A screening method for microbial genome insertion sites and its application

Abstract

The present invention discloses a method for screening microbial genome insertion sites and applications. Through the method, the present invention screened 21 insertion sites in the halophilic mononas genome, which can be used for the stable integration and expression of exogenous genes, especially for the stable integration and expression of high-value compound biosynthesis pathways, and has a good effect. The microbial genome insertion site developed by the present invention can provide a powerful tool for the stable integration and expression of exogenous genes in microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology and metabolic engineering, and in particular to a screening method and application of microbial genome insertion sites. Background Art

[0002] Compared with traditional chemical and enzymatic methods, it is simpler and more cost-effective to introduce specific genes into microorganisms and produce large quantities of products through fermentation. Many methods have been developed in the past to transform the genome of cells (such as yeast or bacteria). However, traditional transformation methods rely on random insertion of genes into the cell genome, which may interrupt the expression of endogenous traits or inhibit the expression of inserted genes in transgenic cells. Therefore, targeted genome modification of cells is the preferred method for applied and basic research. Targeted insertion of transgenes into specific locations in the cell genome will effectively improve the quality of transgenic events, reduce related costs and provide new methods for manufacturing transgenic products.

[0003] Compared with traditional microorganisms that grow in mild environments (such as Escherichia coli) as chassis bacteria, microorganisms that survive in extreme environments, especially halomonas that can perform open fermentation without sterilization, can enable the fermentation industry to achieve open production and significantly reduce production costs. However, as non-model organisms, microorganisms, especially halomonas, are still relatively poorly studied and understood, especially the research and progress of gene editing tools, which limits researchers' genetic manipulation of halomonas and the production of metabolites. Therefore, it is particularly important to develop insertion sites and methods for inserting heterologous or exogenous sequences in the genome of microorganisms, especially halomonas. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a method and application for screening microbial genome insertion sites. The present invention screens the intergenic spacer region of microorganisms through bioinformatics and transcriptome sequencing, and uses the construction of fluorescent recombinant vectors to verify the expression intensity and stability of the intergenic spacer region genome. The obtained intergenic spacer region provides a powerful tool for constructing recombinant microorganisms, especially halophilic monocytic genes.

[0005] The present invention provides a method for screening a microbial insertion site, wherein the insertion site is an intergenic spacer region, and the screening method comprises the following steps:

[0006] (1) Eliminate intergenic regions less than 200 bp in length and those containing tRNA, rRNA, sRNA, and TRF;

[0007] (2) Eliminate intergenic regions containing predicted promoter sequences and find promoters located on IGRs through promoter prediction. If the upstream gene direction is “-” or the downstream gene direction is “+”, subtract an additional 25 bp upstream or downstream corresponding to IGR;

[0008] (3) Eliminate intergenic regions where the distance between the -35 region in the upstream gene promoter and the -35 region in the downstream gene promoter is less than or equal to 50 bp;

[0009] (4) Screening out the intergenic spacer region containing the Escherichia coli genome transcription factor binding site in the 1-50 bp position of the -35 region of the promoter of the upstream gene or downstream gene;

[0010] (5) Screening out the intergenic spacer regions whose upstream or downstream gene transcriptional activities are not in the top 50% of all gene transcriptional activities to obtain the insertion site;

[0011] (6) constructing two fluorescent protein plasmids respectively, wherein the first fluorescent protein plasmid comprises a fluorescent protein and the insertion site described in step (5), and the second fluorescent protein plasmid comprises the fluorescent protein in the first fluorescent protein plasmid;

[0012] (7) transferring the first fluorescent protein plasmid described in step (6) into a microorganism to obtain a recombinant bacterium containing the first fluorescent protein;

[0013] (8) transferring the second fluorescent protein plasmid described in step (6) into a microorganism to obtain a recombinant bacterium containing the second fluorescent protein;

[0014] (9) Cultivate the recombinant bacteria obtained in fermentation steps (7) and (8) and perform fluorescence measurement.

[0015] Through the above screening method, the present invention screened 21 insertion sites (intergenic spacer regions) in Halomonas, and the nucleotide sequences thereof are any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21. Wherein, the intergenic spacer region refers to the intergenic spacer region on the genome of Halomonas; wherein, the recombinant Halomonas refers to the insertion of foreign genes in the intergenic spacer region on the genome of Halomonas, and the foreign genes include but are not limited to: fluorescent protein, P34HB synthetic gene cluster. Preferably, the nucleotide sequence of the intergenic spacer region is any one of SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.15.

[0016] Furthermore, in step (2), screening is performed by salt concentration, urea concentration or fermentation cycle.

[0017] Further, the salt concentration is 10-100 g / L sodium chloride. Preferably, the salt concentration is 60 g / L sodium chloride.

[0018] Further, the urea concentration is 0-10 g / L urea. Preferably, the urea concentration is 3.6 g / L urea.

[0019] Furthermore, the fermentation period is 12-72 h. Preferably, the fermentation period is 9-30 h.

[0020] Furthermore, the fluorescent proteins in the first fluorescent protein plasmid and the second fluorescent protein plasmid are regulated by a promoter.

[0021] Further, the promoter is any one of porin58, porin140, and porin141. Among them, porin58, porin140, and porin141 represent promoters of different strengths. The strengths of the three promoters are compared, and the strongest promoter is porin141, followed by porin140, and finally porin58.

[0022] The present invention also provides an application of an intergenic spacer region for stably synthesizing P34HB in halomonas, comprising the following steps:

[0023] (1) inserting the P34HB synthetic gene cluster into the intergenic spacer region to obtain a P34HB synthetic gene cluster integration module, and inserting the P34HB synthetic gene cluster integration module into a plasmid to obtain an integration plasmid containing the P34HB synthetic gene cluster;

[0024] Wherein, the P34HB synthetic gene cluster is aldD-dhaT-orfz, and the nucleotide sequence of aldD-dhaT-orfz is shown in SEQ ID NO.274;

[0025] (2) The integration plasmid containing the P34HB synthetic gene cluster described in step (1) is conjugated with Halomonas to obtain a recombinant Halomonas containing the P34HB synthetic gene cluster, and the recombinant Halomonas is cultured and fermented.

[0026] Preferably, the nucleotide sequence of the intergenic spacer region is any one of SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.15.

[0027] The present invention also provides an integration plasmid, which comprises any one of the nucleotide sequences of the intergenic spacer region.

[0028] The Halomonas sp. LY01 strain used in the present invention has been disclosed in patent CN116396886A, and its deposit number is GDMCC No: 62635; the Halomonas sp. LY03 strain has been disclosed in patent CN116970538A, and its deposit number is GDMCC NO. 63382.

[0029] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0030] The genomic insertion sites screened out by the screening method of the present invention have strong expression intensity and stability, wherein the expression intensity of different insertion sites is diverse, and the expression intensity of the gene can be changed by selecting different genomic insertion sites, avoiding the method of using promoters of different strengths to change the expression intensity of the plasmid. At the same time, the insertion site can provide a powerful tool for the stable integration and expression of exogenous genes in microorganisms, especially halophilic monocytosing bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 This is the information diagram of the insertion site fragment plasmid in Example 2 of the present invention;

[0033] Figure 2 This is a gel image verifying the successful insertion of fluorescent protein into 21 insertion sites in Example 2 of the present invention;

[0034] Figure 3 This is a fluorescence expression intensity diagram of 21 insertion sites in Example 2 of the present invention;

[0035] Figure 4 This is a plasmid information diagram of the fluorescent protein fragment and the pSEVA321 backbone reconstructed in Example 3 of the present invention;

[0036] Figure 5 This is a comparative analysis diagram of the fluorescence expression intensity of the plasmid and the fluorescence expression intensity of the genome in Example 3 of the present invention;

[0037] Figure 6 This is a plasmid information diagram of the reconstruction of three fluorescent proteins with different expression intensities and the pSEVA321 backbone in Example 4 of the present invention;

[0038] Figure 7 This is a verification gel image of Example 4 of the present invention in which different expression fluorescence intensities are inserted into the LOCUS-9, LOCUS-10, and LOCUS-15 insertion sites, respectively;

[0039] Figure 8 A comparative analysis diagram of the fluorescence expression intensities of three fluorescent plasmids with different expression intensities and three templates with different expression intensities inserted into three different insertion sites in Example 4 of the present invention;

[0040] Fig. 9 This is a plasmid information diagram of the reconstructed promoters with three different expression strengths and the P34HB synthetic gene cluster in Example 5 of the present invention;

[0041] Fig.10 This is a gel image verifying that the P34HB synthetic gene cluster in Example 5 of the present invention was successfully inserted into the genome of Halobacterium spp. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0043] Example 1 Screening Method for Microbial Genome Insertion Sites

[0044] In order to avoid the influence on regulation, the intergenic spacer region (IGR) with a length of less than 200 bp and IGR containing tRNA, rRNA, sRNA sequences and tandem repeat sequences (TRF) can be screened out. In addition, the influencing factors of the promoter need to be considered. Since the upstream and downstream genes of IGR have four different possible direction combinations, if the upstream gene direction is "-" or the downstream gene direction is "+", an additional 25 bp is subtracted from the upstream or downstream corresponding to the IGR. Under the conditions of 60g / L NaCl, 3.6 g / L Urea, and three different fermentation cycles (9 h, 19 h, and 30 h), 50 IGRs that meet the conditions were screened out. In order to avoid the effect of regulatory sequences, IGRs whose -35 region in the upstream gene promoter is less than or equal to 50 bp from the -35 region in the downstream gene promoter were further screened out. Through the prediction of the promoter online software, the spacer that affects the expression of the adjacent gene was removed, and 32 IGRs that meet the conditions were screened out. Because Halomonas sp The binding of transcription factors in the LY03 genome may interfere with the expression module inserted in the IGR, so it is necessary to sp . The transcription factor binding sites on the LY03 genome were analyzed, and the transcription factor binding sites on LY03 were found through sequence alignment and the homology was calculated. If the IGR region contained a TF Bindingsites sequence with a similarity of 100% with the transcription factor binding sites (TF Bindingsites) in the Escherichia coli genome, the IGR was eliminated. Through the above screening, 21 IGRs that met the above conditions were finally obtained, and the IGRs were named LOCUS-1~21, and the sequences (5'-3') of LOCUS-1~21 are as follows:

[0045] LOCUS-1: see SEQ ID NO.1; LOCUS-2: see SEQ ID NO.2; LOCUS-3: see SEQ ID NO.3; LOCUS-4: see SEQ ID NO.4; LOCUS-5: see SEQ ID NO.5; LOCUS-6: see SEQ ID NO.6; LOCUS-7: see SEQ ID NO.7; LOCUS-8: see SEQ ID NO.8; LOCUS-9: see SEQ ID NO.9; LOCUS-10: see SEQ ID NO.10; LOCUS-11: see SEQ ID NO.11; LOCUS-12: see SEQ ID NO.12; LOCUS-13: see SEQ ID NO.13; LOCUS-14: see SEQ ID NO.14; LOCUS-15: see SEQ ID NO.15; LOCUS-16: see SEQ ID NO.16; LOCUS-17: see SEQ ID NO.17; LOCUS-18: see SEQ ID NO.18; LOCUS-19: see SEQ ID NO.19; LOCUS-20: see SEQ ID NO.20; LOCUS-21: see SEQ ID NO.21.

[0046] Example 2 Verification of expression intensity of screening sites

[0047] In order to check the expression intensity of the screening site inserted into the genomic position, fluorescent protein (GFP) was inserted into the 21 intergenic regions (IGRs) finally screened in Example 1 by homologous recombination. The specific operations are as follows:

[0048] 1. Construction of plasmid for insertion site fragment

[0049] The GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and plasmid pRE112 backbone were amplified by PCR with porin58 promoter as template. Under the action of Gibson ligase, the GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and plasmid pRE112 backbone were recombined to form a new plasmid named pRE112-LOCUS-1-GFP. The desired inserted gene was obtained by overlap extension PCR with pRE112-LOCUS-1-GFP as template. Part of the product was sent to a biological company for sequencing. The plasmid information is as follows: Figure 1 shown.

[0050] The primer sequences (5'-3') for amplifying the GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and pRE112 plasmid backbone are as follows:

[0051] GFP fragment amplification primers: LOCUS-1R-GFP-R: SEQ ID NO.22; LOCUS-1L-GFP-F: SEQ ID NO.23;

[0052] Primers for amplifying LOCUS-1L fragment: pRE112-LOCUS-1L-F: SEQ ID NO.24; LOCUS-1L-GFP-R: SEQ ID NO.25;

[0053] Primers for amplifying LOCUS-1R fragment: pRE112-LOCUS-1R-R: SEQ ID NO.26; LOCUS-1R-GFP-F: SEQ ID NO.27;

[0054] Primers for amplifying the backbone of pRE112 plasmid: pRE112-LOCUS-1R-F: SEQ ID NO.28; pRE112-LOCUS-1L-R: SEQ ID NO.29.

[0055] The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0056] Table 1 Amplification system

[0057]

[0058] Table 2 Amplification program

[0059]

[0060] After the PCR reaction is completed, prepare agarose gel of corresponding concentration and perform electrophoresis to observe the size of DNA bands. Place the gel under ultraviolet light and quickly cut the gel of the target DNA fragment, cutting off the excess gel as much as possible. Detect the concentration of the recovered DNA, and then calculate the proportion of DNA added according to the length and concentration of the target fragment and the pRE112 backbone, and connect with Gibson mixed enzyme. The Gibson Assembly connection system and procedure are shown in Table 3 and Table 4:

[0061] Table 3 Gibson Assembly connection system

[0062]

[0063] Table 4 Gibson Assembly connection procedure

[0064]

[0065] The ligation product was then transformed into S17-1 competent cells, placed on ice for 30 min, heat-shocked in a 42°C water bath for 2 min, and immediately placed on ice for 2 min. 400 μL LB medium (without antibiotics) was added to the centrifuge tube, mixed and placed in a 37°C shaker at 200 rpm for 60 min for recovery; then centrifuged at 5000 rpm for 5 min to collect the bacteria, and spread them on an LB plate containing the corresponding antibiotics; cultured in a 37°C incubator for 12-16 h; after colony PCR verification, the PCR product with the correct band size was sent to a biological company for sequencing.

[0066] According to the method of Example 1 above, the remaining 20 insertion site fragments (LOCUS-2-21) were constructed to insert into the plasmid, and the primer sequences (5'-3') were as follows:

[0067] Insertion site LOCUS-2 plasmid construction primers: LOCUS-2L-GFP-F: see SEQ ID NO.30; LOCUS-2L-GFP-R: see SEQ ID NO.31; LOCUS-2R-GFP-F: see SEQ ID NO.32; LOCUS-2R-GFP-R: see SEQ ID NO.33; PRE112-LOCUS-2L-F: see SEQ ID NO.34; PRE112-LOCUS-2L-R: see SEQ ID NO.35; PRE112-LOCUS-2R-F: see SEQ ID NO.36; PRE112-LOCUS-2R-R: see SEQ ID NO.37;

[0068] Insertion site LOCUS-3 plasmid construction primers: LOCUS-3L-GFP-F: see SEQ ID NO.38; LOCUS-3L-GFP-R: see SEQ ID NO.39; LOCUS-3R-GFP-F: see SEQ ID NO.40; LOCUS-3R-GFP-R: see SEQ ID NO.41; PRE112-LOCUS-3L-F: see SEQ ID NO.42; PRE112-LOCUS-3L-R: see SEQ ID NO.43; PRE112-LOCUS-3R-F: see SEQ ID NO.44; PRE112-LOCUS-3R-R: see SEQ ID NO.45;

[0069] Insertion site LOCUS-4 plasmid construction primers: LOCUS-4L-GFP-F: see SEQ ID NO.46; LOCUS-4L-GFP-R: see SEQ ID NO.47; LOCUS-4R-GFP-F: see SEQ ID NO.48; LOCUS-4R-GFP-R: see SEQ ID NO.49; PRE112-LOCUS-4L-F: see SEQ ID NO.50; PRE112-LOCUS-4L-R: see SEQ ID NO.51; PRE112-LOCUS-4R-F: see SEQ ID NO.52; PRE112-LOCUS-4R-R: see SEQ ID NO.53;

[0070] Insertion site LOCUS-5 plasmid construction primers: LOCUS-5L-GFP-F: see SEQ ID NO.54; LOCUS-5L-GFP-R: see SEQ ID NO.55; LOCUS-5R-GFP-F: see SEQ ID NO.56; LOCUS-5R-GFP-R: see SEQ ID NO.57; PRE112-LOCUS-5L-F: see SEQ ID NO.58; PRE112-LOCUS-5L-R: see SEQ ID NO.59; PRE112-LOCUS-5R-F: see SEQ ID NO.60; PRE112-LOCUS-5R-R: see SEQ ID NO.61;

[0071] Insertion site LOCUS-6 plasmid construction primers: LOCUS-6L-GFP-F: see SEQ ID NO.62; LOCUS-6L-GFP-R: see SEQ ID NO.63; LOCUS-6R-GFP-F: see SEQ ID NO.64; LOCUS-6R-GFP-R: see SEQ ID NO.65; PRE112-LOCUS-6L-F: see SEQ ID NO.66; PRE112-LOCUS-6L-R: see SEQ ID NO.67; PRE112-LOCUS-6R-F: see SEQ ID NO.68; PRE112-LOCUS-6R-R: see SEQ ID NO.69;

[0072] Insertion site LOCUS-7 plasmid construction primers: LOCUS-7L-GFP-F: see SEQ ID NO.70; LOCUS-7L-GFP-R: see SEQ ID NO.71; LOCUS-7R-GFP-F: see SEQ ID NO.72; LOCUS-7R-GFP-R: see SEQ ID NO.73; PRE112-LOCUS-7L-F: see SEQ ID NO.74; PRE112-LOCUS-7L-R: see SEQ ID NO.75; PRE112-LOCUS-7R-F: see SEQ ID NO.76; PRE112-LOCUS-7R-R: see SEQ ID NO.77;

[0073] Insertion site LOCUS-8 plasmid construction primers: LOCUS-8L-GFP-F: see SEQ ID NO.78; LOCUS-8L-GFP-R: see SEQ ID NO.79; LOCUS-8R-GFP-F: see SEQ ID NO.80; LOCUS-8R-GFP-R: see SEQ ID NO.81; PRE112-LOCUS-8L-F: see SEQ ID NO.82; PRE112-LOCUS-8L-R: see SEQ ID NO.83; PRE112-LOCUS-8R-R: see SEQ ID NO.84; PRE112-LOCUS-8R-F: see SEQ ID NO.85;

[0074] Insertion site LOCUS-9 plasmid construction primers: LOCUS-9L-GFP-F: see SEQ ID NO.86; LOCUS-9L-GFP-R: see SEQ ID NO.87; LOCUS-9R-GFP-F: see SEQ ID NO.88; LOCUS-9R-GFP-R: see SEQ ID NO.89; PRE112-LOCUS-9L-F: see SEQ ID NO.90; PRE112-LOCUS-9L-R: see SEQ ID NO.91; PRE112-LOCUS-9R-F: see SEQ ID NO.92; PRE112-LOCUS-9R-R: see SEQ ID NO.93;

[0075] Insertion site LOCUS-10 plasmid construction primers: LOCUS-10L-GFP-F: see SEQ ID NO.94; LOCUS-10L-GFP-R: see SEQ ID NO.95; LOCUS-10R-GFP-F: see SEQ ID NO.96; LOCUS-10R-GFP-R: see SEQ ID NO.97; PRE112-LOCUS-10L-F: see SEQ ID NO.98; PRE112-LOCUS-10L-R: see SEQ ID NO.99; PRE112-LOCUS-10R-F: see SEQ ID NO.100; PRE112-LOCUS-10R-R: see SEQ ID NO.101;

[0076] Insertion site LOCUS-11 plasmid construction primers: LOCUS-11L-GFP-F: see SEQ ID NO.102; LOCUS-11L-GFP-R: see SEQ ID NO.103; LOCUS-11R-GFP-F: see SEQ ID NO.104; LOCUS-11R-GFP-R: see SEQ ID NO.105; PRE112-LOCUS-11L-F: see SEQ ID NO.106; PRE112-LOCUS-11L-R: see SEQ ID NO.107; PRE112-LOCUS-11R-F: see SEQ ID NO.108; PRE112-LOCUS-11R-R: see SEQ ID NO.109;

[0077] Insertion site LOCUS-12 plasmid construction primers: LOCUS-12L-GFP-F: see SEQ ID NO.110; LOCUS-12L-GFP-R: see SEQ ID NO.111; LOCUS-12R-GFP-F: see SEQ ID NO.112; LOCUS-12R-GFP-R: see SEQ ID NO.113; PRE112-LOCUS-12L-F: see SEQ ID NO.114; PRE112-LOCUS-12L-R: see SEQ ID NO.115; PRE112-LOCUS-12R-F: see SEQ ID NO.116; PRE112-LOCUS-12R-R: see SEQ ID NO.117;

[0078] Insertion site LOCUS-13 plasmid construction primers: LOCUS-13L-GFP-F: see SEQ ID NO.118; LOCUS-13L-GFP-R: see SEQ ID NO.119; LOCUS-13R-GFP-F: see SEQ ID NO.120; LOCUS-13R-GFP-R: see SEQ ID NO.121; PRE112-LOCUS-13L-F: see SEQ ID NO.122; PRE112-LOCUS-13L-R: see SEQ ID NO.123; PRE112-LOCUS-13R-F: see SEQ ID NO.124; PRE112-LOCUS-13R-R: see SEQ ID NO.125;

[0079] Insertion site LOCUS-14 plasmid construction primers: LOCUS-14L-GFP-F: see SEQ ID NO.126; LOCUS-14L-GFP-R: see SEQ ID NO.127; LOCUS-14R-GFP-F: see SEQ ID NO.128; LOCUS-14R-GFP-R: see SEQ ID NO.129; PRE112-LOCUS-14L-F: see SEQ ID NO.130; PRE112-LOCUS-14L-R: see SEQ ID NO.131; PRE112-LOCUS-14R-F: see SEQ ID NO.132; PRE112-LOCUS-14R-R: see SEQ ID NO.133;

[0080] Insertion site LOCUS-15 plasmid construction primers: LOCUS-15L-GFP-F: see SEQ ID NO.134; LOCUS-15L-GFP-R: see SEQ ID NO.135; LOCUS-15R-GFP-F: see SEQ ID NO.136; LOCUS-15R-GFP-R: see SEQ ID NO.137; PRE112-LOCUS-15L-F: see SEQ ID NO.138; PRE112-LOCUS-15L-R: see SEQ ID NO.139; PRE112-LOCUS-15R-F: see SEQ ID NO.140; PRE112-LOCUS-15R-R: see SEQ ID NO.141;

[0081] Insertion site LOCUS-16 plasmid construction primers: LOCUS-16L-GFP-F: see SEQ ID NO.142; LOCUS-16L-GFP-R: see SEQ ID NO.143; LOCUS-16R-GFP-F: see SEQ ID NO.144; LOCUS-16R-GFP-R: see SEQ ID NO.145; PRE112-LOCUS-16L-F: see SEQ ID NO.146; PRE112-LOCUS-16L-R: see SEQ ID NO.147; PRE112-LOCUS-16R-F: see SEQ ID NO.148; PRE112-LOCUS-16R-R: see SEQ ID NO.149;

[0082] Insertion site LOCUS-17 plasmid construction primers: LOCUS-17L-GFP-F: see SEQ ID NO.150; LOCUS-17L-GFP-R: see SEQ ID NO.151; LOCUS-17R-GFP-F: see SEQ ID NO.152; LOCUS-17R-GFP-R: see SEQ ID NO.153; PRE112-LOCUS-17L-F: see SEQ ID NO.154; PRE112-LOCUS-17L-R: see SEQ ID NO.155; PRE112-LOCUS-17R-F: see SEQ ID NO.156; PRE112-LOCUS-17R-R: see SEQ ID NO.157;

[0083] Insertion site LOCUS-18 plasmid construction primers: LOCUS-18L-GFP-F: see SEQ ID NO.158; LOCUS-18L-GFP-R: see SEQ ID NO.159; LOCUS-18R-GFP-F: see SEQ ID NO.160; LOCUS-18R-GFP-R: see SEQ ID NO.161; PRE112-LOCUS-18L-F: see SEQ ID NO.162; PRE112-LOCUS-18L-R: see SEQ ID NO.163; PRE112-LOCUS-18R-F: see SEQ ID NO.164; PRE112-LOCUS-18R-R: see SEQ ID NO.165;

[0084] Insertion site LOCUS-19 plasmid construction primers: LOCUS-19L-GFP-F: see SEQ ID NO.166; LOCUS-19L-GFP-R: see SEQ ID NO.167; LOCUS-19R-GFP-F: see SEQ ID NO.168; LOCUS-19R-GFP-R: see SEQ ID NO.169; PRE112-LOCUS-19L-F: see SEQ ID NO.170; PRE112-LOCUS-19L-R: see SEQ ID NO.171; PRE112-LOCUS-19R-F: see SEQ ID NO.172; PRE112-LOCUS-19R-R: see SEQ ID NO.173;

[0085] Insertion site LOCUS-20 plasmid construction primers: LOCUS-20L-GFP-F: see SEQ ID NO.174; LOCUS-20L-GFP-R: see SEQ ID NO.175; LOCUS-20R-GFP-F: see SEQ ID NO.176; LOCUS-20R-GFP-R: see SEQ ID NO.177; PRE112-LOCUS-20L-F: see SEQ ID NO.178; PRE112-LOCUS-20L-R: see SEQ ID NO.179; PRE112-LOCUS-20R-F: see SEQ ID NO.180; PRE112-LOCUS-20R-R: see SEQ ID NO.181;

[0086] Insertion site LOCUS-21 plasmid construction primers: LOCUS-21L-GFP-F: see SEQ ID NO.182; LOCUS-21L-GFP-R: see SEQ ID NO.183; LOCUS-21R-GFP-F: see SEQ ID NO.184; LOCUS-21R-GFP-R: see SEQ ID NO.185; PRE112-LOCUS-21L-F: see SEQ ID NO.186; PRE112-LOCUS-21L-R: see SEQ IDNO.187; PRE112-LOCUS-21R-F: see SEQ ID NO.188; PRE112-LOCUS-21R-R: see SEQ ID NO.189.

[0087] 2. Gene Integration

[0088] (1) The integration plasmids constructed with the 21 insertion site fragments were cultured separately, and after 12-16 hours, they were conjugated with HalomonasLY 03 in a 20LB plate. After 8 hours, a small amount of conjugated bacteria was picked and spread on a 60LB plate with the corresponding resistance. After 36-48 hours, the single clone colony was verified again;

[0089] (2) Antibiotic-free verification can be performed after a single colony grows on the plate;

[0090] (3) Select the colonies that have successfully removed the antibodies for double verification (universal F1 / R and specific F2 / R), such as Figure 2 As shown in the figure, when the target band of 3000 bp appears, it proves that GFP has been successfully inserted into the 21 insertion sites on the genome. The PCR reaction solution with successful double verification was selected for testing, and after successful sequencing, the strains YG-1, YG-2, YG-3, YG-4, YG-5, YG-7, YG-8, YG-9, YG-10, YG-11, YG-12, YG-13, YG-14, YG-15, YG-16, YG-17, YG-18, YG-19, YG-20, and YG-21 were obtained.

[0091] 3. Fluorescence Measurement

[0092] (1) Streak the 21 prepared strains containing fluorescent protein-expressing plasmids on 60LB solid plates for activation culture and incubate them inverted at 37°C for 24 h.

[0093] (2) On a clean bench, pick a single colony and inoculate it into a 1 mL deep-well plate containing 60 LB. Incubate at 37°C and 220 rpm for 12 h to obtain the seed solution.

[0094] (3) Take 1 μL of each of the above seed solutions and seed them into 1 mL of deep-well plates containing 60 LB and make three sets of parallel samples. Continue to culture for 12 h.

[0095] (4) Take 20 μL of bacterial solution and add it to 180 μL of PBS solution. Mix well and place it in a 96-well plate with a lid. 60 LB is used as a blank control. The fluorescence intensity is measured using a Thermo Varioskan Lux microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The fluorescence intensity of the 21 insertion sites is shown in Table 5:

[0096] Table 5 Fluorescence intensity of 21 insertion sites

[0097]

[0098] The fluorescence expression intensity of the plasmid constructed with 21 insertion sites is as follows Figure 3As shown in the figure, the average fluorescence intensity of GFP inserted into LOCUS1-5 sites is about 200, while the average fluorescence intensity of GFP inserted into LOCUS-7, LOCUS-9, LOCUS-10, LOCUS-14, LOCUS-15 and other sites is basically over 500. Among them, the fluorescence expression intensity at LOCUS-9, LOCUS-10, and LOCUS-15 sites is relatively strong, and their average fluorescence values ​​are 771.7, 791.7, and 796.7, respectively.

[0099] Example 3 Comparative analysis of genomic locus expression intensity and plasmid expression intensity

[0100] The data obtained in Example 2 were compared with the fluorescence intensity obtained from the plasmid constructed using porin58-GFP as a template to illustrate the characteristics of the expression intensity of different insertion sites on the genome and the expression intensity of the plasmid.

[0101] 1. Construction of GFP gene expression plasmid

[0102] The specific implementation of the plasmid is the same as in Example 2-1, and the plasmid pSEVA321 porin58-GFP is constructed with the GFP fragment. Figure 4 shown.

[0103] The amplification primer sequences (5′-3′) are as follows: GFP-R: see SEQ ID NO.190; PORIN58-F: see SEQ ID NO.191; PORIN58-GFP-F: see SEQ ID NO.192; PORIN58-R: see SEQ ID NO.193.

[0104] 2. Comparative analysis of plasmid fluorescence assay and genomic fluorescence expression

[0105] The fluorescence measurement operation was the same as in Example 2-3. The fluorescence expression intensity was monitored. The average fluorescence value of porin58-GFP when expressed in pSEVA321porin58-GFP plasmid was 391.7. The fluorescence expression intensity of porin58-GFP inserted at 21 sites was compared with that of the original porin58-GFP. Figure 5As shown, the fluorescence intensity expressed at different sites has a certain proportional relationship with the fluorescence intensity expressed by the plasmid itself, and can also be roughly classified according to the proportional relationship. The plasmid expression of porin58-GFP is basically 4 times that of its genomic expression at the LOCUS-13 site; the sites where the plasmid expression of porin58-GFP is about 2 times that of the genomic expression are LOCUS-1, LOCUS-4 and LOCUS-11; and the fluorescence expression intensity at the insertion sites such as LOCUS-6 and LOCUS-12 is basically equivalent to the fluorescence intensity expressed on the plasmid; There are also sites where the fluorescence intensity of genomic expression is twice that of plasmid expression, such as LOCUS-9, LOCUS-10 and LOCUS-15. In addition, there are other proportional relationships between plasmid expression and expression at different genomic sites, which explains the uncertainty and diversity of expression intensity at different insertion sites on the genome compared with plasmids, thereby clarifying the rules of genomic expression and establishing a library of genomic sites with different expression intensities. Therefore, the expression intensity of genes can be changed by selecting insertions at different genomic sites, which avoids the use of methods to change the expression intensity of plasmids through promoters of different strengths.

[0106] Example 4 Stability Analysis of Genomic Expression

[0107] According to the data obtained in Example 2, three insertion sites with strong fluorescence expression intensity (LOCUS-9, LOCUS-10, LOCUS-15) were selected to replace templates with different expression intensities (porin58-GFP, porin140-GFP, porin141-GFP), and their fluorescence expression intensity was monitored to verify the stability of the screening site.

[0108] 1. Construction of GFP gene expression plasmids with different expression intensities

[0109] Specific implementation of plasmid construction: Referring to Example 2-1, GFP fragments with different expression intensities were amplified using plasmids pSEVA321 porin58-GFP, pSEVA321 porin140-GFP, and pSEVA321 porin141-GFP templates, respectively. The plasmid information is as follows: Figure 6 shown.

[0110] The amplification primer sequences (5′-3′) are as follows: GFP-R: see SEQ ID NO.194; PORIN58-F: see SEQ ID NO.195; PORIN58-GFP-F: see SEQ ID NO.196; PORIN58-R: see SEQ ID NO.197; PORIN140-F: see SEQ ID NO.198; PORIN140-GFP-F: see SEQ ID NO.199; PORIN140-R: see SEQ ID NO.200; PORIN141-F: see SEQ ID NO.201; PORIN141-GFP-F: see SEQ ID NO.202; PORIN141-R: see SEQ ID NO.203.

[0111] 2. Gene Integration

[0112] The specific operation of gene integration was referred to Example 2-2, and finally strains (LOCUS-9-porin58-GFP, LOCUS-9-porin140-GFP, LOCUS-9-porin141-GFP, LOCUS-10-porin58-GFP, LOCUS-10-porin140-GFP, LOCUS-10-porin141-GFP, LOCUS-15-porin58-GFP, LOCUS-15-porin140-GFP, LOCUS-15-porin141-GFP) with different expression intensity templates (porin58-GFP, porin140-GFP, porin141-GFP) at three different sites (LOCUS-9, LOCUS-10, LOCUS-15) were obtained.

[0113] The gene integration primer sequences (5′-3′) are as follows:

[0114] Insertion site LOCUS-9 gene integration primers: LOCUS-9L-GFP-F: see SEQ ID NO.204; LOCUS-9L-GFP-R: see SEQ ID NO.205; LOCUS-9R-GFP-F: see SEQ ID NO.206; LOCUS-9R-GFP-R: see SEQ ID NO.207; PRE112-LOCUS-9L-F: see SEQ ID NO.208; PRE112-LOCUS-9L-R: see SEQ ID NO.209; PRE112-LOCUS-9R-F: see SEQ ID NO.210; PRE112-LOCUS-9R-R: see SEQ ID NO.211;

[0115] Insertion site LOCUS-10 gene integration primers: LOCUS-10L-GFP-F: see SEQ ID NO.212; LOCUS-10L-GFP-R: see SEQ ID NO.213; LOCUS-10R-GFP-F: see SEQ ID NO.214; LOCUS-10R-GFP-R: see SEQ ID NO.215; PRE112-LOCUS-10L-F: see SEQ ID NO.216; PRE112-LOCUS-10L-R: see SEQ ID NO.217; PRE112-LOCUS-10R-F: see SEQ ID NO.218; PRE112-LOCUS-10R-R: see SEQ ID NO.219;

[0116] Insertion site LOCUS-15 gene integration primers: LOCUS-15L-GFP-F: see SEQ ID NO.220; LOCUS-15L-GFP-R: see SEQ ID NO.221; LOCUS-15R-GFP-F: see SEQ ID NO.222; LOCUS-15R-GFP-R: see SEQ ID NO.223; PRE112-LOCUS-15L-F: see SEQ ID NO.224; PRE112-LOCUS-15L-R: see SEQ IDNO.225; PRE112-LOCUS-15R-F: see SEQ ID NO.226; PRE112-LOCUS-15R-R: see SEQ ID NO.227.

[0117] PCR was used to verify whether GFP with different expression intensities was successfully inserted into the LOCUS-9, LOCUS-10, and LOCUS-15 sites. The results are shown in Figure 7 As shown, when the target fragment is 3000 bp, it proves that GFP with different expression intensities have been successfully inserted into the LOCUS-9, LOCUS-10, and LOCUS-15 sites on the genome.

[0118] 3. Fluorescence Measurement

[0119] The GFP plasmids with different expression intensities obtained in Example 4-1 (pSEVA321 porin58-GFP, pSEVA321porin140-GFP, pSEVA321 porin141-GFP) were measured for their fluorescence expression intensities according to Example 2-3, and the fluorescence expression intensities of GFP with different expression intensities inserted at three different sites were measured, and compared with the fluorescence expression intensities of the plasmids. Figure 8As shown in the figure, the fluorescence expression intensity of the plasmid is shown in porin58-GFP, porin140-GFP and porin141-GFP. The fluorescence intensity ratio of GFP expressed by promoters with different expression strengths in the three plasmids is about 1:4:8. Compared with the insertion of promoters with different strengths at the same site in the genome, such as inserting three GFPs with different expression strengths of porin58-GFP, porin140-GFP and porin141-GFP into LOCUS-9, the ratio of their expression fluorescence intensity is also about 1:4:8, and the same results are obtained in LOCUS-10 and LOCUS-15. This shows that the promoters with different strengths at a certain site on the genome keep the ratio of their expression intensity consistent with the intensity ratio when expressed in the plasmid, thereby indicating that the genome expression is stable. However, from the perspective of overall expression intensity, for the promoter with porin58 strength, the fluorescence intensity of GFP expressed at LOCUS-9, LOCUS-10 and LOCUS-15 sites is basically twice the intensity of GFP expressed by the plasmid, and when the promoter strength at these three sites is replaced with porin140-GFP and porin141-GFP, the fluorescence intensity of GFP expressed at LOCUS-9, LOCUS-10 and LOCUS-15 sites is also basically twice the intensity of GFP expressed by the plasmid.

[0120] Example 5 Verification of the P34HB synthetic gene cluster genome expression application

[0121] According to Examples 3 and 4, the fluorescence expression of the three sites LOCUS-9, LOCUS-10, and LOCUS-15 is relatively stable. Therefore, promoter-induced gene clusters with different expression strengths (porin58-aldD-dhaT-orfz, porin140-aldD-dhaT-orfz, porin141-aldD-dhaT-orfz) were inserted into these three sites respectively, and the yield of poly 3-hydroxybutyrate-co-4-hydroxybutyrate (P34HB) obtained by these three genomic expression gene clusters was compared with the plasmid expression, so as to compare the stability of the genomic expression and plasmid expression gene clusters.

[0122] 1. Construction of P34HB synthetic gene cluster expression plasmid

[0123] The specific implementation of plasmid construction is as shown in Example 2-1. The P34HB synthetic gene cluster is amplified using plasmids pSEVA321porin58-P34HB, pSEVA321 porin140-P34HB, and pSEVA321 porin141-P34HB as templates. The plasmid information is as follows: Fig. 9 shown.

[0124] The amplification primer sequences (5′-3′) are as follows: GFP-R: see SEQ ID NO.228; PORIN58-F: see SEQ ID NO.229; PORIN58-GFP-F: see SEQ ID NO.230; PORIN58-R: see SEQ ID NO.231; PORIN140-F: see SEQ ID NO.232; PORIN140-GFP-F: see SEQ ID NO.233; PORIN140-R: see SEQ ID NO.234; PORIN141-F: see SEQ ID NO.235; PORIN141-GFP-F: see SEQ ID NO.236; PORIN141-R: see SEQ ID NO.237.

[0125] 2. Genomic Integration of the P34HB Synthetic Gene Cluster

[0126] The specific operation of gene integration was referred to Example 2-2, and different strength templates (porin58-P34HB, porin140-P34HB, The strains obtained were named P9-58, P9-140, P9-141, P10-58, P10-140, P10-141, P15-58, P15-1410, and P15-141 respectively.

[0127] The genomic integration primer sequences (5′-3′) are as follows: 9L-58-P34HB-F: see SEQ ID NO.238; 9L-58-P34HB-R: see SEQ ID NO.239; 9R-58-P34HB-F: see SEQ ID NO.240; 9R-58-P34HB-R: see SEQ ID NO.241; PRE112-LOCUS-9L-F: see SEQ ID NO.242; PRE112-LOCUS-9L-R: see SEQ ID NO.243; PRE112-LOCUS-9R-F: see SEQ ID NO.244; PRE112-LOCUS-9R-R: see SEQ ID NO.245; 9L-140-P34HB-F: see SEQ ID NO.246; 9L-140-P34HB-R: see SEQ ID NO.247; 9L-141-P34HB-F: see SEQ ID NO.248; 9L-141-P34HB-R: see SEQ ID NO.249; 10L-58-P34HB-F: see SEQ ID NO.250; 10L-58-P34HB-R: see SEQ ID NO.251; 10R-58-P34HB-F: see SEQ ID NO.252; 10R-58-P34HB-R: see SEQ ID NO.253; PRE112-LOCUS-10L-F: see SEQ ID NO.254; PRE112-LOCUS-10L-R: see SEQ ID NO.255; PRE112-LOCUS-10R-F: see SEQ ID NO.256; PRE112-LOCUS-10R-R: see SEQ ID NO.257; 10L-140-P34HB-F: see SEQ ID NO.258; 10L-140-P34HB-R: see SEQ ID NO.259; 10L-141-P34HB-F: see SEQ ID NO.260; 10L-141-P34HB-R: see SEQ ID NO.261; 15L-58-P34HB-F: see SEQ ID NO.262; 15L-58-P34HB-R: see SEQ ID NO.263; 15R-58-P34HB-F: see SEQ ID NO.264; 15R-58-P34HB-R: see SEQ ID NO.265; PRE112-LOCUS-15L-F: see SEQ ID NO.266; PRE112-LOCUS-15L-R: see SEQ ID NO.267; PRE112-LOCUS-15R-F: see SEQ ID NO.268; PRE112-LOCUS-15R-R: see SEQ ID NO.269; 15L-140-P34HB-F: see SEQ ID NO.270; 15L-140-P34HB-R: see SEQ ID NO.271; 15L-141-P34HB-F: see SEQ ID NO.272; 15L-141-P34HB-R: see SEQ ID NO.273.

[0128] PCR was used to verify whether the P34HB synthetic gene clusters with different expression intensities were successfully inserted into the LOCUS-9, LOCUS-10, and LOCUS-15 sites. The synthetic gene cluster was aldD-dhaT-orfz, and the nucleotide sequence of aldD-dhaT-orfz was shown in SEQ ID NO.274. The results were as follows Fig.10 As shown, when the target fragment is 5000 bp, it proves that the LOCUS-9, LOCUS-10, and LOCUS-15 sites on the genome have been successfully inserted into P34HB synthetic gene clusters with different expression intensities.

[0129] 3.P34HB synthetic gene cluster expression plasmid fermentation test

[0130] (1) Culture medium:

[0131] 60LB plate culture medium: yeast extract powder 0.5%; tryptone 1%; sodium chloride 60%, agar powder 1.8 g / 100 mL, pH 8.5.

[0132] Component I: magnesium sulfate: 0.2 g / L; urea: 1.0 g / L; (prepared with 50 times concentrated mother liquor: 10 g / L magnesium sulfate, 3 g / L urea);

[0133] Component II: Potassium dihydrogen phosphate (5.2 g / L), prepared with 50 times mother solution of 260 g / L; Glucose solution (30 g / L): prepared with 500 g / L glucose mother solution;

[0134] Component III (10 mL / L): 5 g / L ammonium ferric citrate, 1.5 g / L anhydrous calcium chloride, 41.7 ml / L 12 mol / L hydrochloric acid; Component IV (1 mL / L): 100 mg / L zinc sulfate heptahydrate, 30 mg / L manganese sulfate tetrahydrate, 300 mg / L boric acid, 10 mg / L copper sulfate pentahydrate, 30 mg / L sodium molybdate.

[0135] Fermentation medium (50 MM): glucose 30 g / L, 10 g / L 1,4-butanediol; sodium chloride 50 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, component III: Fe(III)-NH4-Citrate 5 g / L, CaCl2•2H2O 2 g / L, HCl 12 mol / L, component IV: ZnSO4•7H2O 0.1 g / L, MnCl2•4H2O 0.03 g / L, H2BO3 0.3 g / L, CoCl2•6H2O 0.2 g / L, CuSO4•5H2O 0.01 g / L, NiCl2•6H2O 0.02 g / L.

[0136] (2) Strain activation:

[0137] Take the bacterial strain from the -80℃ refrigerator in the laboratory, pick up the bacterial liquid with a pipette tip, streak it on a plate solid culture medium (yeast powder 5 g / L; tryptone 10 g / L; sodium chloride 60 g / L, pH 8.5), and culture it at 37℃ for 24 h.

[0138] (3) Primary seed cultivation:

[0139] Pick a single colony and inoculate it into a 12 mL shaking tube (5 mL 60LB medium: 5 g / L yeast powder; 10 g / L tryptone; 60 g / L sodium chloride; pH 8.5), and culture the culture in a shaker at 37°C and 220 rpm for 12 h.

[0140] (4) Preparation of fermentation medium:

[0141] Fermentation medium (50 MM): glucose 30 g / L, 10 g / L 1,4-butanediol; sodium chloride 50 g / L, yeast powder 1.2 g / L, urea 0.2~3 g / L, anhydrous magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 1.5~5.5 g / L, Fe(III)-NH4-Citrate 5 g / L, CaCl2•2H2O 2 g / L, HCl 12 mol / L, ZnSO4•7H2O 0.1g / L, MnCl2•4H2O 0.03g / L, H2BO30.3 g / L, CoCl2•6H2O 0.2 g / L, CuSO4•5H2O 0.01 g / L, NiCl2•6H2O 0.02 g / L, NaMoO4•2H2O 0.03 g / L.

[0142] (4) Fermentation culture:

[0143] Inoculate 5% of the seed solution (2.5 mL) into a 500 ml conical flask and culture it in a shaker at 37°C and 220 rpm for 48 h.

[0144] (5) Determination of cell dry weight, PHA content and 4HB molar fraction:

[0145] Cell dry weight (CDW): Place 30-35 mL of fermented bacterial liquid in a 50 mL centrifuge tube, centrifuge at room temperature for 6 minutes at 8000 rpm, and discard the supernatant; add appropriate amount of deionized water to restore the original volume, resuspend, ensure that the precipitate disappears completely, centrifuge under the same conditions, and discard the supernatant; seal the centrifuge tube with sealing film and freeze it in a -80℃ refrigerator for 2 h; place the centrifuge tube in a vacuum freeze dryer and dry it for 12-16 hours; weigh and calculate the cell dry weight (g / L).

[0146] Determination of PHA content and 4HB molar fraction: Weigh 0.05 g of the dried bacteria obtained by grinding and fermentation, place in a well-sealed esterification tube, add 2 mL of chloroform, 1700 μL of methanol and 300 μL of concentrated sulfuric acid, react in an oil bath at 100°C for 1 hour, cool to room temperature, add 1 mL of ddH2O, shake and mix thoroughly, and let stand to separate. After the aqueous phase and the organic phase are completely separated, take the chloroform layer (usually the lower layer) and filter it into a liquid phase bottle using a 0.22 μm organic filter membrane. GC detection is performed using a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, 30 m long, 0.25 mm inner diameter and 0.25 μm stationary phase) and a hydrogen flame ionization detector (FID). High-purity nitrogen is used as the carrier gas.

[0147] The program temperature setting is shown in Table 6:

[0148] Table 6 Program temperature settings

[0149]

[0150] The injection volume was 1 μL, and the PHA content and 4HB mole fraction were quantitatively analyzed by the external standard method, and the PHA content and 4HB mole fraction were calculated based on the peak area.

[0151] The fermentation results are shown in Table 7:

[0152] Table 7 Fermentation results of plasmid strains

[0153]

[0154] 4. Fermentation test of P34HB synthetic gene cluster genome integrated strain

[0155] The specific operation steps refer to Example 5-3, and the fermentation results are shown in Table 8:

[0156] Table 8 Fermentation results of genome-integrated strains

[0157]

[0158] According to the data analysis of Tables 7 and 8, whether the P34HB gene cluster is expressed in a plasmid or at different sites in the genome, promoters of different strengths basically do not affect the cell dry weight and PHA content. The dry weight is basically maintained at about 11 g / L, and the PHA content expressed by the genome is almost the same as that expressed by the plasmid. Therefore, according to the fermentation data of the P34HB gene cluster in the strain and the genome-integrated strain, the growth and PHA accumulation of the gene cluster in the genome are almost unaffected, reflecting the stability of the gene cluster expressed at the genome site. However, the proportion of 4HB in the genome-integrated strain is about 5 mol% higher than that fermented in the plasmid. Therefore, these data show that expression through genome integration is more stable than plasmid expression, and the expression intensity at different sites in the genome may be higher.

[0159] Example 6 Testing and characterization of the screening method of the present invention on other microorganisms (Escherichia coli Nissel, Escherichia coli BL21, Halomonas LY01, Halomonas LY03)

[0160] The genome annotation of LY01 was sorted out, and the positions and lengths of all intergenic regions (IGRs) on the LY01 genome were calculated, and 3756 IGRs were obtained. In order to avoid the impact on regulation, the intergenic regions (IGRs) with a length of less than 200 bp and IGRs containing tRNA, rRNA, sRNA sequences and tandem repeat sequences (TRF) were first screened out, and 511 IGRs were obtained. Then the LY01 genome sequence and the transcription factor binding site sequence (TF binding site) of E. coli were compared. The sequences with more than 85% similarity on the LY01 genome were marked as the transcription factor binding sites of LY01, and the intergenic regions containing transcription factor binding sites were screened out, resulting in 123 intergenic regions. Then, in order to avoid the influence of promoters on regulation, the intergenic regions containing promoter sequences were screened out by combining local and online promoter prediction methods, resulting in 120 intergenic regions. Finally, the transcriptomics data of LY01 were compared, and the intergenic regions whose expression intensities of upstream and downstream adjacent genes were in the top 50% of all genes at 9h and 22h were selected, resulting in 11 intergenic regions.

[0161] The nucleotide sequence of the LY01 intergenic spacer region is any one of SEQ ID NO.275, SEQ ID NO.276, SEQ ID NO.277, SEQ ID NO.278, SEQ ID NO.279, SEQ ID NO.280, SEQ ID NO.281, SEQ ID NO.282, SEQ ID NO.283, SEQ ID NO.284, and SEQ ID NO.285, and is named LY01_1 to LY01_11.

[0162] The genome annotation of LY03 was sorted out, and the positions and lengths of all intergenic regions (IGRs) on the LY03 genome were calculated, and 3903 IGRs were obtained. In order to avoid the impact on regulation, the intergenic regions (IGRs) with a length of less than 200 bp and the IGRs containing tRNA, rRNA, sRNA sequences and tandem repeat sequences (TRF) were first screened out, and 428 IGRs were obtained. Then the LY03 genome sequence was compared with the transcription factor binding site sequences (TF bindings) of E. coli, and the sequences on the LY03 genome with a similarity of more than 85% were marked as TF binding sites of LY03, and the IGRs containing transcription factor binding sites were screened out, and 9 IGRs were obtained. Finally, in order to avoid the impact of promoters on regulation, the IGRs containing promoter sequences were screened out by combining local and online promoter prediction methods, and 9 IGRs were still obtained.

[0163] The nucleotide sequence of the LY03 intergenic spacer region is any one of SEQ ID NO.286, SEQ ID NO.287, SEQ ID NO.288, SEQ ID NO.289, SEQ ID NO.290, SEQ ID NO.291, SEQ ID NO.292, SEQ ID NO.293, and SEQ ID NO.294, and is named LY03_1 to LY03_9.

[0164] The genome annotations of Nissel were sorted out, and the positions and lengths of all intergenic regions (IGRs) on the Nissel genome were calculated, resulting in 9713 IGRs. In order to avoid the impact on regulation, the intergenic regions (IGRs) with a length of less than 200 bp and the IGRs containing tRNA, rRNA, sRNA sequences and tandem repeat sequences (TRF) were first screened out to avoid the impact on regulation, and 945 IGRs were obtained. Then the Nissel genome sequence and the transcription factor binding site sequence (TF bindings) of E. coli were compared, and the sequences on the Nissel genome with a similarity of more than 85% were marked as Nissel's transcription factor binding sites, and the IGRs containing transcription factor binding sites were screened out, resulting in 6 IGRs. Finally, in order to avoid the impact of promoters on regulation, the IGRs containing promoter sequences were screened out by combining local and online promoter prediction methods, and 6 IGRs were still obtained.

[0165] The nucleotide sequence of the Nissel intergenic spacer region is any one of SEQ ID NO.295, SEQ ID NO.296, SEQ ID NO.297, SEQ ID NO.298, SEQ ID NO.299, and SEQ ID NO.300, and is named Nissel_1 to Nissel_6.

[0166] The genome annotation of BL21 was sorted out, and the positions and lengths of all intergenic regions (IGRs) on the BL21 genome were calculated, and 8834 IGRs were obtained. In order to avoid the impact on regulation, the intergenic regions (IGRs) with a length of less than 200 bp and the IGRs containing tRNA, rRNA, sRNA sequences and tandem repeat sequences (TRF) were first screened out, and 823 IGRs were obtained. Then the BL21 genome sequence and the transcription factor binding site sequences (TF bindings) of E. coli were compared, and the sequences on the BL21 genome with a similarity of more than 85% were marked as TF binding sites of BL21. The IGRs containing transcription factor binding sites were screened out, and 11 IGRs were obtained. Finally, in order to avoid the impact of promoters on regulation, the IGRs containing promoter sequences were screened out by combining local and online promoter prediction methods, and 4 IGRs were still obtained.

[0167] The nucleotide sequence of the BL21 intergenic spacer region is any one of SEQ ID NO.301, SEQ ID NO.302, SEQ ID NO.303, and SEQ ID NO.304, and is named BL21_1 to BL21_4.

[0168] Table 9 Fluorescence intensity of 28 insertion sites

[0169]

[0170] According to the data analysis of Table 9, the fluorescence intensity of the 28 insertion sites ranged from 123.0 to 711.0, with a median of 380.5 and a mean of 468.6, of which 22 sites (FI / OD600) had a mean greater than 300 and 6 sites (FI / OD600) had a mean greater than 600, indicating that the expression intensity of the introduced genes was higher on the basis of reducing the effect on cell proliferation. The overall screened sites had a good effect, and a few were more prominent. Therefore, these data show the applicability and effectiveness of the screening method for the genomic insertion site.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A microbial genome insertion site, characterized in that: The insertion site is an intergenic spacer region, and the nucleotide sequence of the microbial genome insertion site is shown in any one of SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO.

15.

2. A use of the insertion site as claimed in claim 1 for the stable synthesis of P34HB in halomonas, characterized in that: The following steps are involved: (1) inserting the P34HB synthetic gene cluster into the insertion site of claim 1 to obtain a P34HB synthetic gene cluster integration module, and inserting the P34HB synthetic gene cluster integration module into a plasmid to obtain an integration plasmid containing the P34HB synthetic gene cluster; The P34HB synthetic gene cluster is aldD-dhaT-orfz; (2) The integration plasmid containing the P34HB synthetic gene cluster obtained in step (1) is conjugated with Halomonas to obtain a recombinant Halomonas containing the P34HB synthetic gene cluster, and the recombinant Halomonas is cultured and fermented.

Images