阅读说明：本技术 一种重组质粒和重组工程菌及其构建方法和提高d-阿洛酮糖的产量的应用 (Recombinant plasmid, recombinant engineering bacterium, construction methods of recombinant plasmid and recombinant engineering bacterium, and application of recombinant plasmid and recombinant engine) 是由 谭丹 刘禹佳 房欣蕾 樊沛瑶 余韫 卢晓云 于 2020-05-08 设计创作，主要内容包括：本发明公开了一种重组质粒和重组工程菌及其构建方法和提高D-阿洛酮糖的产量的应用,属于酶定向进化技术领域。本发明设计了定向进化的重组质粒,该定向进化的重组质粒便可视作一基因电路,即DTE酶效率的基因电路。与此同时,并本发明还公开了基于上述重组质粒构建得到的重组工程菌,具体为大肠杆菌E.coli DH5αpSB1C3-psiR-pPsi-EGFP-cp-kanR-DTE。本发明的上述基因电路使用阻遏蛋白PsiR与其对应启动子pPsi实现了D-阿洛酮糖的生物传感器；使得菌体可感应体内D-阿洛酮糖的生产量以表达不同水平的抗生素抗性基因,从而将DTE酶的效率转化为菌体的生存优势。(The invention discloses a recombinant plasmid, a recombinant engineering bacterium, a construction method thereof and application of the recombinant plasmid and the recombinant engineering bacterium in improving the yield of D-psicose, and belongs to the technical field of enzyme directed evolution. The invention designs the recombinant plasmid of directed evolution, and the recombinant plasmid of directed evolution can be regarded as a gene circuit, namely the gene circuit of DTE enzyme efficiency. Meanwhile, the invention also discloses a recombinant engineering bacterium constructed based on the recombinant plasmid, in particular to escherichia coli E.coli DH5 alpha pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE. The gene circuit realizes a D-psicose biosensor by using the repressor protein PsiR and the corresponding promoter pPsi; the cells can sense the production of D-psicose in vivo to express antibiotic resistance genes at different levels, thereby converting the efficiency of DTE enzyme into the survival advantage of the cells.)

1. A recombinant plasmid is characterized in that a skeleton sequence is inserted into a pSB1C3 vector plasmid, and a coding sequence of a D-psicose-induced repressor psiR factor, a promoter pPsi induced by D-psicose, a fluorescent protein EGFP coding sequence, a coupling element cp and a kanamycin resistance gene fragment kanR are sequentially carried on the skeleton or inserted into a restriction endonuclease site, so that the plasmid pSB1C3-psiR-pPsi-EGFP-cp-kanR is obtained; and then the DTE enzyme library is connected into the skeleton by using restriction endonuclease to obtain a recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE.

2. The recombinant plasmid of claim 1 wherein the nucleotide sequence of the backbone is as set forth in sed.id.no. 1; the nucleotide sequence of the D-psicose induced repressor psiR factor is shown in SED.ID.NO. 2; the sequence of the fluorescent protein EGFP, the coupling element cp and the kanamycin resistance gene fragment kanR is shown in SED.ID.NO. 3; the original sequence of the DTE enzyme library is shown in sed.id.no. 4.

3. The recombinant plasmid of claim 1, wherein the D-psicose-inducible repressor psiR factor gene is derived from Agrobacterium tumefaciens, iGEM element No. BBa _ K2448006; the DTE zymogen start sequence is derived from Pseudomonas cichororii, iGEM element number BBa _ K2791019.

4. The recombinant plasmid of claim 1 wherein the D-psicose-inducible promoter, pPsi, is a. tumefactens, where the native promoter is hybridized to the strong promoter, pTac, and the iGEM element is numbered BBa _ K2448016; the RBSs downstream of pPsi and pLac are both strong RBSs, iGEM element number BBa _ B0034.

5. The method for constructing a recombinant plasmid according to any one of claims 1 to 4, comprising the steps of:

(1) ligating the backbone into the vector pSB1C3 using restriction enzymes to obtain plasmid pSB1C 3-pPsi;

(2) connecting a D-psicose induced repressor psiR sequence, a fluorescent protein EGFP, a coupling element cp and a kanamycin resistance gene fragment kanR into a skeleton in sequence by adopting a Golden Gate connection mode to obtain a plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR;

(3) the DTE enzyme library is connected into a framework by using restriction enzymes in a BioBrick assembly mode to obtain a recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE.

6. The recombinant engineering bacterium constructed based on the recombinant plasmid of any one of claims 1 to 4, wherein the recombinant engineering bacterium is Device-D-cp0-w/o-dte, which is preserved in China general biological center of culture Collection of microorganisms with the strain preservation number of CGMCC NO. 18931.

7. The use of the recombinant engineered bacterium of claim 6 in the production of D-psicose, wherein the recombinant engineered bacterium can be directed evolved to improve the efficiency of the DTE enzyme in producing D-psicose.

8. The method for directionally evolving the DTE enzyme by using the recombinant engineered bacterium as claimed in claim 6, wherein the recombinant engineered bacterium Device-D-cp0-w/o-DTE is transferred gradually into a solid medium applied with D-fructose or kanamycin (2 μ g/ml) for culture after inducing the expression of the DTE enzyme in LB medium, and the surviving colonies are screened to prepare for the library construction of the next round.

9. The method for directed evolution of DTE enzymes by recombinant engineered bacteria of claim 8, wherein library construction comprises: surviving colonies were collected, plasmid extracted after washing with buffer, and the library was subsequently amplified with primer pairs complementary to the DTE coding sequence.

10. The method for the directed evolution of DTE enzymes by recombinant engineered bacteria of claim 8, comprising the steps of:

1) inoculating the recombinant engineering bacterium Device-D-cp0-w/o-dte into an LB culture medium in an inoculation amount of 2%, and adding chloramphenicol and IPTG for culture;

2) collecting bacterial liquid, centrifuging, resuspending, coating on a solid culture medium plate, culturing at 37 deg.C for 12 hr, transferring thallus Porphyrae to another solid LB culture medium, and adding kanamycin; wherein the solid culture medium is formed by adding 15g/L agar powder and 50g/L fructose into an MM culture medium;

3) picking the surviving colony on the solid culture medium and culturing in a shake bacteria tube added with LB culture medium, adding chloramphenicol, and then washing and extracting plasmid;

4) the DTE library was amplified using a PCR system constructed with specific primers complementary to the DTE coding sequence.

Technical Field

The invention belongs to the technical field of enzyme directed evolution, and relates to a recombinant plasmid, a recombinant engineering bacterium, a construction method of the recombinant plasmid and the recombinant engineering bacterium, and application of the recombinant plasmid and the recombinant engineering bacterium in improving the yield of D-psicose.

Background

D-psicose is a rare monosaccharide occurring in a small amount in nature, and is a C-3 epimer of fructose. As a rare sugar, the sugar has good taste and no bitter aftertaste, and is a novel sweetener. At a 10% dilution, D-psicose is 70% of the sweetness of sucrose, but only 0.3% of its calories. Is an ideal substitute for sucrose. The D-psicose can inhibit intestinal canal alpha-glycosidase and liver lipase production, and inhibit fat accumulation. The D-psicose has blood sugar inhibiting effect. Supplementation of the diet with psicose can relieve postprandial blood glucose elevation and enhance insulin sensitivity. Animal experiments also report that D-psicose has protective effects on type II diabetes. Compared with D-fructose and D-glucose, the D-psicose has higher reducibility and is easier to initiate Maillard reaction so as to adjust the color of the pastry and increase the flavor. The D-psicose also has a neuroprotective effect and has potential medical value for neurodegenerative diseases. In the mouse experiment, D-psicose was absorbed by the small intestine but was hardly metabolized in the body, and was excreted with urine. In the mouse experiments, long-term administration of 3% psicose did not produce significant side effects. D-psicose was approved by the FDA as "GRAS" (generally regarded as safe) in 2011.

Industrial production of D-psicose generally uses a biological fermentation method due to very low natural yield and complicated chemical production process. The family of D-tagatose epimerase (EC 5.1.3.31, D-tagatose-3-epimerase, DTE enzyme) catalyzes the conversion between D-fructose and D-psicose, and is a key enzyme for industrial production of D-psicose. The DTE enzyme family comprises several sources of enzymes, which vary in their substrate optima, enzyme efficiency, pH and temperature optima, and metal ion cofactors. Among them, the first one was found to be derived from Pseudomonas cichororii ST-24 strain, whose optimum substrate was D-tagatose, and thus was named DTE enzyme. Subsequently discovered DTE enzyme family members derived from Agrobacterium tumefaciens specifically catalyze the conversion between D-fructose and D-psicose and are named DPE enzymes (D-psicose epimerase). The DTE enzyme family is also found and characterized in Rhodobacterium sphaeroides SK011, Clostridium cellulolyticum H10 and other strains. The natural DTE enzyme without modification is used for producing D-psicose by taking D-fructose as a substrate, the equilibrium conversion rate is about 30 percent, and therefore, the yield has larger promotion space. And the optimum pH of the natural DTE enzyme is alkalescent, but the fermentation production of D-psicose requires an acidic environment to reduce the occurrence of Maillard reaction, so how to engineer the optimum pH of the DTE enzyme is an important direction.

Synthetic biology is a subject of research on engineering of biological systems. The subject focuses on introducing concepts in rational design, system modeling and engineering, such as modularization, standardized design, etc., into traditional genetic engineering. Directed evolution and biosensors are two important enabling technologies in synthetic biology. Directed evolution mimics Darwinian natural selection for the optimization of a particular biological property, such as an enzyme, a metabolic pathway, or even the optimization of the entire organism. The advantage of directed evolution is that the characteristics of the system are optimized under the condition of insufficient knowledge of the target system, thereby greatly accelerating the speed of the cycle of synthetic biology design, test and construction. However, the construction mode, the phenotype presentation mode, the screening flux and the like of the mutation library in the directed evolution often restrict the application of the technology; how to screen the most suitable individual from a diverse population is one of the core problems of directed evolution. In recent years, the development of synthetic biology techniques and concepts has allowed researchers to construct more complex engineering bacteria and engineering cells than ever before. With the increase of complexity of engineering bacteria or engineering cells, the inserted DNA fragments, such as plasmids, etc., become more complex, and they tend to be composed of several modular DNA fragments which have been reported in research. Thus, these inserted DNA fragments or inserted plasmids inspired by synthetic biology are often referred to figuratively as "genetic circuits".

According to different screening platforms, the directed evolution can be divided into in vivo, in vitro and in vitro modes. With the in vivo approach closest to darwinian evolution. MAGE (multiplex automated genome engineering) is an in vivo directed evolution method that utilizes the Red recombination system of lambda phage to recombine single-stranded DNA libraries into host genomes. The method greatly improves the capacity of the mutant library, but lacks an efficient screening means and is easy to cause the waste of the library. PACE (phage accelerated continuous evolution) is a milestone directed evolution technology. The technology can complete continuous multi-round (hundreds of rounds) directed evolution without manual intervention, greatly improves the efficiency of evolution, and realizes the originally intractable evolution target. However, the technology requires continuous culture, has high control requirement on the flowing of a culture system, and is easy to cause the failure of evolution due to complete washing of phage. At the same time, this technique uses in vivo mutagenesis and does not allow effective control of library formation. In addition to these novel approaches, directed evolution can also be performed using traditional methods. If the production efficiency of a certain enzyme is directionally evolved by using a traditional method, quantitative methods such as HPLC (high performance liquid chromatography) and the like are often used for detecting the activity of the enzyme in each mutant strain, and the method is time-consuming and labor-consuming and has low screening flux. If the phenotype to be evolved can be characterized by a fluorescent protein, the mutants can be screened traditionally in 96-well plates, thereby boosting the screening throughput to 10²Orders of magnitude, but still far from meeting the needs of library screening. The invention converts the enzymatic activity of DTE enzyme to be optimized into the survival advantage of thalli through the biosensor, so that antibiotics with certain concentration can be directly used for screening on an agar plate, and the flux can reach 10⁸The method is of order of magnitude, simple to operate and does not require continuous culture and other technologies.

The living being is receiving the environmental signal and responding. Evolution has conferred different properties to these response mechanisms: combine different signal molecules and even be optical and electric signals; with appropriate thresholds for triggering the corresponding cellular responses. When these naturally occurring signal response mechanisms are applied to artificially constructed genetic circuits, they are called biosensors. Many biosensors have transcriptional regulation as a mechanism: the induced promoter binds to different activators and repressors under the action of different environmental signals to change the transcription level of downstream genes. In addition, there are biosensors of translation level or posttranslational level, etc. Biosensors often play a central role in signal receiving and signal transmitting in synthetic biology.

With the development of new synthetic biology techniques and new concepts in recent years, it is becoming possible to apply biosensors to the directed evolution of DTE enzymes to improve the production activity of DTE enzymes. With the continuous expansion of synthetic biological communities, the size of induced promoter databases is also increasing. From this database, a D-psicose-inducible promoter was selected and an antibiotic resistance gene was ligated downstream thereof. This gene line was transferred into a strain producing D-psicose, so that the production of D-psicose in the strain could be characterized by the production of antibiotic resistance protein. An error-prone PCR and PCR shuffling technology is used for constructing a DTE enzyme gene library in vitro, and the DTE enzyme gene library is cloned into a gene circuit. Strains carrying at least a certain enzyme efficiency can be quantitatively selected by inducing the expression of DTE enzyme and subsequently culturing the strains carrying the library with different concentrations of the corresponding antibiotic. The DTE enzyme in the surviving strain was amplified, and the in vitro library construction method described above was repeated and screened. Thus, the efficiency of DTE enzyme can be repeatedly improved.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a recombinant plasmid and a recombinant engineering bacterium, a construction method thereof and application of the recombinant plasmid and the recombinant engineering bacterium in improving the yield of D-psicose.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a recombinant plasmid, wherein a skeleton sequence is inserted into a pSB1C3 vector plasmid, and the skeleton is sequentially provided with or inserted into a coding sequence of a D-psicose induced repressor psiR factor, a promoter pPsi induced by D-psicose, a fluorescent protein EGFP coding sequence, a coupling element cp and a kanamycin resistance gene fragment kanR at a restriction endonuclease site to obtain the plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR; and connecting the DTE enzyme library into the skeleton by using restriction endonuclease to obtain a recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE.

Preferably, the nucleotide sequence of the backbone is as set forth in sed.id.no. 1; the nucleotide sequence of the D-psicose induced repressor psiR factor is shown in SED.ID.NO. 2; the sequence of the fluorescent protein EGFP, the coupling element cp and the kanamycin resistance gene fragment kanR is shown in SED.ID.NO. 3; the original sequence of the DTE enzyme library is shown in sed.id.no. 4.

Preferably, the D-psicose-induced repressor psiR factor gene is derived from Agrobacterium tumefaciens, and the iGEM element is numbered BBa _ K2448006; the DTE zymogen start sequence is derived from Pseudomonas cichorii, iGEM element number BBa _ K2791019.

Preferably, the promoter pPsi induced by D-psicose is formed by hybridizing a natural promoter in A.tumefaciens and a strong promoter pTac, and the iGEM element is numbered as BBa _ K2448016; the RBSs downstream of pPsi and pLac are both strong RBSs, iGEM element number BBa _ B0034.

The invention also discloses a construction method of the recombinant plasmid, which comprises the following steps:

(1) ligating the backbone into the vector pSB1C3 using restriction enzymes to obtain plasmid pSB1C 3-pPsi;

(2) connecting a D-psicose induced repressor psiR sequence, a fluorescent protein EGFP, a coupling element cp and a kanamycin resistance gene fragment kanR into a skeleton in sequence by adopting a Golden Gate connection mode to obtain a plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR;

(3) the DTE enzyme library is connected into a framework by using restriction enzymes in a BioBrick assembly mode to obtain a recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE.

The invention also discloses a recombinant engineering bacterium constructed based on the recombinant plasmid, wherein the recombinant engineering bacterium is Device-D-cp0-w/o-dte, is preserved in the China general biological center of the Committee for culture Collection of microorganisms, and has the strain preservation number of CGMCC NO. 18931.

The invention also discloses application of the recombinant engineering bacteria in production of D-psicose, and the recombinant engineering bacteria can be subjected to directed evolution to improve the efficiency of producing D-psicose by using DTE enzyme.

The invention also discloses a method for directionally evolving the DTE enzyme by utilizing the recombinant engineering bacteria, which comprises the steps of inducing the DTE enzyme expression of the recombinant engineering bacteria Device-D-cp0-w/o-DTE in an LB culture medium, gradually transferring the recombinant engineering bacteria Device-D-cp0-w/o-DTE into a solid culture medium respectively applied with D-fructose or 2 mu g/ml kanamycin for culture, and screening surviving colonies to prepare for library construction of the next round.

Further, library construction includes: surviving colonies were collected, plasmid extracted after washing with buffer, and the library was subsequently amplified with primer pairs complementary to the DTE coding sequence.

Further, the method specifically comprises the following steps:

1) inoculating the recombinant engineering bacterium Device-D-cp0-w/o-dte into an LB culture medium in an inoculation amount of 2%, and adding chloramphenicol and IPTG for culture;

2) collecting bacterial liquid, centrifuging, resuspending, coating on a solid culture medium plate, culturing at 37 deg.C for 12 hr, transferring thallus Porphyrae to another solid LB culture medium, and adding kanamycin; wherein the solid culture medium is formed by adding 15g/L agar powder and 50g/L fructose into an MM culture medium;

3) picking the surviving colony on the solid culture medium and culturing in a shake bacteria tube added with LB culture medium, adding chloramphenicol, and then washing and extracting plasmid;

4) the DTE library was amplified using a PCR system constructed with specific primers complementary to the DTE coding sequence.

Further, the specific steps are as follows:

1) inoculating 2% of recombinant engineering bacteria Device-D-cp0-w/o-dte into a 200mL conical flask filled with 100mL LB culture medium, adding 100mg/L chloramphenicol and 0.2g/L IPTG into the conical flask, and culturing for 10 hours at 37 ℃ and 200 rpm/min;

2) collecting 6mL of bacterial liquid, centrifuging and resuspending to 200. mu.L, coating on a solid culture medium plate, culturing at 37 ℃ for 12 hours after coating, and then transferring the lawn to another solid LB culture medium to which kanamycin with the concentration of 2. mu.g/mL is added; after a plurality of rounds of evolution, the concentration of kanamycin in the culture medium can be properly increased; wherein the solid culture medium is formed by adding 15g/L agar powder and 50g/L fructose into an MM culture medium;

3) surviving colonies on the solid medium were picked and cultured in a shake tube containing LB medium to which 100mg/L chloramphenicol was added. The plasmid was then extracted by washing twice with 1 × PBS. The DTE library was amplified using a PCR system constructed with specific primers complementary to the DTE coding sequence.

Compared with the prior art, the invention has the following beneficial effects:

the invention designs the recombinant plasmid of directed evolution by applying the synthetic biology idea, and the recombinant plasmid of directed evolution can be used as a gene circuit, namely the gene circuit for improving the efficiency of DTE enzyme. Meanwhile, the invention also discloses a recombinant engineering bacterium constructed based on the recombinant plasmid, in particular to escherichia coli E, coli DH5 alpha pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE. The gene circuit realizes a D-psicose biosensor by using the repressor protein PsiR and the corresponding promoter pPsi; the cells can sense the production of D-psicose in vivo to express antibiotic resistance genes at different levels, thereby converting the efficiency of DTE enzyme into the survival advantage of the cells. Compared with single screening, the process of constructing the library, screening the strain carrying the high-efficiency DTE, and constructing the library again based on the high-efficiency strain can obtain a more high-efficiency DTE sequence. The method has the following advantages:

1. the characteristics of the DTE enzyme can be optimized under the condition that the research on the active site of the DTE enzyme and the action mechanism of the DTE enzyme is insufficient;

2. in vitro library construction was used. Increased control over library construction compared to in vivo mutations;

3. the antibiotic and the antibiotic resistance gene are used for applying screening pressure, and compared with continuous culture methods such as PACE (phage accelerated continuous evolution) and the like, the operation difficulty is reduced;

4. the plasmid construction process is a hierarchical design, so that elements can be replaced conveniently to promote the directional evolution platform;

5. the directions of adjacent transcription units are opposite so as to reduce the risk of read-through and reduce the probability of modifying a gene circuit by homologous recombination of thalli under pressure;

6. the plasmid is loaded with a copy of a transcription factor LacI to supplement the expression of genome LacI and reduce the leakage expression of pLac downstream genes;

7. the sensitivity of the D-psicose biosensor is enhanced by using a promoter hybridized with pTac by the D-psicose-induced promoter pPsi;

8. a translational coupling element is linked upstream of the antibiotic resistance gene. This element reduces the expression level of the resistance gene and increases the sensitivity of the bacterial cells to survival stress.

Strain preservation

The invention discloses a recombinant engineering bacterium, which is named as Device-D-cp0-w/o-dte, is suggested to be classified and named as Escherichia coli, is preserved in the common biological center of China Committee for culture Collection of microorganisms at 11, 8 and 2019, and is addressed to No.3 Hokko No.1 of North Chen West Lu of the Yangyang district in Beijing City of China, and the preservation number of the strain is CGMCC No. 18931.

Drawings

FIG. 1 is a schematic diagram of the structure of a recombinant plasmid of the present invention;

FIG. 2 is a flow chart of the method for improving the efficiency of DTE enzyme by using the directed evolution platform;

FIG. 3 is a technical scheme for the construction of the recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE;

FIG. 4 shows the results of an orthogonality test for the sense circuitry portion of the exemplary embodiment to detect pPsi;

FIG. 5 is a graph showing the results of an experiment in which the selection circuit portion detects the interaction of kanamycin and a kanamycin resistance protein in an embodiment;

FIG. 6 shows the results of an embodiment of the coupling circuit portion in detecting the relationship between RFP, EGFP fluorescence intensity and IPTG concentration; (a) and (b) the variation of the fluorescence intensity of RFP (a) and GFP (b) with the concentration of inducer IPTG in the bacterial population containing the coupled circuit after induction; (c) correlation of RFP expressed by bacteria with GFP fluorescence intensity;

FIG. 7 shows the results of an experiment in which the evolution circuit portion of the embodiment detects the change in fluorescence intensity of E.coli cells containing DTE enzymes of different activities.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the invention discloses a construction method of a recombinant plasmid, which comprises the following steps:

(1) the backbone for the directed evolution platform plasmid was synthesized and digested and ligated with the vector pSB1C3 using the restriction enzymes EcoRI and SpeI. Wherein the skeleton is sequentially connected with lacI gene, pPsi promoter, RBS and terminator, pLac promoter, RBS and terminator, BbsI site, BsaI site and the like. Wherein the lacI gene expresses LacI repressor protein for complementing genome-expressed LacI. The pPsi promoter is repressed by the repressor PsiR and is expressed by induction with the inducer D-psicose. The pLac promoter is expressed under IPTG induction. The recombinant plasmid pSB1C3-pPsi was obtained. The promoter pPsi induced by D-psicose is formed by hybridizing a natural promoter in Agrobacterium tumefaciens and a strong promoter pTac, and the iGEM element is numbered BBa _ K2448016. The RBSs downstream of pPsi and pLac (ribosome binding site) are both strong RBSs, and the iGEM element number BBa _ B0034. Both the BbsI site and BsaI site were used for Golden Gate assembly in subsequent steps.

(2) The sequence of the psiR gene with BsaI site was synthesized, and the PsiR repressor encoding gene was inserted into pSB1C3-pPsi by the GoldGate assembly method using BsaIIs type restriction enzyme. The PsiR repressor gene is derived from Agrobacterium tumefaciens, and the iGEM element is numbered BBa _ K2448006. The recombinant plasmid pSB1C3-psiR-pPsi was obtained.

(3) And (3) amplifying the green fluorescent protein EGFP fragment and the antibiotic resistance gene fragment by PCR, and introducing BbsI sites into the two fragments. A green fluorescent protein EGFP coding gene, a translation coupling element cp and a kanamycin resistance gene kanR are simultaneously inserted into pSB1C3-psiR-pPsi by a Golden Gate assembly method by using BbsIIs type restriction endonuclease to obtain a recombinant plasmid pSB1C 3-psiR-pPsi-EGFP-cp-kanR. Wherein the green fluorescent protein iGEM element is numbered BBa _ K2791050.

(4) The recombinant plasmid pSB1C3-psiR-pPsi-EGFP-cp-kanR-DTE was obtained by ligating into a DTE enzyme library in a BioBrick assembly manner using SpeI and PstI restriction enzymes. Wherein the DTE zymogen initial sequence is derived from Pseudomonas cichorii, and the iGEM element number is BBa _ K2791019.

The nucleotide sequence of the framework is shown in SED.ID.NO. 1; the nucleotide sequence of the D-psicose induced repressor psiR factor is shown in SED.ID.NO. 2; the sequence of the fluorescent protein EGFP, the coupling element cp and the kanamycin resistance gene fragment kanR is shown in SED.ID.NO. 3; the original sequence of the DTE enzyme library is shown in sed.id.no. 4.

The method for transforming the plasmid pSB1C3-psiR-pPsi-EGFP-cp-kanR-DTE with the directed evolution function into E.coli to obtain the strain Device-D-cp0-w/o-DTE comprises the following steps:

add 10. mu.l ligation product to 100. mu.l competent cells, mix gently, and stand on ice for 30 min. The tube was heat-shocked at 42 ℃ for 90 s. The centrifuge tube was quickly placed on ice and allowed to cool for 2 min. 900. mu.l of SOC medium without antibiotics was added to each tube and incubated at 37 ℃ for 1h on a shaker. The cells were centrifuged at a low speed, about 800. mu.l of the supernatant was discarded, and the remaining 200. mu.l of the cell suspension was applied directly to LB solid culture plates containing 100. mu.g/ml chloramphenicol. The plate was placed in the forward direction at 37 ℃ until the liquid was absorbed, and the plate was incubated at 37 ℃ for 18-24 h. Selecting single clone for enzyme digestion verification, and sending the single clone to the Scophthiria organism for sequencing identification.

Referring to FIG. 2, the invention also discloses a DTE enzyme directed evolution method based on the recombinant Escherichia coli strain disclosed above. Recombinant E.coli strain E.coli DH 5. alpha. pSB1C3-psiR-pPsi-EGFP-cp-kanR-DTE obtained after ligation of the library by BioBrick assembly. After DTE enzyme expression is induced in LB culture medium, gradually transferring into solid culture medium respectively applied with D-fructose or 2 mug/ml kanamycin for culture, and collecting survival colonies; the subsequent washing with buffer and extraction of plasmid, and final amplification of the library with primer pairs complementary to the DTE coding sequence.

The method comprises the following steps of inducing and expressing a DTE enzyme in an LB culture medium by using an Escherichia coli strain E.coli DH5 alpha pSB1C3-psiR-pPsi-EGFP-cp-kanR-DTE which realizes an oriented evolution platform, wherein the specific operation comprises the following steps: the above strain was inoculated into a 200mL Erlenmeyer flask containing 100mL of LB medium at an inoculum size of 2%. The Erlenmeyer flask was further charged with 100mg/L chloramphenicol and 0.2g/L IPTG. The culture was carried out at 37 ℃ and 200rpm/min for 10 hours. The solid culture medium is transferred into the culture medium for culture, and the specific operation is as follows:

6mL of the broth was collected, centrifuged, resuspended to 200. mu.L, and plated onto solid media plates. The solid culture medium is composed of MM culture medium added with 15g/L agar powder and 50g/L fructose. After coating, the cells were incubated at 37 ℃ for 12 hours. Subsequently, the lawn was transferred to another solid LB medium to which kanamycin was added at a concentration of 2. mu.g/ml.

Screening surviving colonies and constructing the library again, and specifically operating as follows:

surviving colonies were picked from the center and cultured in a shake tube with LB medium added to 100mg/L chloramphenicol. Specific primers are used to form a PCR system, and the DTE enzyme coding sequence is amplified after thalli are cracked at 95 ℃.

The platform set up by the invention can also be used for directed evolution of other enzymes. When directed evolution techniques are used to improve the properties of an enzyme, researchers often desire to convert the quantitative phenotype of the expression of the enzyme, e.g., the amount of product produced by the enzyme, into a strain's survival advantage. Based on the above example of improving DTE enzyme activity, the invention discloses a general strategy for converting the amount of a certain enzyme product into the survival advantage of a strain based on the directed evolution gene circuit. Referring to fig. 3, the specific implementation is:

(1) for a certain enzyme to be optimized, Enz encodes Enz. Biosensors exist that respond to their enzymatic reaction products. This biosensor consists of a repressor Rep (coding sequence Rep) induced by this product and an inducible promoter pRep that binds to and is repressed by the Rep.

(2) For the backbone plasmid pSB1C3, the induced repressor coding sequence rep and its corresponding promoter pRep were ligated into the BsaI site by Golden Gate assembly to obtain plasmid pSB-rep-pRep.

(3) A green fluorescent protein coding sequence EGFP, a translation coupling element cp and a kanamycin resistance gene kanR are simultaneously connected to the BbsI site of the plasmid pSB1C3-rep-pRep in a Golden Gate assembly mode to obtain the plasmid pSB1C 3-rep-pRep-EGFP-cp-kanR.

(4) A library of coding sequence enz was constructed using error-prone PCR and ligated into the library in a BioBrick assembly using an XbaI cleavage site and a PstI cleavage site. Obtaining the plasmid pSB1C 3-rep-pRep-EGFP-cp-kanR-enz.

(5) The promoter strength and the promoter threshold were different for different induced promoters pRep. The appropriate kanamycin concentration is selected accordingly and the library-carrying strains are screened for this antibiotic concentration to obtain the enzyme Enz which is optimized in yield.

The invention aims to improve the enzyme activity of the DTE, and designs an automatic, high-flux and repeatable direct evolution platform to ensure that the DTE can be conveniently self-evolved. In order to design a gene circuit for directionally evolving DTE enzyme efficiency and achieve the aim of representing the yield of D-psicose by the expression quantity of antibiotic resistance protein, the invention designs 4 genetic units of the gene circuit in total, and tests each unit to ensure that the function of finally screening DTE with high enzyme activity can be realized, wherein the four units are respectively an induction circuit, a selection circuit, a coupling circuit and an evolution circuit.

1. Induction circuit

The sensing circuit converts the concentration of D-psicose into the expression level of downstream gene EGFP. The circuit plasmid is pSB1C3-psiR-pPsi-EGFP, the circuit is based on a repressor PsiR and an inducible promoter pPsi corresponding to the repressor PsiR, and various regulating elements are designed in the circuit to realize stable biosensing. The repressor, inducible promoter and downstream elements in this universal loop have different enzyme cleavage sites, the repressor and inducible promoter use two different Golden Gate recognition sites to achieve seamless ligation of the regulatory system, and the downstream elements are ligated using standard bioBrick assembly.

To test the efficiency of both pPsi and PsiR, we performed two experiments of orthogonality of pPsi and efficiency of pPsi. The results of the experiment are shown in FIG. 4. FIG. 4 shows that the fluorescence intensity detected by the experimental groups for different types of sugars is different, whereas the fluorescence intensity of the allulose group is always higher than that of the other sugar experimental groups. Thus, it can be concluded that: the pPsi promoter is specific for D-psicose and insensitive to other sugars.

The efficiency test of pPsi aims at detecting the effect of the allulose concentration on the expression intensity of pPsi, and the results show that the fluorescence intensity induced by 50mM allulose is the highest, and the higher the allulose concentration is, the stronger the fluorescence intensity will be. We can conclude that the efficiency of pPsi and the allulose concentration in the medium are positively correlated.

2. Selection circuit

The selection circuit converts the expression level of the antibiotic resistance gene into different growth rates of the thallus under the condition of gradient concentration antibiotic, and takes the fluorescence intensity as a quantitative index. The circuit plasmid is pSB1C 3-lacI-pLac-EGFP-kanR. The circuit characterizes three antibiotic resistance proteins including kanamycin, streptomycin and ampicillin, and finally kanamycin is selected due to the wider dynamic range of kanamycin.

We have designed a series of experiments to characterise the function of the selection circuit, exemplified by the selection circuit-KanR, the results of which are shown in FIGS. 5 (a) and (b), and show that when the concentration of IPTG is within a certain range, the final OD600 of the bacteria will decrease with increasing kanamycin concentration. This means that if the expression level of KanR is high, the bacteria have a high probability of survival because higher IPTG concentrations can induce more KanR expression. In addition, bacteria expressing more KanR survived more.

3. Coupling circuit

The coupling circuit controls the expression level of both ends of the stem-loop through a coupling element cp so as to improve the sensitivity to the survival pressure of the antibiotic. The plasmid of the circuit is pSB1C 3-lacI-pLac-RFP-cp-EGFP. This part is designed to take advantage of the helicase activity of the ribosome, which will unwind the hairpin between the two coding sequences for expression of the downstream sequence when the ribosome approaches the end of the upstream coding sequence. The length of the hairpin corresponds to the optimal distance the ribosome can unwind. By this hairpin structure, the level of downstream conversion can be correlated with upstream quantitation. The problem solved by this coupling element is one of the most critical problems in directed evolution and synthetic biology: how to control the ratio of input to output.

To test whether downstream translation levels correlate quantitatively with upstream expression by hairpin structure. We designed the experiment, and the results are shown in FIG. 6 (a), (b) and (c). The results show that the fluorescence intensity of GFP is obviously stronger than that of RFP, and the linear relation of the fluorescence intensity of GFP and RFP can also be preliminarily determined.

4. Evolutionary circuit

The evolutionary circuit summarizes all the regulatory units used above and quantitatively converts the DTE enzyme activity into the anti-pressure growth capacity of the strain in an antibiotic environment with a specific concentration. The circuit plasmid is pSB1C 3-psiR-pPsi-EGFP-cp-kanR-DTE. By taking advantage of the properties of the selection and coupling circuit, we obtained the appropriate antibiotic concentration to exert growth pressure during evolution.

We know that the activity of each enzyme in the DTE enzyme family is different and that the DTE enzyme activity is significantly higher than the DPE enzymes. In order to make the system really play a role, DTE enzyme and DPE enzyme with obvious difference in enzyme activity are respectively used for constructing evolution circuit plasmids and converting the evolution circuit plasmids into escherichia coli.

First, according to the sensing circuit, the efficiency of pPsi and the concentration of psicose in the medium are positively correlated. Second, if the activity of DTE is higher, more psicose in the medium results in more gene expression. Third, the fluorescence intensity is qualitatively correlated with the expression of the resistance gene according to the coupling circuit. To characterize the expression of the antibiotic resistance gene, we designed an experiment and the results are shown in figure 7. FIG. 7 shows that the antibiotic gene expressed in E.coli containing DTE is always higher than DPE, which means that the activity of DTE is higher than DPE. This demonstrates that our system can distinguish between DTEs with different activities, i.e. that the system is indeed efficient.

Kanamycin is preferred over other antibiotics according to the selection circuit. We set up three kanamycin concentration gradients and measured the changes in E.coli cell growth at these concentrations. The results show that the bacterial concentration gradually decreases with increasing antibiotic concentration. I.e. increasing antibiotic concentration means increasing the evolution pressure, the inhibition of bacterial growth is also more pronounced. All the above experimental results show that the evolutionary circuit can work normally.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

<110> university of west ampere traffic

<120> recombinant plasmid, recombinant engineering bacterium, construction methods thereof and application of recombinant plasmid and recombinant engineering bacterium in improving yield of D-psicose

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1648

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gaattcgcgg ccgcttctag aggagatcta cactagcact atcagagtta ttaagctact 60

aaagcgtagt tttcgtcgtt tgcagcctgc ccgctttcca gtcgggaaac ctgtcgtgcc 120

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg 180

gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc 240

tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg 300

atggtggtta acggcgggat ataacatgag ctatcctcgg tatcgtcgta tcccactacc 360

gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc 420

atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg 480

gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt 540

tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt 600

aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc 660

agtcgcgtac catcctcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca 720

tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca 780

tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc 840

gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt 900

tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg 960

gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg 1020

ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg 1080

tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg 1140

acatcgtata acgttactgg tttcacattc accattagtt ttctcctctt tggcgctatc 1200

atgccatacc gcgaaaggtt ttgcaccagg cacgtaagag gttccaactt tcaccataat 1260

gaaactcaca caggaaagga gaccgtacca tggtctccgc aaaaaacccc gcttcggcgg 1320

ggttttttcg ctataaacgc agaaaggccc acccgaaggt gagccagtgt gactctagta 1380

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 1440

tcgttttatt tgatgcctgg gtcttcactg tttgaagact ttctcctctt tttgcaccat 1500

cgattgtgca atccacacat tatacgagcc gatgattaat tgtcaacagc tcaaattgtg 1560

agcggataac aattgacatt gtgagcggat aacaagatac tgagcacaga attgtaaaga 1620

ggagaaatac tagtagcggc cgctgcag 1648

<210>2

<211>1108

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ggtctcggaa agactaatga ccggtatctc ttctaaaaaa gctaccatct acgacctgtc 60

tatcctgtct ggtgcttctg cttctaccgt ttctgctgtt ctgaacggtt cttggcgtaa 120

acgtcgtatc tctgaagaaa ccgctgacaa aatcctgtct ctggctaaag ctcagcgtta 180

caccaccaac ttacaggctc gtggtctgcg ttcttctaaa tctggtctgg ttggtctgct 240

ggttccggtt tacgacaacc gtttcttctc ttctatggct cagaccttcg aaggtcaggc 300

tcgtaaacgt ggtctgtctc cgatggttgt ttctggtcgt cgtgacccgg aagaagaacg 360

tcgtaccgtt gaaaccctga tcgcttactc tatcgacgct ctgttcatcg ctggtgttac 420

cgacccggac ggtgttcacc aggtttgcgc tcgtgctgct ctgccgcacg ttaacatcga 480

cctgccgggt aaattcgctt cttctgttat ctctaacaac cgtcacggtg ctgaaatcct 540

gaccgctgct atcctggctc acgctgctaa aggtggttct ctgggtccgg acgacgttat 600

cctgttcggt ggtcacgacg accacgcttc tcgtgaacgt atcgacggtt tccacgctgc 660

taaagctgac tacttcggtg ttgaaggtgg tgacgacatc gaaatcaccg gttactctcc 720

gcacatgacc gaaatggctt tcgaacgttt cttcggtcgt cgtggtcgtc tgccgcgttg 780

cttcttcgtt aactcttcta tcaacttcga aggtctgctg cgtttcatgg gtcgtcacga 840

cggtgaagct ttcggtgaca tcgttgttgg ttgcttcgac tacgacccgt tcgcttcttt 900

cctgccgttc ccggtttaca tgatcaaacc ggacatcgct cagatgctgg aaaaaggttt 960

cgaactgctg gaagaaaacc gtaccgaacc ggaagttacc atcatcgaac cgcagctgat 1020

cccgccgcgt accgctctgg aaggtccgct ggacgacatc tgggacccgg ttgctctgcg 1080

tcgtatggct aaataacgca aagagacc 1108

<210>3

<211>744

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gaagacagga gaaaactaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 60

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 120

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 180

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 240

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 300

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 360

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 420

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 480

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 540

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 600

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 660

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 720

gacgagctgt acaagtaagt cttc 744

<210>4

<211>914

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gaattcgcgg ccgcttctag atgaacaagg tgggcatgtt ttatacctat tggagcaccg 60

aatggatggt ggattttccg gcgaccgcga aacgtattgc gggcctgggc tttgatctga 120

tggaaattaa cctggaggag tttcataacc tggcggatgc gaaaaaacgc gaactgaaag 180

cggtggcgga tgatttaggc ttaaccgtga tgtgctgcat tggcctgaaa agcgaatatg 240

attttgcgag cccggataaa agcgttcgtg atgcgggcac cgaatatgtg aaacgcctgc 300

tggatgattg ccatttactg ggtgcgccgg tttttgcggg cctgaacttt tgtgcgtggc 360

cgcagcatcc tcctctggat atggtggata aacgcccgta tgtggatcgc gcgattgaat 420

cagttcgccg cgtgattaaa gtggcggaag actatggcat tatttatgcg ctggaagtgg 480

tgaaccgcta tgaacagtgg ctgtgcaacg atgcgaaaga agcgattgcg tttgcggatg 540

cggttgatag cccggcgtgc aaagttcagc tggatacctt tcatatgaac atcgaggaaa 600

acagctttcg cgatgcgatt ctggcgtgca aaggcaaagt gggccatttt catattggcg 660

aacagaaccg cttacctcct ggtgaaggcc gtttaccgtg ggatgaaatt tttggcgcgc 720

tgaaagaaat tggctatgat ggcaccattg cgatggaacc gtttatgcgc accggtggtt 780

cagttggccg cgatgtttgt gtttggcgcg atctgtcaaa tggcgcgacc gatgaagaaa 840

tggatgaacg cgcgcgtcgt agcttacagt ttgtgcgcga taaattagcg tgatactagt 900

agcggccgct gcag 914