Efficient gene transcription activation system and application thereof

Abstract

The invention relates to an efficient gene transcription activation system and application thereof. The system comprises a positive regulatory gene, a DNA sequence which is located at the upstream of the positive regulatory gene and is combined with a positive regulatory protein expressed by the positive regulatory gene, and a corresponding promoter of the DNA sequence. According to the system, a UAS sequence combined with a positive regulatory protein expressed by a positive regulatory gene and a corresponding promoter are utilized to express the positive regulatory gene, the just synthesized positive regulatory protein can be immediately combined to an upstream UAS sequence, and the positive regulatory protein interacts with a sigma 54 factor to stimulate the expression of the downstream positive regulatory gene of the corresponding promoter, so that more positive regulatory proteins are synthesized, and the positive regulatory protein is expressed by the corresponding promoter. A positive feedback transcriptional regulation expression cycle is formed. The invention provides a gene transcription activation system taking the nifA gene as a positive regulation gene, so that the enzyme activity of nitrogenase and the yield of indole-3-acetic acid and prodigiosin are effectively improved. The invention further provides a gene transcription activation system taking the slnR gene as a positive regulation gene, and the salinomycin yield is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an efficient gene transcription activation system and application thereof. Background Art

[0002] Organisms adapt to complex external environments by regulating their own gene expression. There are many ways to regulate gene expression, including regulation at the transcriptional level, regulation at the translational level, and post-translational modification. Transcriptional regulation is usually one of the most important and effective ways to regulate gene expression. Bacterial transcription initiation is a highly regulated process involving the interaction between RNA polymerase and promoter. Sigma factors determine the promoter sequence specificity of RNA polymerase and the efficiency of transcription initiation. In addition to the housekeeping sigma factor responsible for recognizing most promoters, bacteria have also evolved selective sigma factors to guide RNA polymerase to different promoters. σ 54 Alternative sigma factor-mediated transcription initiation requires an activator protein that typically binds to an activation sequence (UAS) upstream of the promoter.

[0003] Natural products from microorganisms are usually produced by the joint action of multiple genes (biosynthetic gene clusters) that exist in clusters in the genome. These natural products are a huge treasure trove of biological resources. Generally, there is at least one regulatory protein in these biosynthetic gene clusters, among which positive regulatory proteins are often favored by researchers. The traditional method is to overexpress positive regulatory genes through strong promoters to increase the yield of corresponding natural products. This traditional method usually has limited effect on the increase of natural product yields, and a new gene transcription activation method is urgently needed to maximize the synthesis of target products. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a highly efficient gene transcription activation system and its application.

[0005] The technical solution of the present invention is as follows:

[0006] A highly efficient gene transcription activation system comprises a positive regulatory gene, a DNA sequence (UAS) located upstream of the positive regulatory gene and bound by a positive regulatory protein expressed by the positive regulatory gene, and a corresponding promoter (P).

[0007] Preferably, according to the present invention, the positive regulatory gene is the nifA gene, and the DNA sequence (UAS) located upstream of the nifA gene and bound by the positive regulatory protein (NifA) expressed by the nifA gene and the corresponding promoter (P) are UAS-P nifH 、UAS-P nifE 、UAS-PnifB 、UAS-P nifU 、UAS-P rnf or UAS-P nifF .

[0008] Further preferably, the Genbank ID of the nifA gene is 12528487 (locus_tag: PSTAA_RS06800); the DNA sequence bound by the positive regulatory protein and its corresponding promoter UAS-P nifH 、UAS-P nifE 、UAS-P nifB 、UAS-P nifU 、UAS-P rnf and UAS-P nifF The sequences are shown in SEQ ID NO.1 to 6.

[0009] Preferably, according to the present invention, the positive regulatory gene is the slnR gene, and the DNA sequence (UAS) located upstream of the slnR gene and bound by the positive regulatory protein (SlnR) expressed by the slnR gene and the corresponding promoter (P) are UAS-P slnQ 、UAS-P slnA1 、UAS-P slnF 、UAS-P slnT1 、UAS-P slnB3 or UAS-P slnC .

[0010] Further preferably, the Genbank ID of the slnR gene is 54209321 (locus_tag: SLNWT_RS01160); the DNA sequence bound by the positive regulatory protein and its corresponding promoter UAS-P slnQ 、UAS-P slnA1 、UAS-P slnF 、UAS-P slnT1 , UAS-P slnB3 and UAS-P slnC The sequences are shown in SEQ ID NOs. 7 to 12 respectively.

[0011] The application of the above gene transcription activation system in improving the activity of nitrogenase.

[0012] Application of the above gene transcription activation system in increasing the yield of indole-3-acetic acid.

[0013] The application of the gene transcription activation system in improving the production of prodigiosin.

[0014] The application of the above gene transcription activation system in improving the production of salinomycin.

[0015] The present invention also provides a recombinant strain, which contains the gene transcription activation system.

[0016] The steps not described in detail in the present invention are all performed according to the prior art.

[0017] Technical features and beneficial effects of the present invention:

[0018] 1. The present invention provides an efficient gene transcription activation system, which expresses the positively regulated gene by utilizing the UAS sequence and its corresponding promoter bound by the positively regulated protein expressed by the positively regulated gene. The newly synthesized positively regulated protein can immediately bind to the upstream UAS sequence and activate the gene through the σ 54 The interaction of factors stimulates the expression of positive regulatory genes downstream of the corresponding promoter, thereby synthesizing more positive regulatory proteins, forming a positive feedback transcriptional regulation expression cycle. This positive feedback transcriptional regulation expression cycle can synthesize a large amount of positive regulatory proteins, thereby promoting the synthesis of corresponding natural products. In addition, the present invention is a novel and efficient gene transcription activation strategy. Any UAS sequence and its corresponding promoter region upstream of a gene containing a positive regulatory protein and the target of the positive regulatory protein is a positive regulatory protein. Then the efficient gene transcription activation strategy of the present invention can play a role, providing a simple and effective method for the efficient synthesis of certain natural products.

[0019] 2. The present invention provides a gene transcription activation system using the nifA gene as a positive regulatory gene, the system contains 6 UAS sequences regulated by the positive regulatory protein (NifA) expressed by the nifA gene and its corresponding promoter, and the nitrogenase activity expressed by the gene transcription activation system reaches 368 times the activity of the wild-type nitrogenase. The present invention also successfully utilizes the gene transcription activation system to efficiently synthesize indole-3-acetic acid (IAA) and prodigiosin, and increases the yield of these two products.

[0020] 3. The present invention provides a gene transcription activation system using the slnR gene as a positive regulatory gene, the system contains 6 UAS sequences regulated by the positive regulatory protein (SlnR) expressed by the slnR gene and their corresponding promoters. The present invention also successfully used the gene transcription activation system to increase the production of salinomycin by 3.19 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of NifA's efficient gene transcription activation strategy;

[0022] Figure 2 Schematic diagram of DNA construction for feasibility verification of NifA efficient gene transcription activation system;

[0023] In the figure, P12445 is a strong constitutive promoter screened in P. stutzeri DSM4166, P nifH is the promoter of the nitrogen fixation gene nifH (encoding nitrogenase ferritin), UAS ( u pstream a Activating s sequence) is P nifH Promoter upstream activation sequence.

[0024] Figure 3 The recombinant plasmids p15A-apra-oriT-phiC31-P12445-firefly, p15A-apra-oriT-phiC31-P nifH -firefly and p15A-apra-oriT-phiC31-P nifH -Firefly-nifA enzyme digestion verification electrophoresis results;

[0025] Figure 4 The results of luciferase activity detection of different recombinant bacteria are shown in Figure 2.

[0026] In the figure, P12445 represents the endogenous strong constitutive promoter P12445 overexpressing the nifA gene; -nifA represents the expression of nifA gene only by UAS-P nifH The promoter controls the expression of firefly, and +nifA represents that the NifA efficient gene transcription activation system controls the expression of firefly. The efficient gene transcription activation system was verified under normal air conditions (aerobic) and nitrogen-fixing conditions (microaerobic).

[0027] Figure 5 Schematic diagram of the structure of the nif gene cluster.

[0028] Figure 6 For the promoter UAS-P nifB , UAS-P rnf , UAS-P nifE , UAS-P nifU , UAS-P nifF Luciferase activity detection results.

[0029] Figure 7 Schematic diagram of nitrogenase activity assay and characterization of different recombinant bacteria;

[0030] In the figure, WT is the wild-type strain of P. stutzeri DSM4166, P12445 represents the recombinant strain P12445-nifA overexpressing the nifA gene with the strong constitutive promoter P12445, and UAS-P nifH, UAS-P nifE , UAS-P nif , UAS-P nifU , UAS-P rnf Represent different recombinant bacteria UAS-P nifH -nifA,UAS-P nifE -nifA,UAS-P nifB -nifA,UAS-P nifU -nifA and UAS-P rnf -nifA.

[0031] Figure 8 The tryptophan-dependent synthesis pathway (IAM pathway) of IAA selected for the present invention: IaaM: tryptophan 2-monooxygenase, IaaH: IAM-specific amidohydrolase;

[0032] Fig. 9 Schematic diagram of the synthesis pathway for heterologous synthesis of IAA using the NifA efficient transcriptional activation system;

[0033] In the figure, IAM-head, IAM-middle, and IAM-tail represent the positions of nifA in the synthesis pathway, respectively. nifH represents the control group without nifA, IAM-P12445 represents the strong constitutive promoter overexpressing the IAM pathway;

[0034] Fig.10 is the yield of IAA in the fermentation broth of different recombinant bacteria;

[0035] Fig.11 This is a line graph showing the changes in IAA production in the fermentation broth of the recombinant bacteria IAM-head after culture medium optimization under different concentrations of L-Trp as substrate.

[0036] Fig.12 Schematic diagram of the direct cloning of the prodigiosin biosynthetic gene cluster.

[0037] Fig.13 This is the electrophoresis gel image of the prodigiosin biosynthesis gene cluster after direct cloning.

[0038] Fig.14 This is a schematic diagram of the modification of the prodigiosin biosynthesis gene cluster;

[0039] In the figure, Pig-1 is only UAS-P nifH The control group of the promoter, Pig-2 is the control group in which the P12445 strong promoter overexpressed the entire prodigiosin biosynthesis gene cluster, and Pig-3 is the experimental group in which the NifA efficient transcriptional activation system was used to express the entire prodigiosin biosynthesis gene cluster;

[0040] Fig.15 This is a diagram of the color changes of different recombinant bacteria after successful construction.

[0041] Fig.16 The bar graph is a graph showing the production of prodigiosin in fermentation broths of different recombinant bacteria;

[0042] Fig.17 This is a bar graph of prodigiosin production in the fermentation broth of the recombinant strain Pig-3 after fermentation medium optimization.

[0043] Fig.18 The salinomycin biosynthesis gene cluster and the corresponding positive regulatory protein SlnR and its regulated location (the location indicated by the dotted arrow).

[0044] Fig.19 Schematic diagram for constructing the SlnR efficient gene transcription activation system.

[0045] Fig. 20 The BPC diagram and the corresponding secondary mass spectrum of S.albus DSM41398 detected by LC-MS after fermentation;

[0046] Fig.21 This is a bar graph showing the production of salinomycin after fermentation of different strains;

[0047] In the figure, WT represents the fermentation yield of the wild-type strain DSM41398; kasOp* represents the fermentation yield of the recombinant kasOp*-slnR; Q, A1, F, T1, B3, and C represent the fermentation yield of the recombinant UAS-P slnQ -slnR,UAS-P slnA1 -slnR,UAS-P slnF -slnR,UAS-P slnT1 -slnR,UAS-P slnB3 -slnR and UAS-P slnC -slnR fermentation yield.

[0048] Fig. 22 The results of qPCR determination of slnR gene transcription levels in different strains.

[0049] In the figure, WT represents the slnR transcription level of the wild-type strain DSM41398; kasOp* represents the slnR transcription level of the recombinant strain kasOp*-slnR; T1 represents the recombinant strain UAS-P slnT1 -slnR transcript levels.

[0050] Fig.23 It is a schematic diagram of the efficient gene transcription activation system of the present invention.

[0051] In the figure, UAS stands for upstream activation sequence and PTR stands for gene encoding positive transcriptional regulatory protein.

[0052] All bar graphs and line graphs in the above figures are calculated by standard deviations of 3 groups of parallel experiments. Results of the significant difference analysis of the mean values: *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0053] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are provided in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0054] The Pseudomonas stutzeri (DSM4166) and Streptomyces albus (DSM41398) are available from DSMZ / German Collection of Microorganisms. Serratia marcescens (ATCC13880) is available from ATCC / American Type Culture Collection.

[0055] Example 1: Construction of NifA efficient gene transcription activation system

[0056] 1. Nitrogen fixation positive regulatory gene nifA (Genbank ID 12528487) is a pathway-specific positive regulatory gene in the nitrogen fixation gene cluster (nif) from Pseudomonas stutzeri (DSM4166). Its expression product NifA protein can bind to the UAS region upstream of the promoter of each nif gene (such as nifH gene) in the nif gene cluster ( u pstream a Activating s NifA protein can bind to nifH promoter (P nifH ) Upstream UAS binds and catalyzes σ 54 The conformational change of 54 -RNA polymerase holoenzyme switches from a closed state to an open promoter complex with high transcriptional activity, thereby enhancing the transcription of the nifH gene.

[0057] 2. If Figure 1 As shown, UAS-P nifH The background expression of NifA can synthesize a small amount of NifA protein; this small amount of NifA protein positively regulates its own UAS-P nifH, synthesizing more NifA protein, thus forming a positive feedback transcriptional regulation cycle, and then synthesizing a large amount of NifA protein.

[0058] 3. Based on this feature, we designed and verified the NifA efficient gene transcription activation system

[0059] like Figure 2 As shown in the figure, three groups of expression cassettes were constructed. The first group was the overexpression of the firefly gene controlled by the endogenous strong constitutive promoter P12445 of DSM4166, which was P12445-firefly; the second group was the overexpression of the firefly gene controlled by the endogenous strong constitutive promoter P12445 of DSM4166, which was P12445-firefly; nifH (SEQ ID NO.1) controlled firefly gene, as a detection of UAS-P nifH The control group of the original transcription intensity of the promoter is P nifH -firefly; the third group is based on the second group and adds the positive regulatory gene nifA downstream of the firefly gene, which is P nifH -firefly-nifA. The three groups of expression cassettes were constructed into the p15A-apra-oriT-phiC31 vector to obtain the recombinant plasmids: p15A-apra-oriT-phiC31-P12445-firefly, p15A-apra-oriT-phiC31-P nifH -firefly and p15A-apra-oriT-phiC31-P nifH -firefly-nifA.

[0060] The specific construction method is as follows:

[0061] (1) Using p15A-apra-oriT-phiC31-amp-ccdB (SEQ ID NO. 13) as a template, PCR amplification was performed to obtain the vector fragments p15A-apra-oriT-phiC31, promoter P12445, promoter UAS-P nifH , firefly gene and nifA gene; the 40bp homology arm is introduced by PCR primers, and the PCR is completed by ApexHFHS DNA polymerase premix-FS (Aikerui Bio) kit, and the specific operations and conditions are carried out according to the instructions of the kit; the required primers are shown in Table 1.

[0062] (2) The PCR fragments were recovered by gel extraction and then recombined in Escherichia coli GB05-dir expressing RecE, RecT, Redγ and RecA by Red / ET homologous recombination technology according to the gene combinations of the three groups. After extraction, the recombinant plasmids p15A-apra-oriT-phiC31-P12445-firefly, p15A-apra-oriT-phiC31-P nifH -firefly and p15A-apra-oriT-phiC31-P nifH -firefly-nifA; then the recombinant plasmid p15A-apra-oriT-phiC31-P12445-firefly was double-digested with AfeI and MfeI to verify that p15A-apra-oriT-phiC31-P nifH -firefly and p15A-apra-oriT-phiC31-P nifH -firefly-nifA was double-digested with AfeI and PstI, and the results of enzyme electrophoresis were as follows Figure 3 As shown. Figure 3 It can be seen that the recombinant plasmid was successfully constructed.

[0063] (3) In order to avoid the influence of the nifA gene and the related promoter regulated by nifA in the nif gene cluster, the nif gene cluster was knocked out by homologous recombination: 1 kb sequences upstream and downstream of the chloramphenicol resistance gene cm and the nif gene cluster (HAL and HAR, as homologous arms for homologous recombination) were amplified by PCR, and then the three fragments were amplified by tandem PCR to obtain the HAL-cm-HAR fragment. In P. stutzeri DSM4166 containing the recombinase (pBBR1-kan-Rha-BAS-redγ), the nif gene cluster was knocked out by homologous recombination using the cm gene to obtain the nif knockout strain DSM4166-nifko;

[0064] In order to eliminate the influence of the integration position of the expression cassette on the expression of the target gene, the expression cassette was site-specifically integrated by attB / attP site-specific recombination. Since the wild-type DSM4166 genome does not contain attB site, the attB site was integrated into the specific position PSTAA_RS19520 on the genome by single exchange, and the nif gene cluster was knocked out and Pseudomonas stutzeri containing attB site (DSM4166-nifko-attB) was obtained. The specific method is as follows: firstly, the pK18mob vector containing 40bp homologous arms and 1kb single exchange homologous arms (PSTAA_RS19520) were obtained by PCR amplification. The 40bp homologous arms were introduced by PCR primers, and then the attB site knock-in plasmid (pK18mob-19520-attB, kanamycin resistance) was constructed by Red / ET homologous recombination technology. The P. stutzeri was used for conjugation transfer. The DSM4166 endogenous recombination system inserted the plasmid into the wild-type DSM4166 and DSM4166-nifko without the attB site by single crossover to obtain Pseudomonas stutzeri containing the attB site (DSM4166-attB) and Pseudomonas stutzeri with the nif gene cluster knocked out and containing the attB site (DSM4166-nifko-attB). The required primers are shown in Table 2. The vector template is pK18mobSacB, and the Genebank ID is FJ437239.1.

[0065] (4) Through conjugation transfer and attB / attP site-specific recombination, the recombinant plasmids p15A-apra-oriT-phiC31-P12445-firefly and p15A-apra-oriT-phiC31-P nifH -firefly and p15A-apra-oriT-phiC31-P nifH -firefly-nifA was integrated into the genome of Pseudomonas stutzeri (DSM4166-nifko-attB) with the nif gene cluster knocked out and containing the attB site, and the recombinant bacteria P12445-firefly, UAS-P nifH -firefly and UAS-P nifH -firefly-nifA.

[0066] Table 1. Primers used to verify the feasibility of constructing a NifA efficient gene transcription activation system

[0067]

[0068]

[0069] In the table: the underlined sequences are homology arms used for recombination, and the lowercase letters are artificially added ribosome binding (RBS) regions.

[0070] Table 2. Primers required for constructing DSM4166-nifko and attB site knock-in plasmid pK18mob-19520-attB

[0071]

[0072] In the table: the underlined sequences are homology arms used for tandem PCR or recombination. The attB sequence is short (90 bp) and therefore introduced on the primer.

[0073] 4. Firefly luciferase activity detection

[0074] The recombinant bacteria P12445-firefly, UAS-P nifH -firefly and UAS-P nifH -firefly-nifA were cultured under normal air conditions (aerobic), and the Firefly luciferase activity in the bacterial solution was determined; then the recombinant bacteria P nifH -firefly and P nifH -firefly-nifA were cultured under nitrogen-fixing conditions (microaerobic) and the Firefly luciferase activity in the bacterial solution was measured. The results are as follows Figure 4 shown.

[0075] The normal air condition is aerobic overnight culture in LB medium at 30°C; the nitrogen fixation condition is slightly aerobic overnight culture in Medium 460 at 30°C (99% argon + 1% oxygen). Single-Luciferase (Firefly) Reporter Assay Kit) was used to measure the Firefly luciferase activity, and the specific operation and conditions were carried out according to the instructions of the kit. The composition of the medium Medium 460 was: K2HPO4 0.5g / L, MgSO4×7H2O 0.2g / L, NaCl 0.1g / L, Yeast extract 0.2g / L, FeCl3×6H2O 15mg / L, Na2MoO4×2H2O 12mg / L, DL-Na-malate 6.6g / L, pH 7.0.

[0076] Depend on Figure 4 It can be seen that the second group of UAS-P nifHThe original expression intensities of the three groups were very weak (the luciferase activity of the -nifA bar graph was low), while the luciferase activity of the third group using the efficient gene transcription activation system was significantly improved (the luciferase activity of the +nifA bar graph was very high), and its activity was much higher than the luciferase activity of the first group under the control of the endogenous strong constitutive promoter P12445. This shows that the efficient gene transcription activation system of the present invention can effectively promote the expression of Firefly luciferase. Under normal air conditions (aerobic), the third group (UAS-P nifH , + nifA) was 42 times higher than that of the first group (P12445) and 42 times higher than that of the second group (UAS-P nifH , -nifA) is more than 1000 times.

[0077] Example 2: NifA high-efficiency gene transcription activation system significantly improves nitrogenase activity

[0078] 1. If Figure 5 As shown, except for the promoter UAS-P nifH In addition, there is the promoter UAS-P nifB (SEQ ID NO.3), UAS-P rnf (SEQ ID NO.5), UAS-P nifE (SEQ ID NO.2), UAS-P nifU (SEQ ID NO.4) and UAS-P nifF (SEQ ID NO.6) is regulated by the positive regulatory protein expressed by the nifA gene, and its feasibility is verified respectively. The results are as follows Figure 6 As shown, the method is the same as that for verifying UAS-P nifH Consistent under normal air conditions (aerobic).

[0079] Depend on Figure 6 It can be seen that the promoter UAS-P nifB 、UAS-P rnf 、UAS-P nifE 、UAS-P nifU and UAS-P nifF Both can be used to construct a NifA efficient gene transcription activation system.

[0080] 2. According to the method described in Example 1, promoter P12445, UAS-P nifH 、UAS-P nifE 、UAS-P nifB 、UAS-P nifU and UAS-P rnfPlaced upstream of the nifA gene, the recombinant plasmids p15A-apra-oriT-phiC31-P12445-nifA and p15A-apra-oriT-phiC31-P nifH -nifA, p15A-apra-oriT-phiC31-P nifE -nifA, p15A-apra-oriT-phiC31-P nifB -nifA, p15A-apra-oriT-phiC31-P nifU -nifA and p15A-apra-oriT-phiC31-P rnf -nifA, the required primers are shown in Table 3. Then the above expression cassette plasmid was integrated into the genome of Pseudomonas stutzeri (DSM4166-attB) containing the attB site by conjugation transfer and attB / attP site-specific recombination to obtain the recombinant bacteria P12445-nifA, UAS-P nifH -nifA、UAS-P nifE -nifA、UAS-P nifB -nifA,UAS-P nifU -nifA,UAS-P rnf -nifA.

[0081] Table 3. Primers required for constructing a NifA efficient gene transcription activation system to improve nitrogenase activity

[0082]

[0083]

[0084] In the table: the underlined sequences are the homology arms used for recombination.

[0085] 3. For wild Pseudomonas stutzeri (P. stutzeri DSM4166), recombinant bacteria P12445-nifA, UAS-P nifH -nifA,UAS-P nifE -nifA,UAS-P nifB -nifA,UAS-P nifU -nifA and UAS-P rnf -nifA was used to determine the nitrogenase activity. The specific method is as follows:

[0086] (1) The above strains were inoculated into 1.8 mL of LB liquid medium, wherein, except for the wild-type Pseudomonas stutzeri (P. stutzeri DSM4166) which was inoculated into a non-resistant LB liquid medium, the remaining recombinant bacteria were inoculated into an LB liquid medium containing apramycin, and then cultured overnight at 30° C. and 950 rpm;

[0087] (2) Collect the cells by centrifugation at 4°C and 6000 rpm for 5 min, wash once with Medium 460, resuspend the cells in 1 ml of Medium 460, measure OD600, and dilute to OD 600 =1.0;

[0088] (3) Add 18 ml of Medium 460 to the anaerobic flask (100 ml) and 2 ml of OD 600 = 1.0 bacterial solution, mix well;

[0089] (4) A mixed gas (99% argon + 1% oxygen) was passed into the bottle for 3 min, and then the bottle cap was tightly closed and cultured at 30°C and 200 rpm for 16 h;

[0090] (5) Then, 10 ml of gas was taken out from the anaerobic bottle and 10 ml of acetylene gas was injected into it. The culture was continued at 30 °C and 200 rpm for 4 h.

[0091] (6) The gas in the bottle was taken for gas chromatography detection (KB-Al2O3 / Na2SO4 capillary column, length: 30.0 m, inner diameter: 0.53 mm, film thickness 20.0 μm; vaporization chamber temperature: 100°C; chromatographic column program: 100°C, 5 min; FID detector);

[0092] (7) After GC detection, the protein content of the bacterial solution was measured, and the nitrogenase activity was characterized by ethylene production / protein amount / time. Protein concentration determination method: 1) Centrifuge to collect the bacteria in the anaerobic bottle, wash with ddH2O, and discard the supernatant; 2) Add 500μl 0.5MNaOH solution to resuspend, boil for 5 minutes to lyse the bacteria; 3) Add 500μl 0.5M HCl to mix, and centrifuge at 13900rpm for 10 minutes; 4) Take the supernatant and dilute it appropriately to determine the protein concentration (Coomassie Brilliant Blue standard curve method). The results of nitrogenase activity determination are as follows: Figure 7 shown.

[0093] Depend on Figure 7 It can be seen that the nitrogenase activity of the recombinant bacteria P12445-nifA constructed by the traditional strong constitutive promoter overexpression gene method is 23 times higher than that of the wild-type strain; while the recombinant bacteria UAS-P nifH-nifA、UAS-P nifE -nifA、UAS-P nifB -nifA、UAS-P nifU -nifA and UAS-P rnf The nitrogenase activity of -nifA was increased by 109 to 368 times compared with the wild-type strain, and by about 5 to 16 times compared with the P12445-nifA group. The experimental results show that the efficient gene transcription activation system of the present invention has obvious advantages over the traditional method of overexpressing genes with strong promoters.

[0094] Example 3: NifA high-efficiency gene transcription activation system efficiently synthesizes indole-3-acetic acid and prodigiosin

[0095] 1. The NifA efficient gene transcription activation system of the present invention is similar in function to the strong constitutive promoter, but is more efficient and can divert the synthetic resources in the host to the promoter regulated by the positive regulatory protein. In addition to the host's original synthetic pathway being greatly overexpressed, it is also expected that the NifA efficient gene transcription activation system can have a better expression effect on the exogenous synthetic pathway. Therefore, we selected two synthetic pathways of exogenous compounds to explore the application performance of the efficient gene transcription activation system of the present invention.

[0096] 2. Select a synthetic pathway for indole-3-acetic acid (IAA) that is relatively simple in its synthetic pathway. IAA is the most common and important plant hormone that can promote plant growth. If IAA can be efficiently synthesized through the strategy of the present invention, a multifunctional bacterial agent that can both efficiently fix nitrogen and promote plant growth can be obtained. The IAA synthetic pathway used in the present invention is a tryptophan-dependent indole-3-acetamide (IAM) synthetic pathway (e.g. Figure 8 The pathway uses L-tryptophan (L-Trp) as the starting compound, which is oxidized to an IAM intermediate under the catalysis of tryptophan 2-monooxygenase (encoded by iaaM); IAM is then hydrolyzed to IAA by an IAM-specific amidohydrolase (encoded by iaaH).

[0097] The specific synthesis and determination are as follows:

[0098] (1) The iaaM gene (Genbank ID 3367035) and iaaH gene (Genbank ID 3367036) from Pseudomonas syringae were artificially synthesized by Ruibo Biotechnology Co., Ltd.

[0099] (2) Fig. 9As shown in the figure, five IAM pathway expression cassettes were constructed: IAM-head (using the NifA efficient gene transcription activation system, the nifA gene is located at the most upstream of the entire operon), IAM-middle (using the NifA efficient gene transcription activation system, the nifA gene is located in the middle of the entire operon), IAM-tail (using the NifA efficient gene transcription activation system, the nifA gene is located at the most downstream of the entire operon), IAM-P nifH (Directly using UAS-P nifH Promoter expression IAM pathway, no transcriptional activation effect), IAM-P12445 (IAM pathway expression controlled by P12445 strong promoter, as a control group of traditional overexpressed genes). According to the method described in Example 1, these five expression cassettes were constructed into the p15A-apra-oriT-phiC31 vector respectively to obtain five recombinant plasmids. The primers used to construct the recombinant plasmids are shown in Table 4;

[0100] (3) These five recombinant plasmids were integrated into the genome of Pseudomonas stutzeri (DSM4166-nifko-attB) with the nif gene cluster knocked out through conjugation transfer and attB / attP site-specific recombination to obtain recombinant bacteria IAM-head, IAM-middle, IAM-tail, IAM-P nifH and IAM-P12445;

[0101] (4) The recombinant bacteria IAM-head, IAM-middle, IAM-tail, and IAM-P nifH and IAM-P12445 in the presence of 1.0 g L -1 The fermentation was carried out in a standard succinate medium (SSM) containing L-Trp for 4 days, and the IAA production of the fermentation broth was determined. Fig.10 As shown; the standard succinate medium components are: K2HPO4 6.0g / L, KH2PO4 3.0g / L, (NH4)2SO41.0g / L, MgSO4·7H2O 0.2g / L, sodium succinate 5.5g / L, pH 7.0).

[0102] Table 4. Primers used to construct the indole-3-acetic acid synthesis pathway in Example 3

[0103]

[0104]

[0105]

[0106] In the table, the underlined sequences are the homology arms used for recombination.

[0107] Depend on Fig.10 It can be seen that the IAA production of IAM-head and IAM-tail groups is higher than that of IAM-P nifH There is a significant improvement compared with the IAM-P12445 group, while the yield of the IAM-middle group is lower than that of the IAM-P12445 group. The possible reason is that when the nifA gene is inserted into the middle of the synthesis pathway, the expression ratio of the upstream and downstream genes may be changed, resulting in the synthesis efficiency of IAA being affected and the IAA yield being reduced. Despite this, the IAM-head and IAM-tail groups also have higher IAA yields than IAM-P12445. The results show that the efficient gene transcription activation strategy has great advantages over the traditional strong promoter overexpression strategy in expressing simple biosynthetic pathways. At the same time, the results also show that the relative position of nifA has a great influence on the efficient gene transcription activation system. The optimal efficient gene transcription activation expression pattern is to place the positively regulated nifA gene in the regulated promoter UAS-P nifH The first gene downstream. The IAA yield of the recombinant bacteria IAM-head in SSM medium can reach 864 mg / L.

[0108] We then optimized the fermentation medium based on the recombinant bacteria IAM-head. We selected LB liquid medium that is more suitable for the growth of P. stutzeri for fermentation, and increased the amount of L-Trp initially added to the medium. The recombinant bacteria IAM-head was fermented in LB liquid medium with an initial L-Trp concentration of 1.0, 2.0, and 10.0 g / L, respectively. The fermentation conditions were the same as above, and samples were taken at different time points to measure the IAA production. The results are shown in Figure 2. Fig.11 As shown,

[0109] Depend on Fig.11 It can be seen that the IAA yield under the culture condition of 1.0 g / L L-Trp can reach 1.2 g / L; the IAA yield under the culture condition of 2.0 g / L L-Trp can reach 2.0 g / L; the IAA yield under the culture condition of 10.0 g / L L-Trp can reach 9.0 g / L, which is the highest yield of IAA synthesized using tryptophan as a precursor.

[0110] 3. The application potential of the NifA efficient gene transcription activation system in expressing simple biosynthetic pathways has been verified by heterologous expression of the IAM pathway for IAA synthesis. The application of this system in expressing complex biosynthetic pathways has become a further exploration of the capabilities of this system.

[0111] The prodigiosin (2-methyl-3-pentyl-6-methoxyalanine) biosynthesis pathway was selected to explore the application capabilities of the NifA efficient gene transcription activation system. The prodigiosin biosynthesis gene cluster (pig) contains a total of 14 genes (whose Genbank locus_tags are J0F95_RS05340, J0F95_RS05345, J0F95_RS05350, J0F95_RS05355, J0F95_RS05360, J0F95_RS05365, J0F95_RS05370, J0F95_RS05375, J0F95_RS05380, J0F95_RS05385, J0F95_RS05390, J0F95_RS05395, J0F95_RS05400, and J0F95_RS05405), with a length of 21 kb. Prodigiosin is a promising drug candidate with multiple biological activities, including anticancer, antimicrobial, antimalarial, antifungal, and immunosuppressive effects. At the same time, as it is a red pigment compound, it provides an easy-to-detect phenotype for early screening of prodigiosin-producing strains.

[0112] The specific synthesis and determination are as follows:

[0113] (1) Fig.12 As shown in the figure, the genome of Serratia marcescens ATCC13880 was first extracted, and the genome was digested with NdeⅠ restriction endonuclease to obtain a genomic fragment containing the pig gene cluster. Subsequently, the pig gene cluster was directly cloned into the p15A-cm vector in E. coli GB05-dir by ExoCET direct cloning to obtain the plasmid p15A-cm-prodigiosin. The recombinant after direct cloning was extracted from the plasmid and verified by AflⅢ restriction enzyme digestion. The electrophoresis gel image of enzyme digestion verification is shown in the figure. Fig.13 As shown;

[0114] Depend on Fig.13 It can be seen that a total of 17 recombinants were extracted, of which 8 were correctly verified by enzyme digestion, and the accuracy rate of direct cloning was about 50%.

[0115] (2) We then analyzed the pig gene cluster according to Fig.14 Modification, where Pig-1 represents only UAS-P nifHControl the expression of the entire pig gene cluster; Pig-2 represents the traditional method control group in which the strong promoter P12445 controls the expression of the entire pig gene cluster; Pig-3 represents the use of the NifA efficient gene transcription activation system to express the pig gene cluster, and three expression cassettes are obtained; first, a BstZ17I restriction site is reserved during direct cloning, and the plasmid p15A-cm-prodigiosin is digested with the restriction endonuclease BstZ17I to release the pig gene cluster, and then according to the method described in Example 1, respectively The three expression cassettes were constructed into the p15A-apra-oriT-phiC31 vector to obtain three recombinant plasmids. The primers used to construct the recombinant plasmids are shown in Table 5. Then, the three recombinant plasmids were integrated into the genome of Pseudomonas stutzeri (DSM4166-nifko-attB) with the nif gene cluster knocked out through conjugation transfer and attB / attP site-specific recombination to obtain recombinant bacteria Pig-1, recombinant bacteria Pig-2 and recombinant bacteria Pig-3. After antibiotic screening, the conjugant phenotype was as follows: Fig.15 As shown;

[0116] Depend on Fig.15 It can be seen that the colony color of Pig-1 has no significant difference from the wild-type DSM4166, the colony color of Pig-2 shows a slight red color, and the colony color of Pig-3 shows a distinct dark red color.

[0117] (3) After the three conjugates were verified to be correct by colony PCR, they were fermented in LB liquid medium, and then the fermentation broth was used to extract prodigiosin with acidified ethanol (4% (v / v) 1M HCl ethanol solution). After 3 days of fermentation in LB liquid medium, the cells were collected by centrifugation, and the cells were lysed and prodigiosin was dissolved with acidified ethanol; the fermentation supernatant was used to extract prodigiosin with an equal volume of acidified ethanol. The yield of prodigiosin after extraction was measured, and the results were as follows: Fig.16 shown.

[0118] Depend on Fig.16 It can be seen that no prodigiosin was detected in Pig-1, the prodigiosin yield of Pig-2 was 3.4 mg / L; the prodigiosin yield of Pig-3 can reach 173.0 mg / L. The results are consistent with the IAA yield results. The prodigiosin yield of the engineering strain using the NifA efficient gene transcription activation system is much higher than the yield of the strain overexpressing the gene cluster with the traditional strong promoter (the yield of Pig-3 is much higher than that of Pig-2). This result shows that the NifA efficient gene transcription activation system of the present invention can also efficiently express complex biosynthetic pathways, and its function is much more powerful than the traditional strong promoter overexpression method.

[0119] Subsequently, the fermentation conditions of prodigiosin were optimized, and it was found that the cell density was a factor that had a great influence on the production of prodigiosin. The Pig-3 strain was fermented in LB and TB liquid culture media, and the prodigiosin production was measured after fermentation. The results are as follows: Fig.17 The TB liquid culture medium comprises: 12 g / L Casein, 24 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, and 4 mL / L glycerol.

[0120] Depend on Fig.17 It can be seen that the bacterial density in TB medium is greater and the prodigiosin production is higher. After the fermentation conditions are optimized, the maximum prodigiosin production can reach 1.3 g / L, which is the highest level of prodigiosin production in heterologous expression.

[0121] The NifA efficient gene transcription activation system of the present invention is feasible and powerful. The system can make full use of the host's own resources, so that the gene expression level under the control of the efficient gene transcription activation system is significantly improved. The present invention uses the NifA efficient gene transcription activation system to significantly increase the nitrogenase activity of the DSM4166 strain by 368 times; obtain a heterologous high-yield strain of IAA, with a maximum yield of 9.0g / L, which is the highest yield of heterologous synthesis of IAA at present; obtain a heterologous high-yield strain of prodigiosin, with a maximum yield of 1.3g / L, which is the highest yield of heterologous synthesis of prodigiosin at present.

[0122] Table 5. Primers used to construct the prodigiosin synthesis pathway in Example 3

[0123]

[0124] In the table, the underlined sequences are homology arms used for recombination, the lowercase letters are artificially added ribosome binding (RBS) regions, and the lowercase and italic letters are BstZ17I restriction sites reserved for direct cloning.

[0125] Example 4: Construction of SlnR efficient gene transcription activation system

[0126] This example broadens the application scope of the efficient gene transcription activation system of the present invention on the basis of Examples 1 to 3, verifies the feasibility of the system in Streptomyces, and provides a simple and efficient method for the efficient synthesis of natural products.

[0127] 1. The nitrogenase activity was significantly increased in P. stutzeti DSM4166 through an efficient gene transcription activation system. In this way, the production of salinomycin was significantly increased in Streptomyces S. albus DSM41398. Salinomycin is a polyether antibiotic, which is a promising candidate drug targeting cancer stem cells. The salinomycin biosynthetic gene cluster (sal) is as follows Fig.18 As shown, the positive regulatory gene slnR (Genbank ID is 54209321) is a positive regulatory gene of the salinomycin biosynthesis gene cluster from Streptomyces albus DSM41398, and the overexpression of the positive regulatory gene slnR can effectively increase the production of salinomycin. The SlnR protein can bind to the UAS region upstream of the promoter of the slnQ, slnA1, slnF, slnT1, slnB3, and slnC genes in the sal gene cluster and positively regulate the transcription of the salinomycin synthesis gene.

[0128] 2. If Fig.19 As shown, six SlnR efficient gene transcription activation systems were constructed according to the method described in Example 1, and the promoter UAS-P regulated by the positive regulatory protein of slnR gene expression was activated. slnQ (SEQ ID NO.7), UAS-P slnA1 (SEQ ID NO.8), UAS-P slnF (SEQ ID NO.9), UAS-P slnT1 (SEQ ID NO.10), UAS-P slnB3 (SEQ ID NO.11) and UAS-P slnC (SEQ ID NO.12) were placed upstream of the positive regulatory gene slnR to construct the corresponding expression cassettes, and the strong constitutive promoter kasOp* in Streptomyces was selected to overexpress the slnR gene as a control group experiment of the traditional strong promoter overexpression gene method; then according to the method described in Example 1, kasOp*-slnR, UAS-P slnQ -slnR、UAS-P slnA1 -slnR、UAS-P slnF -slnR、UAS-P slnT1 -slnR、UAS-P slnB3 -slnR、UAS-P slnC-slnR expression cassette was constructed on the p15A-apra-oriT-phiC31 vector to obtain 7 recombinant plasmids. The primers used to construct the recombinant plasmids are shown in Table 6; then the expression cassette was integrated into the genome of DSM41398 by conjugation transfer and attB / attP site-specific recombination to obtain the recombinant bacteria kasOp*-slnR, UAS-P slnQ -slnR、UAS-P slnA1 -slnR、UAS-P slnF -slnR、UAS-P slnT1 -slnR、UAS-P slnB3 -slnR、UAS-P slnC -slnR.

[0129] Table 6. Primers used to construct the SlnR efficient gene transcription activation system in Example 4

[0130]

[0131]

[0132] In the table: the underlined sequences are the homology arms used for recombination.

[0133] 3. Wild-type DSM41398, recombinant bacteria kasOp*-slnR, UAS-P slnQ -slnR、UAS-P slnA1 -slnR、UAS-P slnF -slnR、UAS-P slnT1 -slnR、UAS-P slnB3 -slnR and UAS-P slnC-slnR were inoculated into 30 mL TSBY medium (3% tryptone soy broth, 0.5% high-glucose tolerant Angel yeast powder, 10.3% sucrose), and cultured at 33°C, 220 rpm for 2 days; then 1 mL of the initial culture was transferred to 30 mL of fresh medium (4% glucose, 3% soybean powder, 1% yeast extract, 0.2% CaCO3), and cultured at 33°C for 1 day as a seed culture; finally, 3 mL of the seed culture was transferred to 30 mL of fermentation medium (0.8% germ powder, 0.5% soybean powder, 0.2% KCl, 0.1% NaCl, 0.16% urea, 0.2% tartaric acid, 0.01% MgSO4, 0.01% K2HPO4, 0.5% CaCO3, 15% soybean oil, pH 6.6-6.9), and cultured at 33°C for 9 days. After the fermentation, 1 ml of XAD-16 macroporous resin was added to the fermentation broth to adsorb the fermentation product for 1 day, and then the cells and resin were collected by centrifugation, and the compound was dissolved in 30 ml of methanol for 4 hours. The methanol supernatant in which the compound was dissolved was collected by centrifugation, the extract was concentrated by rotary evaporation, and the crude extract was re-dissolved in HPLC-purified methanol. The crude extract of the fermentation was filtered and purified, and then tested by HPLC-MS (liquid phase mass spectrometry). The results of liquid phase and secondary mass spectrometry are as follows: Fig. 20 shown.

[0134] Depend on Fig. 20 It can be seen that compared with the salinomycin standard, the production of salinomycin can be detected in the fermentation products of all recombinant bacteria.

[0135] 4. Dilute the fermentation crude extract appropriately and determine the yield by HPLC-MS. The relative yield of salinomycin produced by the recombinant strain was compared with that of the wild-type DSM41398. Fig.21 As shown,

[0136] Depend on Fig.21 It can be seen that the salinomycin production of all recombinant bacteria overexpressing the slnR gene using the SlnR efficient gene transcription activation system was significantly increased compared with the wild type. slnQ -slnR,UAS-P slnA1 -slnR,UAS-P slnF -slnR,UAS-P slnT1 -slnR,UAS-P slnB3 -slnR and UAS-P slnC The salinomycin production of the recombinant strains was 1.77, 1.43, 2.16, 3.19, 1.44 and 1.73 times that of the wild-type strain. The salinomycin production caused by overexpression of the slnR gene (kasOp*-slnR) by the traditional strong promoter was 1.70 times that of the wild-type strain. slnF-slnR,UAS-P slnT1 The salinomycin production of -slnR was also significantly improved compared with the salinomycin production caused by overexpression of the traditional strong promoter.

[0137] 4. qPCR was used to analyze the wild-type strain of DSM41398, the recombinant kasOp*-slnR and the recombinant UAS-P slnT1 The transcription level of the slnR gene of -slnR was measured, and the results were as follows Fig. 22 shown.

[0138] Depend on Fig. 22 It can be seen that the recombinant bacteria UAS-P slnT1 -slnR has a significantly higher transcription level than the recombinant bacterium kasOp*-slnR, indicating that the SlnR efficient gene transcription activation system causes gene overexpression at the transcription level, further demonstrating that the efficient gene transcription activation system of the present invention has obvious advantages in overexpressing genes compared with traditional strong promoters.

[0139] In summary, the highly efficient gene transcription activation system of the present invention can be used to express a large amount of positive regulatory proteins, and a large amount of synthesized positive regulatory proteins can act on the UAS region of other related promoters ( Fig.23 Left, such as achieving a significant increase in nitrogenase activity in P. stutzeri DSM4166 and a significant increase in salinomycin production in S. albus DSM41398); at the same time, the novel and efficient gene transcription activation system of the present invention can also perform transcriptional activation of downstream genes while performing a positive feedback effect ( Fig.23 Right, efficient synthesis of indole-3-acetic acid and prodigiosin in P. stutzeri DSM 4166).

[0140] The efficient gene transcription activation system of the present invention is feasible and has advantages over traditional strong promoters. Examples 1 and 2 demonstrate the feasibility of the efficient gene transcription activation system in Gram-negative bacteria and its overexpression effect on the corresponding metabolic pathways; Example 4 demonstrates the feasibility of the efficient gene transcription activation system in Gram-positive bacteria and its overexpression effect on the corresponding metabolic pathways. At the same time, Example 3 further develops the efficient gene transcription activation system of the present invention into a tool for overexpressing metabolic pathways. Compared with the strategy of overexpressing genes with traditional strong promoters, the efficient gene transcription activation system of the present invention has obvious advantages and is a powerful tool in synthetic biology.

Claims

1. A highly efficient gene transcription activation system, characterized in that: The system comprises a positive regulatory gene, a DNA sequence (UAS) located upstream of the positive regulatory gene and bound by a positive regulatory protein expressed by the positive regulatory gene, and a corresponding promoter (P).

2. The high-efficiency gene transcription activation system according to claim 1, characterized in that: The positive regulatory gene is the nifA gene, and the DNA sequence (UAS) located upstream of the nifA gene and bound by the positive regulatory protein (NifA) expressed by the nifA gene and the corresponding promoter (P) are UAS-P nifH 、UAS-P nifE 、UAS-P nifB 、UAS-P nifU 、UAS-P rnf or UAS-P nifF .

3. The high-efficiency gene transcription activation system according to claim 2, characterized in that: The Genbank ID of the nifA gene is 12528487 (locus_tag: PSTAA_RS06800); the DNA sequence bound by the positive regulatory protein and its corresponding promoter UAS-P nifH 、UAS-P nifE 、UAS-P nifB 、UAS-P nifU 、UAS-P rnf and UAS-P nifF The sequences are shown in SEQ ID NO.1 to 6.

4. The high-efficiency gene transcription activation system according to claim 1, characterized in that The positive regulatory gene is the slnR gene, and the DNA sequence (UAS) located upstream of the slnR gene and bound by the positive regulatory protein (SlnR) expressed by the slnR gene and the corresponding promoter (P) are UAS-P slnQ 、UAS-P slnA1 、UAS-P slnF 、UAS-P slnT1 、UAS-P slnB3 or UAS-P slnC .

5. The high-efficiency gene transcription activation system according to claim 4, characterized in that: The Genbank ID of the slnR gene is 54209321 (locus_tag: SLNWT_RS01160); the DNA sequence bound by the positive regulatory protein and its corresponding promoter UAS-P slnQ 、UAS-P slnA1 、UAS-P slnF 、UAS-P slnT1 , UAS-P slnB3 and UAS-P slnC The sequences are shown in SEQ ID NOs. 7 to 12.

6. Use of the efficient gene transcription activation system according to claim 1, claim 2 or claim 3 in improving the activity of nitrogenase.

7. Use of the high-efficiency gene transcription activation system according to claim 1, claim 2 or claim 3 in increasing the production of indole-3-acetic acid.

8. Use of the gene transcription activation system according to claim 1, claim 2 or claim 3 in increasing the production of prodigiosin.

9. Use of the gene transcription activation system according to claim 1, claim 4 or claim 5 in increasing the yield of salinomycin.

10. A recombinant strain, characterized in that The recombinant strain contains the efficient gene transcription activation system according to any one of claims 1 to 5.

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