase (mtase) are two enzymes capable of synthesizing trehalose using glycogen or starch as a substrate.

A linker peptide (linker), which is a polypeptide capable of fusing two enzymes, is used in embodiments of the present invention as a rigid linker peptide that links two key enzymes MTse and MTHase that synthesize trehalose.

Trehalose transporter TRET1 is a channel protein capable of transporting intracellular trehalose out of the cell with high efficiency, and contains 12 transmembrane regions.

Homology arms (homologo arms), two DNA fragments used in genetic engineering of cyanobacteria to integrate a resistance gene, a promoter, and a gene of interest controlled by the promoter into the host cyanobacteria genome by Homologous recombination (Homologous recombination): in this embodiment, the N-terminal and the C-terminal are respectively used.

Definition and abbreviation

The abbreviations and corresponding uses for the names referred to herein are as follows:

host bacterium for constructing genetically engineered bacterium by Syn7942 (Syn 7942) of Syn-type algae

Another host bacterium for constructing genetically engineered bacterium by using Synechococcus PCC7942 mutant Sye7942-C252Y

Extraction of DNA from anabaena PCC7120 as amplification of MTase and MTHase groups

Template for reasons

CyanoQY038 is based on Synechococcus PCC7942 as host for producing trehalose

Engineering bacteria

CyanoQY031 another method for producing trehalose by taking Synechococcus PCC7942 as host

Genetically engineered bacteria of (2)

CyanoQY053 uses Synechococcus Sye7942-C252Y as host to produce trehalose

Genetically engineered bacteria of (2)

Plasmid pQY recombinant plasmid carrying MTase and MTHase genes

Recombinant plasmid carrying TRET 1gene in plasmid pQY165

Plasmid pZZ009 construction of intermediate plasmid of plasmid pQY160

Plasmid pQY construction of an intermediate plasmid (as vector) of plasmid pQY160

Commercial plasmid with plasmid pMV-WHC198677-TRET1 carrying TRET 1gene

Plasmid pNS3-3B with cscB construction of intermediate plasmid of plasmid pQY165 (as vector)

Promoter for PcpcB to start MTse and MTHase gene transcription

Sp ^r Spectacular antibiotic resistance genes

Cm ^r Chloramphenicol resistance gene

P3 connecting peptide

N-terminal sequence of NS3-N neutral site NS3

C-terminal sequence of NS3-C neutral site NS3

SPS-N sucrose synthase SPS upstream N-terminal sequence

SPS-C sucrose synthase SPS downstream C-terminal sequence

The PCR amplification systems involved in the following examples were 50. Mu.L:

5 XBuffer: 10. Mu.L; 2.5mM dNTPs 4. Mu.L; forward primer F (10. Mu.M): 1. Mu.L; reverse primer R (10. Mu.M) 1. Mu.L; 1. Mu.L of Fastpfu Fly DNA polymerase; plasmid/genome template: according to the actual conditions, 100ng of genomic DNA and 5-30ng of plasmid DNA are used; adding ultrapure water to 50 mu L;

PCR amplification conditions:

(1) 95 ℃ C:: 5min, (2) 95 ℃ C.): 30s, (3) annealing temperature (depending on the primer pair case): 30s, (4) 72 ℃ C.): 2-4kb/min (return (2), 30 cycles in total), (5 ℃ C.) (72 ℃ C.): and 10min.

The single cleavage system referred to in the examples below was 50. Mu.L

10 Xbuffer (dye-containing): 5. Mu.L; plasmid dosage: 5 μg; dosage of fast cutting enzyme: 5. Mu.L; ultrapure water was added to 50. Mu.L.

After the system is prepared, the mixture is slightly and uniformly mixed, the mixture is treated for 15min by a warm bath at 37 ℃, the rapid cutting enzyme is deactivated by heating at 65 ℃ for 5min, and the required fragments are recovered by the system after enzyme cutting.

The double cleavage system referred to in the examples below was 50. Mu.L

10 Xbuffer (dye-containing): 5. Mu.L; plasmid dosage: 5 μg; dosage of fast cutting enzyme 1: 5. Mu.L; dosage of fast cutting enzyme 2: 5. Mu.L; ultrapure water was added to 50. Mu.L.

After the system is prepared, the mixture is slightly and uniformly mixed, the mixture is treated for 15min by a warm bath at 37 ℃, the rapid cutting enzyme is deactivated by heating at 65 ℃ for 5min, and the required fragments are recovered by the system after enzyme cutting.

BG11 medium: from 1.5g L ^-1 NaNO ₃ ,40mg L ^-1 K ₂ HPO ₄ ·3H ₂ O,36mg L ^-1 CaCl ₂ ·2H ₂ O,6mg L ^-1 Citric acid, 6mg L ^-1 Ferric ammonium citrate, 1mg L ^-1 EDTA disodium salt, 20mg L ^-1 NaCO ₃ ,2.9mg L ^-1 H ₃ BO ₃ ,1.8mg L ^-1 MnCl ₂ ·4H ₂ O,0.22mg L ^-1 ZnSO ₄ ·7H ₂ O,0.39mg L ^-1 NaMoO ₄ ·2H ₂ O,0.079mg L ^-1 CuSO ₄ ·5H ₂ O and 0.01mg L ^-1 CoCl ₂ ·6H ₂ O composition.

Example 1 construction of trehalose producing genetically engineered bacterium cyanoQY038.

(1) Preparation of recombinant plasmid pQY carrying MTase and MTHase genes 160:

1) Extracting genome DNA of anabaena PCC 7120;

2) Designing and synthesizing a primer pair aiming at the MTase corresponding gene all0167 with a sequence shown as SEQ ID NO.1, wherein the sequence of a primer SP-MTS-P1-F is shown as SEQ ID NO.4, and the sequence of a primer SP-MTS-P3-R is shown as SEQ ID NO. 5;

3) Using the genome DNA of the step 1) as a template, and performing PCR amplification by using the primer synthesized in the step 2) to obtain a DNA fragment of MTase corresponding to the gene all 0167;

4) Designing and synthesizing a primer pair aiming at MTHase corresponding gene all0168 with a sequence shown as SEQ ID NO.2, wherein a primer SP-P3-MTH-F sequence is shown as SEQ ID NO.6, and a primer SP-MTH-P3-R sequence is shown as SEQ ID NO. 7;

5) Using the genome DNA of the step 1) as a template, and carrying out PCR amplification by using the primer synthesized in the step 4) to obtain a DNA fragment of the MTHase corresponding gene all 0168;

6) Plasmid pZZ009, which was digested with NdeI/SpeI to recover large fragments, was digested with NdeI/SpeI, and plasmid pZZ009, which was constructed with pMD18-T as the backbone, was ligated in sequence with the resistance fragment Sp ^r Promoter P _cpcB . Plasmid pZZ009, with sequence information shown in SEQ ID NO.8, was double digested with both NdeI/SpeI enzymes to plasmid pZZ009, resulting in a vector fragment of approximately 5270bp with NdeI and SpeI cohesive ends;

7) The gene fragments of step 3), 5), 6) were seamlessly ligated using Seamless Assembly Cloning Kit to give plasmid pMD18T-Sp ^r -P _cpcB The sequence information of the P3 in the plasmid is shown as SEQ ID NO.16, and the reaction system and conditions are shown as the specification;

8) Designing a synthetic primer pair aiming at the plasmid in the step 7), wherein the sequence of a primer SPS-SPPSPH-F20 is shown as SEQ ID NO.9, and the sequence of the primer SPS-SPPSPH-R20 is shown as SEQ ID NO. 10;

9) Plasmid pMD18T-Sp obtained in step 7) ^r -P _cpcB PCR amplification is carried out by taking MTase-P3-MTHaseDNA as a template and utilizing the primer synthesized in the step 8) to obtain Sp ^r -P _cpcB -a mtase-P3-MTHase fragment;

10 Plasmid pQY was digested with SpeI to obtain a vector fragment of 5802bp size linearized with SpeI cohesive ends, and plasmid pQY was a plasmid with the pEASY-Blunt Simple backbone, with homology arms SPS-N and SPS-C sequences. The sequence information of the plasmid pQY is shown as SEQ ID NO.11, and the reaction system and conditions are shown in the specification;

11 Step 9), 10) were seamlessly ligated using Seamless Assembly Cloning Kit to obtain plasmid pQY160, the vector map of which is shown in FIG. 1.

(2) Preparing a recombinant plasmid pQY165 carrying a TRET1 gene:

1) Carrying out TRET1 complete gene synthesis by Beijing Liuhua large gene technology Co., ltd, carrying out codon optimization on the TRET1 complete gene synthesis, adding XbaI at the N end, adding BamHI at the C end, and obtaining plasmid pMV-WHC198677-TRET1, wherein the optimized sequence information is shown as SEQ ID NO. 3;

2) The plasmid pMV-WHC198677-TRET1 is subjected to XbaI/BamHI digestion to obtain a TRET1 fragment;

3) The plasmid pNS3-3B with cscB was digested with XbaI/BamHI (excision of the sucrose permease cscB gene);

4) Ligating the TRET1 fragment obtained in step 2) with the vector fragment obtained in step 3) to obtain a recombinant plasmid pQY165, wherein the vector map is shown in FIG. 2.

(3) Into Syncs 7942 (Syn 7942) was transferred plasmid pQY and plasmid pQY165;

1) 1mL of logarithmic phase (OD) ₇₃₀ Syn7942, 5000 Xg, about 1) cells were collected by centrifugation for 5min, washed 2 times with fresh BG11 medium, the supernatant discarded and the pellet resuspended in 250. Mu.L BG11 solution;

2) To the resuspended algae solution was added 2. Mu.L of each of two plasmids: plasmids pQY and pQY165 (at a concentration of 100. Mu.g/mL);

3) Wrapping the EP added with the plasmid by using tinfoil paper, and incubating for 20 hours at a temperature of 30 ℃ by using a shaking table;

4) The incubated transformation products were spread on BG11 plates (Sp ^r ：10μg/mL，Cm ^r ：5μg/mL)，30℃，100μmol photons m ^-2 s ^-1 Culturing under light intensity for 4-5 days to allow the transformant to grow out, and picking up the transformant and fresh BG11 plate (Sp ^r ：10μg/mL，Cm ^r :5 μg/mL) streaking;

(4) Screening and identifying complete integrated transformant culture to obtain cyanoQY038;

the result of screening and identifying the transformant with complete integration after cell enrichment is shown in FIG. 3, wherein the engineering strain is completely integrated, namely in the genome of the engineering strainThe SPS site series bands shown in FIG. 3 are obtained by PCR amplification using engineering strain and Wild Type (WT) genomes as templates and primers 0808-T-Flong (sequence shown as SEQ ID NO. 12) and 0808-T-Rlong (sequence shown as SEQ ID NO. 13), wherein the size of the engineering strain bands is 8085bp, and the size of the wild type bands is 2997bp; the NS3 site series band is obtained by PCR amplification by using engineering strains and Wild Type (WT) genomes as templates and using primers NS3-T1-F (the sequence of which is shown as SEQ ID NO. 14) and NS3-T1-R (the sequence of which is shown as SEQ ID NO. 15), wherein the size of the engineering strains band is 4024bp, and the size of the wild type band is 289bp. Liquid BG11 (Sp) ^r ：10μg/mL，Cm ^r :5. Mu.g/mL) to obtain cyanoQY038.

The SPS site series 20 mu L PCR amplification system is as follows: 2 XVazyme

Master Mix (Dye Plus): 10. Mu.L; forward primer F (10. Mu.M): 2. Mu.L; reverse primer R (10. Mu.M) 2. Mu.L; genome template: 5ng; ultrapure water was added to 20. Mu.L.

PCR amplification conditions: (1) 94 ℃ C:: 3min, (2) 94℃:10s, (3) 68 ℃ C.): 30-60sec/kb (return (2), 35 cycles of total cycles), (5 ℃ C.) (68 ℃ C.): and thoroughly extending for 7 min.

The NS3 site series 20. Mu.L PCR amplification system is: 2X Rapid Taq Master Mix: 10. Mu.L; forward primer F (10. Mu.M): 2. Mu.L; reverse primer R (10. Mu.M) 2. Mu.L; genome template: 5ng; ultrapure water was added to 20. Mu.L.

PCR amplification conditions: (1) 95 ℃ C:: 5min, (2) 95 ℃ C.): 30s, (3 ℃ C.): 30s, (4) 72 ℃ C.): 15sec/kb (return (2), 30 cycles in total), (5) 72 ℃ C.): and 10min.

Example 2 construction of trehalose-producing genetically engineered bacterium cyanoQY053.

Example 1 was repeated except that step (3) was performed by transferring plasmid pQY and plasmid pQY165 into the high light and high temperature resistant mutant of Synechococcus Sye 7942-C252Y; and (4) screening and identifying the complete integrated transformant to obtain screening and identifying results of the cyanoQY053 are shown in figure 3.

The invention also provides application of the constructed genetically engineered bacterium in production of trehalose.

The promoter active in cyanobacteria drives MTase and MTHase, and the trehalose transport protein TRET1 driven by the promoter is expressed in cyanobacteria, so that the photosynthetic autotrophic microorganism cyanobacteria is utilized to absorb light energy to fix carbon dioxide, and trehalose is synthesized and secreted in a large amount under the condition of no salt stress. The method of producing trehalose by the genetically engineered bacteria obtained in the above examples is specifically described below.

Example 3. Genetically engineered strain CyanoQY038 synthesizes and secretes trehalose.

(1) Culturing the genetically engineered bacterium cyanoQY038 obtained in the example 1 in a column type photoreactor;

the column type photoreactor is a sharp-bottom glass tube made of common glass with the height of 58cm and the diameter of 3.3 cm; the total liquid loading amount of the reactor can reach 300ml, and 200ml of liquid is filled in the experimental process. The seed solution is BG11 culture in logarithmic growth phase (supplemented with corresponding antibiotic: sp ^r ：10μg/mL，Cm ^r :5. Mu.g/mL), initial inoculum concentration OD ₇₃₀ 0.1, BG11 (no antibiotics added) medium, 150. Mu. Mol photons m at 30 ℃ ^-2 s ^- ' light intensity, introducing a mixture of gases (3% CO) ₂ +97% air).

(2) Expression of transport protein TRET1

The culture was continued by adding IPTG to the cyanobacteria culture at a final concentration of 1mM to the post-logarithmic phase (day 4) by the column aeration culture described above.

Example 4 genetically engineered strain CyanoQY053 synthesizes and secretes trehalose.

The present example was the same as example 3, except that the genetically engineered bacterium cultured in step (1) was CyanoQY053.

The growth was smoothed after induction with IPTG for CyanoQY038 and CyanoQY053, and the results are shown in fig. 5. There was no significant difference in the growth of the two strains.

Taking 1mL of algae solution of engineering strain in the culture process, centrifuging at 8000rpm for 10min, and transferring the supernatant into anotherClean 1.5mL EP tube for determination of extracellular trehalose content; the cell pellet was resuspended using 1mL 80% ethanol and placed in a 65℃water bath prepared in advance for 4h. Centrifuging at 8000rpm for 10min, transferring supernatant to new EP tube, N ₂ Drying, adding 1mL of ultrapure water for dissolution, thus preparing an intracellular trehalose sample, diluting the extracellular and intracellular extracted trehalose, and detecting by utilizing ion chromatography or a kit. ICS-5000 Using ion chromatography ⁺ (Dionex, thermo Scientific, USA), PA10 (Dionex carboPac, thermo Scientific, USA) analytical column. The mobile phase was 200mM NaOH solution, the flow rate was 0.9mL/min, the trehalose peak time was about 2.8min as shown in FIG. 4A, the trehalose standard (50 mg/L) peak plot, FIG. 4B was ICS5000 using ion chromatography ⁺ The peak pattern of the supernatant (100-fold dilution) of the CyanoQY038 culture was measured, and the trehalose peak-out time was 2.821min, which was identical to that of the standard.

Quantitative analysis of intracellular and extracellular trehalose content was performed (fig. 5b, c), and after 1mM IPTG induction, the two engineering strains began to secrete large amounts of trehalose extracellular. After 15 days of culture, 1mM IPTG induces the 11 th day, the extracellular secretion of trehalose by CyanoQY038 reaches 1.97g/L, the total amount reaches 2.03g/L, and the content of trehalose per unit OD can reach 414.78mg/L; cyanoQY053 exocytosis trehalose up to 2.66g/L, total amount up to 2.75g/L, unit OD trehalose content up to 541.95mg/L, and both engineering strains have more than 96% trehalose secreted extracellularly (FIG. 5D). These results indicate that the engineered strain into which the TRET1 transporter gene is introduced can secrete extracellular trehalose accumulated during the culture process very efficiently.

Example 5 construction of trehalose-producing genetically engineered bacterium cyanoQY031 and Synthesis of trehalose.

The construction of the trehalose producing genetically engineered bacterium CyanoQY031 was repeated as in example 1, except that in step (3) only plasmid pQY160 was transferred into Syn7942 (Syn 7942) in the Syn.

Synthesis of trehalose example 3 was repeated except that expression was not induced by the transporter TRET1 of step (2).

The growth of the strain is monitored in the column type illumination culture process of the cynaoQY031, a strain growth curve is drawn (figure 6A), meanwhile, the quantitative analysis of the intracellular trehalose content is carried out (figure 6B), and after 12 days of culture, 295mg/L of trehalose can be accumulated in the cell of the cynaoQY 031. These results demonstrate that trehalose can be synthesized under conditions of salt-free stress treatment in engineering strains without the presence of TRET1, as long as trehalose synthase mtase and mtase are present at the same time.

Although the present invention has disclosed an exemplary demonstration, for the synthetic secretion of trehalose, the three proteins MTSase, MTHase and TRET1 required each have key conserved regions for catalysis, and the corresponding proteins can accomplish the catalytic synthesis of trehalose and transport of trehalose as long as the key conserved regions exist.

(1) The similarity of the amino acid sequence of MTse used by the engineering bacteria and the candida albicans (Nostoc flagelliforme) GenBank:EF433294.1 reaches 84 percent, and the annotation is that: malto-oligosyltrehalose synthase, in addition, candida hairlike MTse has been reported to function as maltooligosaccharide-based trehalose synthase. Details are described in j.microbiol.biotechnoll.2010.molecular Cloning and Characterization of Maltooligosyltrehalose Synthase Gene from Nostoc flagelliforme.wu, s., shen, r., zhang, x., wang, Q.

(2) The amino acid sequence comparison result of the MTHase and nostoc flagelliforme (Nostoc flagelliforme) GenBank: HM802214.1 used by the engineering bacteria of the patent is that the sequence similarity is 82%, and the annotation is that: malto-oligosyltrehalose trehalohydrolase, and furthermore Candida hairy-derived MTHase, have been reported to function as maltooligosyl trehalose hydrolase. Details are described in J.Microbiol. Biotechnol.2011.Molecular Cloning of Maltooligosyltrehalose Trehalohydrolase Gene from Nostoc flagelliforme and Trehalose-Related Response to stress. Wu, S., he, L., shen, R., zhang, X., wang, Q.

(3) The amino acid sequence comparison result of TRET1 and anopheles gambiae (Anopheles gambiae) AgTret1 GenBank: AB369548.1 used by the engineering bacteria of the patent shows that the sequence similarity is 80.4%; the amino acid sequence of the polypeptide is aligned with that of silkworm (Bombyx mori) BmTRet1 GenBank: AB369550.1, and the sequence similarity is 58.5%; the amino acid sequence alignment with Apis mellifera AmTret1 GenBank: AB369549.1 shows that the sequence similarity is 58.7%; the amino acid sequence similarity with Drosophila melanogaster (Drosophila melanogaster) DmTret1-1B GenBank:AB369553.1 was 73%. The genes from different sources listed above have been demonstrated in the literature to have the same function as TRET1 used in this patent, and can all achieve trehalose transport. Details are described in Insect Biochem Mol biol.2010.the trehalose transporter 1gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues.Kanamori,Y, saito, a., hagiwara-kooda, y., tanaka, d., mitsunasu, k., kikuta, s., watanabe, m., cornete, r., kikawada, T., okuda, T.

While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.

SEQUENCE LISTING

<110> Qingdao bioenergy and Process institute of China academy of sciences

<120> a genetically engineered bacterium for producing trehalose, and construction method and application thereof

<130>

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 2766

<212> DNA

<213> MTse nucleotide sequence

<400> 1

atgcgaattc ctaaagctac ttatcgtatt cagtttacac cggagtttgg gtttgatgat 60

gctagagcga tcgcatctta cctagcagat ttaggtattt ctgattttta tgcttcgccg 120

atttttaagg ctagaactgg tagtacacat ggttatgatg tggtggatgg gtcacaactc 180

aatccccaat taggaactac tgaggctttt gaggcgttgg tggctgaatt acagtctcta 240

ggtctgggct ggttgcaaga tattgttcct aaccacatgg cttatagtag cgaaaatcca 300

tatttaatgg atgtgctgga acatgggccg gattctagtt ataccgacta ctttgatgtg 360

tgttggaact ctccatttgc taatagtcag gagcgcatct tagctccttt attaggcgat 420

ttttatggtg aatctttaga aaagggagat attgaactgc aatatgagca aaatggttta 480

actgttaact attacagctt aaagttgcca ttgcgcttag aatcttatac taaatttatt 540

agtcacaatt tgggtaaact cacacgtaca ctgggacgga atcaccctga ttttattaaa 600

cttttaggca ttctctacat tctgaaaaat gttccctcgg aagttgtagg aaaacagcga 660

caagaccaaa ttgcttttat caagggtttg atttgggaac tctacaccag taatgatgat 720

atccacgcat tcattgagga gaatattcaa acatttaatg gtgaagcagg taattttgaa 780

agttttaatt tgttagatga cttactcaat gaccaattct atcgcctcgc tttttggaag 840

gtcggggcag aggaaatgaa ctatcgccgc ttctttactg ttaacgaatt gatttctgtg 900

aaagtagaag aaatgcgcgt gtttaacaat acccatagtt taattcacca actaatagaa 960

cagggcatat ttactggttt aagaattgac cacattgacg gactttatca acctacgcag 1020

tatttagaac gtctacgaga aaagatgggg gatgtttata tcaccgttga gaaaattttg 1080

gaattaactg aagatttacc agaaaattgg gatattcaag gtacatctgg atatgatttt 1140

ttgaattacg ttaatggggt attttgtcag tctgcgaatg aatcattgtt tgataatatt 1200

taccataaat ttattgggaa acctgtagat tatccatccc tcgttaatga gaaaaaacac 1260

ctgattttag aaaaaaattt agcgggtgat gtggataact tggcaagctt gctgaagaat 1320

attgccagca aataccgcta tggtaacgat tttacattga atgggttgaa aagagcgatc 1380

gccacagttc tgaccctatt ccccatttac cgcacttata tcacccccga tgggattgga 1440

gatagtgata aagtctgcat tcaaggagtc atcgaggctg cgaaaaaaca agtacctctc 1500

ctacatcacg agatgaactt tatagaaaaa gtaatgctgc tggagttcga tgattctctt 1560

aaccaaacag aacgggaaca atggatatat tttgtcttgc ggatgcagca atacagtggc 1620

ccccttatgg ctaaaggtgt agaagatacc accttatatg tttacaaccg tctgctttcc 1680

ttgaatgaag taggcggaaa tcccagccat ttcggtatta ctgtcgataa attccatcac 1740

tttaaccaac aacaccaagc taactggcct cacaccatga acgccacagc tacccatgac 1800

acaaagcgcg gcgaagacat gagagcaagg ttaaatgtgt tatctgaaat tccccaggag 1860

tgggaagaac agataaatct atggagccaa cttaatcaaa cacatcgtag caataaccaa 1920

cccgatcgca acgacgaata ttttctctat caaactctgg taggtgcgtt tccctttgca 1980

gaacacgaac aagcgtcctt tgtccagagg gtacaagact acatgattaa agccattcgg 2040

gaagcgaaag tacatactgc atggttacgt cccgataatg agtacgaaga agcttgtagc 2100

tcttttattg agaaagtact tgaccccaag gtatcacggc aatttttaga aacattccac 2160

ccctttcaag agaaaattgc cgaatatggc atatttaatt ctctttccca aactcttctg 2220

aaaattgctg ctcctggtgt acccgacttt taccaaggaa cagaactttg ggacttaagc 2280

ttagttgacc ctgacaaccg ccgtcccgtt gattttgaat cacgcgctgc atacttaagc 2340

accattcagg agcaaagcaa aaccgatatt ctcggcttaa tttccgaact aatccatcac 2400

aaaaccgacg gtagaatcaa actattttta acactccaag ccctcaaagc cagaacaaaa 2460

taccttgcat tattccaaga cggcgaatat ctcccgttag aaattcacgg aacccacgcc 2520

aaccatatca tcgcatttgc cagacaaaaa ggtgagcaaa ccgcgatcgc catcgccccc 2580

cgcttcctca ccaccctcac ccccccagga caaacaccac taggcgaaat ctggcaagat 2640

actcacctaa aactccccgc caaaacttgg tacaaccccc tcacccacca aaccctacaa 2700

accgaagaca ccctacccat cgcccaagcc ctcacccact tccccgtagc cctgttaatc 2760

gccgaa 2766

<210> 2

<211> 1863

<212> DNA

<213> MTHase nucleotide sequence

<400> 2

gtgacatttt tcacaacacg ttatttaaga ggaggatact gcatgaaaat tggcgctcac 60

tacttgggta atggagagtg tgaatttaca gtttggtctc caacattaaa cagcgttgca 120

gtgcaaatct taaagccaga ggaaaagtta attccgctta aagctcaagg ggagggatat 180

tggcagataa aagtaaatga tgtgtatcca ggtacgctgt atcgatatca attaaatgac 240

caagaagcct ttgctgatcc tgcttctcaa tatcaaccag aaggagttca cggggcttct 300

caagttgttg atcataaatt tgagtggaca gataaaactt ggtctggaat ttccttagaa 360

tcgatgattt tctatgaact tcatgtaggg acttttacgc ctgaaggaac ctttaccact 420

atcattcccc ggttaccaga actaagggag ttgggtatca atgccattga acttatgccg 480

atcgctcaat ttccgggtga tgatcatatt gagcctgact tagcataccg taactggggt 540

tatgacggtg tttatccgtt tgctgtccaa aattcttatg gtagccccgc agatttaaag 600

aattttgtca atgcttgtca cgaaaatggt attgctgtag tgctggatgt ggtttataac 660

cactttggcc cagaaggtaa ttatatgggt cagttcgcgc cctattttac tagaacatat 720

aagacacctt ggggcaatgc gatgaatttt gacgatgcct atagtcaagg tgtgcggaat 780

tattttattc aaaatgcgct gtattggtta ggagagttcc acatagatgg gttgcggttg 840

gatgcgattc aagcaatcta tgatttgggt gcaaagcatt tcttgtggga attggcggag 900

gttgtacata acttctccca aggaggaaca tggaaacgcc atttaattgc tgaaagtgac 960

ttgaataatc cccaaattat tcgtccggta gaatcgggtg gctatggact tgatgcacag 1020

tggagtgatg actttcacca cgcattacac gcattgttaa caggcgatcg ccaaggttat 1080

tatcaagact tcggtaagtg tgccgattta gctaaagcct acgcagatac ttttgtctat 1140

gattggcggt atgcgccaca ccgtaaacgt tttcatggta tatcatgccg cgatcgccca 1200

ttatctcagt tttcggtatg tatccaaaat catgaccaaa taggtaatca aatgcagggg 1260

gaacgcttat ctgagcgaat ttcctttgca gggttgaagt tagcggctgg tgctgtgttg 1320

ctatcgcctt atttaccgtt gttatttatg ggtgaagaat atggcgaaac tgcacctttt 1380

atatattttg tcagtcattc tgacctcgat ttgattcaag cggtacgtgc tggacgcaag 1440

gaagagtttg aagcgttcca ttatgcggaa gacccaccag accccgaatc ggcggaaact 1500

ttcctacgtt gtaaactcaa ctgggaatta cgtcatcaag gtcagcacaa ggttttatgg 1560

gattggtatc gtcagttaat tcatttacgc aaaactcatc ccgcattgct gaattatgac 1620

cgcgacaata tcgaagcaac tagtgatgag gataagcaaa tagttgtggt acggcgttgg 1680

tgtgagtcaa gggaagtgat attggcgatg aattttaata cgtctccggt gtcgctggtt 1740

ttaacaattg aaaagagtgc gcggaagtta ttagactctg ctgattctga agggtttgag 1800

acgttgtctg tgggggagga ggttgtttta cagcctacaa gtttggtttt gtatgaggtc 1860

tag 1863

<210> 3

<211> 1515

<212> DNA

<213> nucleotide sequence after TRET1 optimization

<400> 3

atggagctga acaacaaaga agatagcccc cgccatactg ttccttttgt gcgccagatc 60

accgaagatg gcaaagccaa actggaaatc taccgcccta ccaccaaccc catctacatc 120

tacacccaga tcctggccgc cattgccgtt agtatgggca gtatggtggt gggttttgcc 180

agcgcctata ctagtcccgc cttggtgagc atgcagaaca ccaccatcac cagctttaaa 240

gtgaccgaac aggaagctag ctgggtgggt ggtattatgc ccctggccgg tttagctggt 300

ggtattgctg gtggcccctt tattgaatac ctgggccgca aaaacaccat tctggccacc 360

gccgtgccct ttattgttgc ctggctgctg attgcctttg ccaacagcat ctggatggtg 420

ctggccggta gagccttgag tggtttttgc gtgggcattg ccagcttaag cctgcccgtg 480

tatctgggtg aaaccgtgca acccgaagtg cgtggtacct tgggtctgtt acccaccgcc 540

tttggcaaca tcggcatcct gatctgcttt gtggccggca aatacgtgaa ttggagcggc 600

ctggccttta ttggtagcat cctgcccatc ccctttatgg tgctgaccct gctgattcct 660

gaaacccccc gctggtttgt tactcgcggt cgcgaagaac gtgcccgcaa agctttgcaa 720

tggctgcgcg gcaaaaaagc cgatgtggaa cccgaactga aaggcatcgt gaaaagccac 780

tgtgaagctg aacgtcacgc cagccagaac gccatctttg atctgatgaa acgcagcaac 840

ctgaaacccc tgctgattgc cctgggcctg atgttttttc agcagctgag cggcattaac 900

gccgtgatct tttacaccgt gagcatcttt aaagatgccg gcagcaccat cgatgaaaac 960

ctgtgcacca tcatcgtggg cgtggtgaat tttggcgcca ccttttttgc caccgtgctg 1020

attgatcgcc tgggccgcaa aatcctgctg tacatcagcg aagtggccat ggtgatcact 1080

ctgctgaccc tgggcacctt tttttactac aagaacagcg gcaacgatgt gagcaacatc 1140

ggctggttac ccctggctag ctttgtgatc tacgtgatcg gctttagcag cggtgtgggt 1200

cctattccct ggctgatgct gggtgaaatc ctgcccggta aaattcgcgg cagcgccgcc 1260

agcgttgcta ccggttttaa ctggacctgc acctttatcg tgaccaaaac ctttgccgat 1320

atcgtggccg ccattggtaa ccatggcgcc ttttggtttt ttggcgtgat ctgcctgatc 1380

ggcctgtttt ttgtgatctt tttcgtgccc gaaacccagg gcaaaagcct ggaagaaatc 1440

gaacgcaaaa tgatgggtcg tgtgcgccgc atgagtagcg tggccaacat gaaacccctg 1500

agctttaaca tgtag 1515

<210> 4

<211> 52

<212> DNA

<213> primer SP-MTS-P1-F

<400> 4

ctcataaagt caagtaggag attaatatgc gaattcctaa agctacttat cg 52

<210> 5

<211> 53

<212> DNA

<213> primer SP-MTS-P3-R

<400> 5

tcttttgccg cagcttcttt tgccgcagct tcttcggcga ttaacagggc tac 53

<210> 6

<211> 62

<212> DNA

<213> primer SP-P3-MTH-F

<400> 6

aaagaagctg cggcaaaaga agctgcggca aaagtgacat ttttcacaac acgttattta 60

ag 62

<210> 7

<211> 53

<212> DNA

<213> primer SP-MTH-P3-R

<400> 7

tcttttgccg cagcttcttt tgccgcagct tcttcggcga ttaacagggc tac 53

<210> 8

<211> 5270

<212> DNA

<213> plasmid pZZ009

<400> 8

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accaattatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 240

ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 300

attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 360

gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagcttgcat gcctgcaggt 420

cgacgatatc actagtcata tgattaatct cctacttgac tttatgagtt gggattttct 480

taaacacaat tcccccggat aaactgaggg agtccaaagt aatgacccta gagttattgt 540

tactgatctc cattaacttt cgttaactac ccggggattt atgagagata ttacctaaat 600

aaatccaggg agaaacacgg aggcagcgac aagggccacc gggatgctca aacagctcag 660

cgcctaggct tgaatgcttt tgcaatccca cagttaactt tatacaacgg tgatgggact 720

tatgtctgtt acatcttgtt aattttattc ctgctttttt gttaagtaat gttgcagggg 780

attctcagat tgtcctggat tgggaaggga agacaaccag tttcgttcag cttatgtttt 840

agggctaaaa ttatgcaatt gatgttcggt gcgaactttt ctcgtttttt tagtttccag 900

tggggtaggg aagactgttg cctagggaac cacagcctac tttccttttt gagcttttta 960

tcccaccatt ttgatattca gggactcttc tctacaggtg ggtatagatt tgttaagttt 1020

attttataac gaaagatcta aatcttggga aaatctctag ttctgccatt catccgctta 1080

ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa 1140

aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg 1200

acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg 1260

tcgccttgcg tataatattt gcccatgaaa ataaaaaagg ggacctctag ggtccccaat 1320

taattagtaa tataatctat taaaggtcat tcaaaaggtc atccaccgga tcagcttagt 1380

aaagccctcg ctagatttta atgcggatgt tgcgattact tcgccaacta ttgcgataac 1440

aagaaaaagc cagcctttca tgatatatct cccaatttgt gtagggctta ttatgcacgc 1500

ttaaaaataa taaaagcaga cttgacctga tagtttggct gtgagcaatt atgtgcttag 1560

tgcatctaac gcttgagtta agccgcgccg cgaagcggcg tcggcttgaa cgaattgtta 1620

gacattattt gccgactacc ttggtgatct cgcctttcac gtagtggaca aattcttcca 1680

actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc tgtctagctt 1740

caagtatgac gggctgatac tgggccggca ggcgctccat tgcccagtcg gcagcgacat 1800

ccttcggcgc gattttgccg gttactgcgc tgtaccaaat gcgggacaac gtaagcacta 1860

catttcgctc atcgccagcc cagtcgggcg gcgagttcca tagcgttaag gtttcattta 1920

gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc gctggaccta 1980

ccaaggcaac gctatgttct cttgcttttg tcagcaagat agccagatca atgtcgatcg 2040

tggctggctc gaagatacct gcaagaatgt cattgcgctg ccattctcca aattgcagtt 2100

cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta 2160

cagcgcggag aatctcgctc tctccagggg aagccgaagt ttccaaaagg tcgttgatca 2220

aagctcgccg cgttgtttca tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac 2280

tgtgtggctt caggccgcca tccactgcgg agccgtacaa atgtacggcc agcaacgtcg 2340

gttcgagatg gcgctcgatg acgccaacta cctctgatag ttgagtcgat acttcggcga 2400

tcaccgcttc cctcatgatg tttaactttg ttttagggcg actgccctgc tgcgtaacat 2460

cgttgctgct ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg 2520

cccgaggcat agactgtacc ccaaaaaaac agtcataaca agccatgaaa accgccactg 2580

cgccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct 2640

acttgcatta cagcttacga accgaacagg cttatgtcca ctgggttcgt gccttcatcc 2700

gtttccacgg tgtgcgtcac ccggcaacct tgggcagcag cgaagtcgag gcatttctgt 2760

cctggctggc gaacgagcgc aaggtttcgg tctccacgca tcgtcaggca ttggcggcct 2820

tgctgttctt ctacggcaag gtgctgtgca cggatctgcc ctggcttcag gagatcggaa 2880

gacctcggcc gtcgcggcgc ttgccggtgg tgctgacccc ggatgaagtg gttcgcatcc 2940

tcggttttct ggaaggcgag catcgtttgt tcgcccagct tctgtatgga acggggatct 3000

gaaatctcta gaggatcccc gggtaccgag ctcgaattcg taatcatggt catagctgtt 3060

tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3120

gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3180

gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3240

ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3300

ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3360

cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 3420

gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3480

tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3540

ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3600

atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 3660

gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3720

tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3780

cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3840

cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 3900

tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3960

cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4020

cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4080

gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4140

gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4200

gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4260

ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4320

atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4380

agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 4440

ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 4500

tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 4560

ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 4620

caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 4680

gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 4740

atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 4800

accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 4860

aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 4920

gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 4980

tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5040

aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5100

ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5160

aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 5220

tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 5270

<210> 9

<211> 47

<212> DNA

<213> primer SPS-SPPSPH-F20

<400> 9

gcccaatctt taactgaaaa gggatttcag atccccgttc catacag 47

<210> 10

<211> 48

<212> DNA

<213> primer SPS-SPPSPH-R20

<400> 10

tttctgtgag gctgactagc gcctagacct catacaaaac caaacttg 48

<210> 11

<211> 5802

<212> DNA

<213> plasmid pQY79

<400> 11

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctgc 240

cctttgcgac actgcccgat ttggtgacgg aactggtgga gggtattccg cccgaggcaa 300

agtaccggtt ttcggcaccg gtggtgaagg cgcaagagtt tcggctagat ggattgctgg 360

agccgatcga agaaggaagc ggctatccaa cagtgttttt agaagcgcag atgcagagcg 420

atcgcggctt ttacagtcgc tactttgcag agttatttgg ttacatccgt cagtatcccg 480

aggcagccaa ttggagaggg ctattgctga tcagggcgcg atcgctggac ttaggtgaag 540

agtcgagttt tacggaactg ctgcagggac gagtgcagcg gctgtatcta gaggatttga 600

tcgggcgatc gctgaattca gtgcgattgc agttgctgaa gctattggtc gtaccagtgg 660

cagcgatagg agagacggcg agagcggtgt tgttgtcagc agaggatgag gatgcgtttg 720

agcagctact cgaactggtg gaggctatag tggcaagtaa gttgcctcag cttgagattg 780

aggagattca tcaaatgctc ggtattgaag tctccgactt aaaacaaacc cgcctctatc 840

aaagcgcggt ggaagaaggt gagaaaaagg gacgccaaga aggtgagctg gctctggtct 900

tgaggcagct gcagcggcga tttgagaact taacggaaga gcaggagcag cagattcgat 960

cgctgtcatt ggaggcgatc gaagccttga gtcttgattg gctcgacttt gagactgtcg 1020

ctgatttaaa tcgctggctc caaaaacatt aaatgcatgg gattgcaaaa aagaccttaa 1080

gcccacgcgc ttctctcagc ccgtcgtgaa aaagactgca atatcgcact gaatactttg 1140

gctgcccgct caagtggaat ggtcaagatt ggcaatcgct acctattgca gcaacgcgat 1200

cgcccaatct ttaactgaaa aggactagtg cgctagtcag cctcacagaa aacgccccca 1260

tcctagtctc cgatccgaaa aggtggggtg gtagcgtagg ggcgttgcat ggctggtatc 1320

gatgttttcg agccgctcac caaggattat gggcccgcgc cgacccgcga tcgcgatcgc 1380

cctcggtctg ctgctgttag ctttcctgat cttggtgggg ttgagtcttg gctcctcaac 1440

aagtctgatg tctcacgatg agggctatta cgccctgcag gcccgttgga ttgtggaaac 1500

gggtgattgg gtaacgccgc gctggtggca agagccactc tacgaccgca caatcggggt 1560

gcaatggctg atcgctgcga gttacaagct gtttggcttc tgcaccactg ccgtccgcct 1620

accggctttg ctcagtggac tggcaacgct ctggttgacc tttgcgattg gcgatcgcct 1680

cttgcctcgt ccccaagccc tgttggcggc gggcattctg ctagtgacgc ccctttggtt 1740

tcagtacgcg caactagcaa cccaagatat gccgttgcta gcggtcgagt tgctctcgat 1800

ttgggcgctc ctacaagccg tctcgggcga tcgccgagct aatctctggg gctttgtggc 1860

gggtttgggg gttggccttg gctttttgat caaaggcttc atgattggcg tgccactgct 1920

tgcgatcgct ccttggtttt tctggtatgc gccgaagcta ctgcgcaatc gtggcctctg 1980

gcttggcctc atcgtcggct ggattccggt cgggatttgg ctctggggca gtcagcagcg 2040

ctggggtgat ctcgcgatcg cccaactctt cgacaaattt ttctttctgg ccagcgaaga 2100

tctctacagc cagccttgga ctttctacct ctggaacttg ccgctcaatg ctttcccatg 2160

gccactgttt gggctaattg gctgggttcg cctctggctg cgaccggaac gcgatcaagg 2220

gcagcttcaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac 2280

gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 2340

tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 2400

gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 2460

tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 2520

cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 2580

tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 2640

tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 2700

cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 2760

ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 2820

gatttaacaa aaatttaacg cgaattttaa caaaattcag ggcgcaaggg ctgctaaagg 2880

aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc 2940

tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt 3000

gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg 3060

ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc 3120

ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac aggatgagga 3180

tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 3240

aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 3300

cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 3360

aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 3420

gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 3480

ccggggcagg atctcctgtc atcccacctt gctcctgccg agaaagtatc catcatggct 3540

gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 3600

aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 3660

ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 3720

atgcccgacg gcgaggatct cgtcgtgacc cacggcgatg cctgcttgcc gaatatcatg 3780

gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 3840

tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 3900

gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 3960

cgccttcttg acgagttctt ctgaattgaa aaaggaagag tatgagtatt caacatttcc 4020

gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 4080

cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 4140

tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 4200

tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 4260

agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 4320

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 4380

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 4440

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 4500

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 4560

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 4620

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 4680

ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 4740

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 4800

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 4860

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 4920

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 4980

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 5040

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 5100

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 5160

cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 5220

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 5280

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5340

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5400

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5460

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5520

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5580

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 5640

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 5700

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 5760

gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag 5802

<210> 12

<211> 42

<212> DNA

<213> primer 0808-T-Flong

<400> 12

ggatgaggat gcgtttgagc agctactcga actggtggag gc 42

<210> 13

<211> 37

<212> DNA

<213> primer 0808-T-Rlong

<400> 13

agcgatcagc cattgcaccc cgattgtgcg gtcgtag 37

<210> 14

<211> 24

<212> DNA

<213> primer NS3-T1-F

<400> 14

tgcgaacctt ctccagcttc agcc 24

<210> 15

<211> 24

<212> DNA

<213> primer NS3-T1-R

<400> 15

ggcggcaatc atcgaagcca gtga 24

<210> 16

<211> 30

<212> DNA

<213> P3 connecting peptide sequence

<400> 16

gaagctgcgg caaaagaagc tgcggcaaaa 30

Claims (9)

1. A genetically engineered bacterium for producing trehalose, wherein the genetically engineered bacterium is obtained by over-expressing a maltooligosyl trehalose synthase (MTse) gene and a maltooligosyl trehalose hydrolase (MTHase) gene by cyanobacteria; the cyanobacteria is Synechococcus;

the nucleotide sequence of the MTse gene is shown as SEQ ID NO. 1;

the nucleotide sequence of the MTHase gene is shown as SEQ ID NO. 2.

2. The genetically engineered bacterium of claim 1, further over-expressing a trehalose transporter TRET1 gene;

the nucleotide sequence of the TRET 1gene is shown as SEQ ID NO. 3.

3. The genetically engineered bacterium of claim 1, wherein the synechococcus is synechococcus PCC7942 or synechococcus Sye7942-C252Y.

4. A method of constructing a genetically engineered bacterium as claimed in any one of claims 1 to 3, characterized by the steps of:

(1) Constructing a recombinant plasmid carrying MTase and MTHase genes;

(2) And (3) introducing the recombinant plasmid obtained in the step (1) into host bacteria to obtain genetically engineered bacteria, wherein the host bacteria are cyanobacteria.

5. The method of claim 4, further comprising the steps of constructing a recombinant plasmid carrying a TRET 1gene, and introducing all of the recombinant plasmid carrying a TRET 1gene and the recombinant plasmid carrying MTase and MTHase genes into a host cell to obtain the genetically engineered bacterium.

6.The method according to claim 4, wherein the MTase and MTHase genes in the recombinant plasmid in the step (1) are linked by a P3 connecting peptide gene, and the P3 connecting peptide gene is shown as SEQ ID NO. 16.

7. Use of a genetically engineered bacterium according to any one of claims 1-3 for the production of trehalose.

8. The use according to claim 7, characterized in that the specific steps for the production of trehalose are as follows:

(1) Culturing genetically engineered bacteria: culturing genetically engineered bacteria in column type photoreactor, inoculating concentration OD ₇₃₀ 0.1, the culture temperature is 28-30deg.C, and the light intensity is 100-200 μm photo ns m ^-2 s ^- The gas used for culturing is 3% CO ₂ A mixture with 97% air;

(2) Transporter TRET1 induced expression: culturing the engineering bacteria in the step (1) until the 4 th day, adding isopropyl thiogalactoside (IPTG) to induce the expression of a transport protein TRET1, and adding the IPTG to induce for 24 hours to separate and purify the trehalose.

9. The use according to claim 8, wherein the cultivation in step (1) is in BG11 medium without antibiotics at 30℃and with a light intensity of 150. Mu. Mol photons m ^-2 s ^-1 。

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