CN119265222B

CN119265222B - A screening method for microbial genome insertion sites and its application

Info

Publication number: CN119265222B
Application number: CN202411788219.2A
Authority: CN
Inventors: 叶健文; 林艺娜; 陈婷婷; 李�浩; 吕金艳; 司徒卫; 余柳松
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2024-12-06
Filing date: 2024-12-06
Publication date: 2025-03-25
Anticipated expiration: 2044-12-06
Also published as: CN119265222A

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 monocytic bacteria 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 sites developed by the present invention can provide a powerful tool for the stable integration and expression of exogenous genes in microorganisms.

Description

Screening method and application of microbial genome insertion sites

Technical Field

The invention relates to the technical fields of synthetic biology and metabolic engineering, in particular to a screening method and application of a microorganism genome insertion site.

Background

The way of introducing specific genes into microorganisms and producing large quantities of products by fermentation is simpler, more cost-effective than traditional chemical and enzymatic methods. Many methods have been developed in the past to transform the genome of a cell (e.g., yeast or bacteria). However, conventional transformation methods rely on random insertion of genes within the genome of the cell, which may terminate expression of endogenous traits, or inhibit expression of inserted genes in transgenic cells, etc. Thus, targeted genomic modification of cells is the first mode of application and basic research, targeting transgenes to specific locations in the cell genome would effectively improve the quality of transgenic events, reduce the costs associated therewith, and provide a new approach to the manufacture of transgenic products.

Compared with the traditional microorganism (such as escherichia coli) growing in a mild environment as chassis bacteria, the microorganism living in an extreme environment, in particular to halophilic monad which can perform open fermentation without sterilization, can realize open production in the fermentation industry, and greatly reduces the production cost. However, microorganisms, particularly halophiles, are currently being studied and understood as non-model organisms, and in particular, gene editing tools have been studied and developed very rarely, which limits the researchers' genetic manipulation and metabolite production of halophiles. Therefore, it is of great importance to develop insertion sites and methods for insertion of heterologous or exogenous sequences in microorganisms, especially in the genome of halophiles.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a screening method and application of a microorganism genome insertion site. The invention screens the inter-gene interval region of the microorganism through bioinformatics and transcriptome sequencing, and verifies the expression intensity and stability of the genome of the inter-gene interval region by constructing a fluorescent recombinant vector, and the obtained inter-gene interval region provides a powerful tool for constructing recombinant microorganism, especially halophila genes.

The invention provides a screening method of microorganism insertion sites, wherein the insertion sites are intergenic interval regions, and the screening method comprises the following steps:

(1) Screening out intergenic spacer regions having a length of less than 200 bp and comprising code tRNA, rRNA, sRNA, TRF;

(2) Screening out inter-gene interval regions containing predicted promoter sequences, finding out promoters positioned on the IGR through promoter prediction, and subtracting 25 bp from the upstream or downstream corresponding to the IGR if the upstream gene direction is "-" or the downstream gene direction is "+";

(3) Screening out intergenic spacer regions having a distance of 50 bp or less from the-35 region in the upstream gene promoter to the-35 region in the downstream gene promoter;

(4) An intergenic spacer region comprising a E.coli genome transcription factor binding site in the 1 st to 50 th bp th sites of the-35 th region of the promoter for screening out the upstream gene or the downstream gene;

(5) Screening out intergenic spacer regions where the transcriptional activity of the upstream gene or the downstream gene is not at the first 50% of the transcriptional activity of all genes, to obtain said insertion sites;

(6) Respectively constructing two fluorescent protein plasmids, wherein the first fluorescent protein plasmid comprises fluorescent proteins and the insertion sites in the step (5), and the second fluorescent protein plasmid comprises the fluorescent proteins in the first fluorescent protein plasmid;

(7) Transferring the first fluorescent protein plasmid in the step (6) into a microorganism to obtain recombinant bacteria containing the first fluorescent protein;

(8) Transferring the second fluorescent protein plasmid in the step (6) into a microorganism to obtain recombinant bacteria containing the second fluorescent protein;

(9) Culturing the recombinant bacteria obtained in the fermentation steps (7) and (8), and performing fluorescence measurement.

By the above screening method, 21 insertion sites (intergenic spacer regions) of any one of nucleotide sequences such as 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、SEQ ID NO.21 are screened in halophila. Wherein the intergenic spacer region refers to an intergenic spacer region on a halophila genome, wherein the recombinant halophila refers to insertion of exogenous genes into the intergenic spacer region on the halophila genome, and the exogenous genes include, but are not limited to, fluorescent proteins, P34HB synthesis gene clusters. 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.

Further, in the step (2), screening is performed by salt concentration, urea concentration or fermentation period.

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

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

Further, the fermentation period is 12-72 h. Preferably, the fermentation period is 9-30 h.

Further, the fluorescent proteins in the first fluorescent protein plasmid and the second fluorescent protein plasmid are regulated and controlled by a promoter.

Further, the promoter is any one of porin, porin, 140 and porin, 141. Wherein porin, porin, 140 and porin represent promoters of different intensities, the intensities of the three promoters are compared, and the strongest promoter is porin141,141, porin140,140 and porin58,58.

The invention also provides an application of the intergenic spacer region in stably synthesizing P34HB in halophila, comprising the following steps:

(1) Inserting the P34HB synthetic gene cluster into the inter-gene interval 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 integrated plasmid containing the P34HB synthetic gene cluster;

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

(2) And (3) ligating the integrated plasmid containing the P34HB synthesis gene cluster in the step (1) with halophiles to obtain recombinant halophiles containing the P34HB synthesis gene cluster, and culturing and fermenting the recombinant halophiles.

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.

The present invention also provides an integrative plasmid comprising any one of the nucleotide sequences of the intergenic spacer region.

The strain Halomonas sp.LY 01 used in the present invention is disclosed in patent CN116396886A under the accession number GDMCC No:62635;Halomonas sp LY03 is disclosed in patent CN116970538A under the accession number GDMCC No. 63382.

In conclusion, compared with the prior art, the invention achieves the following technical effects:

The genome insertion sites screened by the screening method have stronger expression intensity and stability, wherein the expression intensity of different insertion sites has diversity, the expression intensity of genes can be changed by selecting different genome insertion sites, and the method of changing the expression intensity of plasmids by using promoters with different intensities is avoided. Meanwhile, the insertion site can provide a powerful tool for the stable integration and expression of exogenous genes in microorganisms, particularly halophiles.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing information of plasmids of insertion site fragments in example 2 of the present invention;

FIG. 2 is a gel diagram of the invention in example 2, which demonstrates the successful insertion of 21 insertion sites into fluorescent protein;

FIG. 3 is a graph showing fluorescence expression intensities of 21 insertion sites in example 2 of the present invention;

FIG. 4 is a diagram showing plasmid information for the reconstruction of the skeleton of pSEVA321 and a fluorescent protein fragment in example 3 of the present invention;

FIG. 5 is a graph showing the comparative analysis of the fluorescence expression intensity of the plasmid and the fluorescence expression intensity of the genome in example 3 of the present invention;

FIG. 6 is a graph showing plasmid information of three kinds of fluorescent proteins with different expression intensities reconstructed from pSEVA321 skeleton in example 4 of the present invention;

FIG. 7 is a graph showing the gel for verifying the insertion of different expressed fluorescent intensities at LOCUS-9, LOCUS-10 and LOCUS-15 insertion sites, respectively, in example 4 of the present invention;

FIG. 8 is a graph showing the comparison analysis of fluorescence expression intensities of three kinds of fluorescent plasmids with different expression intensities and three kinds of templates with different expression intensities inserted into three different insertion sites in example 4 of the present invention;

FIG. 9 is a graph showing plasmid information reconstructed from three promoters with different expression levels and the P34HB synthetic gene cluster in example 5 of the present invention;

FIG. 10 is a schematic diagram of a verification gel of successful insertion of the P34HB synthesis gene cluster into the genome of halophila in example 5 of the invention.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

Example 1 screening method for genomic insertion sites of microorganisms

To avoid effects on regulation, intergenic spacer regions (IGR) with a length of less than 200 bp and IGR containing the coding tRNA, rRNA, sRNA sequence and tandem repeat sequences (TRF) may be screened. In addition, it is necessary to consider the influence factor of the promoter, because the upstream and downstream genes of the IGR have the possibility of combining in four different directions, if the upstream gene direction is "-" or the downstream gene direction is "+", 25bp is subtracted additionally upstream or downstream of the IGR. 50 qualified IGRs were screened at 60 g/L NaCl, 3.6 g/L Urea, and three different fermentation cycles (9 h, 19 h, 30 h), respectively. In order to avoid the action of regulatory sequences, IGRs are further selected in which the distance of the-35 region in the upstream gene promoter to the-35 region in the downstream gene promoter is 50 bp or less. And removing a spacer region affecting the expression of the adjacent gene by predicting on-line software of the promoter, and screening 32 IGRs meeting the conditions. Because binding of transcription factors in the halomonassp.LY 03 genome may interfere with the inserted expression module in the IGR, analysis of transcription factor binding sites on the halomonassp.LY 03 genome is required, the transcription factor binding site on LY03 was found by sequence alignment and homology was calculated, and if the IGR region contained TF Bindingsites sequence with 100% similarity to the transcription factor binding site (TF Bindingsites) in the E.coli genome, the IGR was eliminated. Through the above screening, 21 IGRs meeting the above conditions are finally obtained, and the IGRs are respectively named as LOCUS-1 to 21, and the sequences (5 '-3') of the LOCUS-1 to 21 are as follows:

LOCUS-1 is shown in SEQ ID No.1, LOCUS-2 is shown in SEQ ID No.2, LOCUS-3 is shown in SEQ ID No.3, LOCUS-4 is shown in SEQ ID No.4, LOCUS-5 is shown in SEQ ID No.5, LOCUS-6 is shown in SEQ ID No.6, LOCUS-7 is shown in SEQ ID No.7, LOCUS-8 is shown in SEQ ID No.8, LOCUS-9 is shown in SEQ ID No.9, LOCUS-10 is shown in SEQ ID No.10, LOCUS-11 is shown in SEQ ID No.11, LOCUS-12 is shown in SEQ ID No.12, LOCUS-13 is shown in SEQ ID No.13, LOCUS-14 is shown in SEQ ID No.14, LOCUS-15 is shown in SEQ ID No.15, LOCUS-16 is shown in SEQ ID No.16, LOCUS-17 is shown in SEQ ID No.17, LOCUS-18 is shown in SEQ ID No.18, LOCUS-19 is shown in SEQ ID No.19, LOCUS-20 is shown in SEQ ID No.21 and 21-20 is shown in SEQ ID No.21.

Example 2 verification of expression intensity of screening sites

To examine the expression intensity of the screening site inserted into the genomic position, fluorescent proteins (GFP) were inserted into the finally selected 21 gene spacer regions (IGRs) of example 1 by homologous recombination, respectively, as follows:

1. Plasmid construction of insertion site fragments

GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and plasmid pRE112 skeleton using porin promoter as template were amplified by PCR, and under the action of Gibson ligase, GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and plasmid pRE112 skeleton were recombined to form a new plasmid, named pRE112-LOCUS-1-GFP, and the desired inserted gene was obtained by overlap extension PCR using pRE112-LOCUS-1-GFP as template, and the partial products were sequenced by Bio-company, and the plasmid information was shown in FIG. 1.

Primer sequences (5 '-3') for amplifying GFP fragment, LOCUS-1L fragment, LOCUS-1R fragment and pRE112 plasmid backbone were as follows:

The GFP fragment amplification primer is LOCUS-1R-GFP-R, SEQ ID NO.22, LOCUS-1L-GFP-F, SEQ ID NO.23;

The LOCUS-1L fragment amplification primer is pRE112-LOCUS-1L-F with SEQ ID NO.24, and LOCUS-1L-GFP-R with SEQ ID NO.25;

The LOCUS-1R fragment amplification primer is pRE112-LOCUS-1R-R with SEQ ID NO.26, LOCUS-1R-GFP-F with SEQ ID NO.27;

pRE112 plasmid skeleton amplification primer pRE112-LOCUS-1R-F SEQ ID NO.28 and pRE112-LOCUS-1L-R SEQ ID NO.29.

The amplification system and amplification procedure are shown in tables 1 and 2:

TABLE 1 amplification System Table

TABLE 2 amplification program Table

After the PCR reaction is completed, agarose gel with corresponding concentration is prepared, electrophoresis is carried out to observe the size of DNA bands, the gel is placed under an ultraviolet lamp, the gel of the target DNA fragment is rapidly cut off, and the redundant gel is cut off as much as possible. The concentration of the recovered DNA was measured, and the addition ratio of the DNA was calculated based on the length and concentration of the target fragment and pRE112 skeleton, and was ligated using Gibson enzyme mix, and the Gibson Assembly ligation system and the procedure are shown in Table 3 and Table 4:

Table 3 Gibson Assembly connection System

Table 4 Gibson Assembly connection procedure

Then the connection product is transformed into S17-1 competent cells, the connection product is placed on ice for cooling 2 min immediately after being subjected to ice bath for 30 min and 42 ℃ water bath heat shock 2 min, 400 mu L of LB culture medium (without antibiotics) is added into a centrifuge tube, the mixture is placed into a 37 ℃ shaking table for 200 rpm resuscitation 60 min after uniform mixing, the mixture is centrifuged for 5min for bacterial collection after 5000 rpm and is coated on LB plates with corresponding antibiotics, the mixture is cultured for 12-16 h in a 37 ℃ incubator, and PCR products with correct strip sizes are sent to a biological company for sequencing after colony PCR verification.

The construction of the remaining 20 insertion site fragments (LOCUS-2-21) into the plasmid was performed according to the method of example 1 described above, and the primer sequences (5 '-3') were as follows:

the construction primer of the LOCUS-2 plasmid at the insertion site is LOCUS-2L-GFP-F shown in SEQ ID NO.30, LOCUS-2L-GFP-R shown in SEQ ID NO.31, LOCUS-2R-GFP-F shown in SEQ ID NO.32, LOCUS-2R-GFP-R shown in SEQ ID NO.34, PRE112-LOCUS-2L-F shown in SEQ ID NO.34, PRE112-LOCUS-2L-R shown in SEQ ID NO.35, PRE112-LOCUS-2R-F shown in SEQ ID NO.36, PRE112-LOCUS-2R-R shown in SEQ ID NO.37;

the construction primer of the insertion site LOCS-3 plasmid is LOCS-3L-GFP-F shown in SEQ ID NO.38, LOCS-3L-GFP-R shown in SEQ ID NO.39, LOCS-3R-GFP-F shown in SEQ ID NO.40, LOCS-3R-GFP-R shown in SEQ ID NO.41, PRE 112-LOCS-3L-F shown in SEQ ID NO.42, PRE 112-LOCS-3L-R shown in SEQ ID NO.43, PRE 112-LOCS-3R-F shown in SEQ ID NO.44, PRE 112-LOCS-3R-R shown in SEQ ID NO.45;

the construction primer of the LOCS-4 plasmid at the insertion site is LOCS-4L-GFP-F shown in SEQ ID NO.46, LOCS-4L-GFP-R shown in SEQ ID NO.47, LOCS-4R-GFP-F shown in SEQ ID NO.48, LOCS-4R-GFP-R shown in SEQ ID NO.49, PRE 112-LOCS-4L-F shown in SEQ ID NO.50, PRE 112-LOCS-4L-R shown in SEQ ID NO.51, PRE 112-LOCS-4R-F shown in SEQ ID NO.52, PRE 112-LOCS-4R-R shown in SEQ ID NO.53;

The construction primer of the insertion site LOCS-5 plasmid is LOCS-5L-GFP-F shown in SEQ ID NO.54, LOCS-5L-GFP-R shown in SEQ ID NO.55, LOCS-5R-GFP-F shown in SEQ ID NO.56, LOCS-5R-GFP-R shown in SEQ ID NO.57, PRE 112-LOCS-5L-F shown in SEQ ID NO.58, PRE 112-LOCS-5L-R shown in SEQ ID NO.59, PRE 112-LOCS-5R-F shown in SEQ ID NO.60, PRE 112-LOCS-5R-R shown in SEQ ID NO.61;

The construction primer of the insertion site LOCS-6 plasmid is LOCS-6L-GFP-F shown in SEQ ID NO.62, LOCS-6L-GFP-R shown in SEQ ID NO.63, LOCS-6R-GFP-F shown in SEQ ID NO.64, LOCS-6R-GFP-R shown in SEQ ID NO.65, PRE 112-LOCS-6L-F shown in SEQ ID NO.66, PRE 112-LOCS-6L-R shown in SEQ ID NO.67, PRE 112-LOCS-6R-F shown in SEQ ID NO.68, PRE 112-LOCS-6R-R shown in SEQ ID NO.69;

The construction primer of the insertion site LOCS-7 plasmid is LOCS-7L-GFP-F shown in SEQ ID NO.70, LOCS-7L-GFP-R shown in SEQ ID NO.71, LOCS-7R-GFP-F shown in SEQ ID NO.72, LOCS-7R-GFP-R shown in SEQ ID NO.73, PRE 112-LOCS-7L-F shown in SEQ ID NO.74, PRE 112-LOCS-7L-R shown in SEQ ID NO.75, PRE 112-LOCS-7R-F shown in SEQ ID NO.76, PRE 112-LOCS-7R-R shown in SEQ ID NO.77;

The construction primer of the insertion site LOCS-8 plasmid is LOCS-8L-GFP-F shown in SEQ ID NO.78, LOCS-8L-GFP-R shown in SEQ ID NO.79, LOCS-8R-GFP-F shown in SEQ ID NO.80, LOCS-8R-GFP-R shown in SEQ ID NO.81, PRE 112-LOCS-8L-F shown in SEQ ID NO.82, PRE 112-LOCS-8L-R shown in SEQ ID NO.83, PRE 112-LOCS-8R-R shown in SEQ ID NO.84, PRE 112-LOCS-8R-F shown in SEQ ID NO.85;

The construction primer of the insertion site LOCS-9 plasmid is LOCS-9L-GFP-F shown in SEQ ID NO.86, LOCS-9L-GFP-R shown in SEQ ID NO.87, LOCS-9R-GFP-F shown in SEQ ID NO.88, LOCS-9R-GFP-R shown in SEQ ID NO.89, PRE 112-LOCS-9L-F shown in SEQ ID NO.90, PRE 112-LOCS-9L-R shown in SEQ ID NO.91, PRE 112-LOCS-9R-F shown in SEQ ID NO.92, PRE 112-LOCS-9R-R shown in SEQ ID NO.93;

The construction primer of the insertion site LOCS-10 plasmid is LOCS-10L-GFP-F, LOCS-10L-GFP-R, LOCS-10R-GFP-F, LOCS-10R-GFP-R, PRE 112-LOCS-10L-F, SEQ ID NO.98, PRE 112-LOCS-10L-R, SEQ ID NO.99, PRE 112-LOCS-10R-F, SEQ ID NO.100, PRE 112-LOCS-10R-R, SEQ ID NO.101;

The construction primer of the insertion site LOCS-11 plasmid is LOCS-11L-GFP-F, LOCS-11L-GFP-R, LOCS-11R-GFP-F, LOCS-11R-GFP-R, PRE 112-LOCS-11L-F, PRE 112-LOCS-11L-R, PRE 112-LOCS-11R, and PRE 112-LOCS-11R-R, respectively, with SEQ ID NO.102, SEQ ID NO.103, SEQ ID NO.105, and SEQ ID NO.109, respectively;

The construction primer of the insertion site LOCS-12 plasmid is LOCS-12L-GFP-F, LOCS-12L-GFP-R, LOCS-12R-GFP-F, LOCS-12R-GFP-R, PRE 112-LOCS-12L-F, SEQ ID NO.114, PRE 112-LOCS-12L-R, SEQ ID NO.115, PRE 112-LOCS-12R-F, SEQ ID NO.116, and PRE 112-LOCS-12R-R, SEQ ID NO.117;

the construction primer of the insertion site LOCS-13 plasmid is LOCS-13L-GFP-F, LOCS-13L-GFP-R, LOCS-13R-GFP-F, LOCS-13R-GFP-R, and PRE 112-LOCS-13L-F, respectively, with the sequence ID NO.118, 119, 121, 122, 123, 124, 125, and 125 respectively;

The construction primer of the insertion site LOCS-14 plasmid is LOCS-14L-GFP-F shown in SEQ ID NO.126, LOCS-14L-GFP-R shown in SEQ ID NO.127, LOCS-14R-GFP-F shown in SEQ ID NO.128, LOCS-14R-GFP-R shown in SEQ ID NO.129, PRE 112-LOCS-14L-F shown in SEQ ID NO.130, PRE 112-LOCS-14L-R shown in SEQ ID NO.131, PRE 112-LOCS-14R-F shown in SEQ ID NO.132, PRE 112-LOCS-14R-R shown in SEQ ID NO.133;

The construction primer of the insertion site LOCS-15 plasmid is LOCS-15L-GFP-F, LOCS-15L-GFP-R, LOCS-15R-GFP-F, LOCS-15R-GFP-R, PRE 112-LOCS-15L-F, SEQ ID NO.138, PRE 112-LOCS-15L-R, SEQ ID NO.139, PRE 112-LOCS-15R-F, SEQ ID NO.140, PRE 112-LOCS-15R-141;

The construction primer of the insertion site LOCS-16 plasmid is LOCS-16L-GFP-F shown in SEQ ID NO.142, LOCS-16L-GFP-R shown in SEQ ID NO.143, LOCS-16R-GFP-F shown in SEQ ID NO.144, LOCS-16R-GFP-R shown in SEQ ID NO.145, PRE 112-LOCS-16L-F shown in SEQ ID NO.146, PRE 112-LOCS-16L-R shown in SEQ ID NO.147, PRE 112-LOCS-16R-F shown in SEQ ID NO.148, PRE 112-LOCS-16R-R shown in SEQ ID NO.149;

The construction primer of the insertion site LOCS-17 plasmid is LOCS-17L-GFP-F, LOCS-17L-GFP-R, LOCS-17R-GFP-F, LOCS-17R-GFP-R, PRE 112-LOCS-17L-F, SEQ ID NO.155, PRE 112-LOCS-17R-F, SEQ ID NO.156, and SEQ ID NO.157 respectively;

The construction primer of the insertion site LOCS-18 plasmid is LOCS-18L-GFP-F shown in SEQ ID NO.158, LOCS-18L-GFP-R shown in SEQ ID NO.159, LOCS-18R-GFP-F shown in SEQ ID NO.160, LOCS-18R-GFP-R shown in SEQ ID NO.161, PRE 112-LOCS-18L-F shown in SEQ ID NO.162, PRE 112-LOCS-18L-R shown in SEQ ID NO.163, PRE 112-LOCS-18R-F shown in SEQ ID NO.164, PRE 112-LOCS-18R-R shown in SEQ ID NO.165;

The construction primer of the insertion site LOCS-19 plasmid is LOCS-19L-GFP-F shown in SEQ ID NO.166, LOCS-19L-GFP-R shown in SEQ ID NO.167, LOCS-19R-GFP-F shown in SEQ ID NO.168, LOCS-19R-GFP-R shown in SEQ ID NO.169, PRE 112-LOCS-19L-F shown in SEQ ID NO.170, PRE 112-LOCS-19L-R shown in SEQ ID NO.171, PRE 112-LOCS-19R-F shown in SEQ ID NO.172, PRE 112-LOCS-19R-R shown in SEQ ID NO.173;

the construction primer of the insertion site LOCS-20 plasmid is LOCS-20L-GFP-F shown in SEQ ID NO.174, LOCS-20L-GFP-R shown in SEQ ID NO.175, LOCS-20R-GFP-F shown in SEQ ID NO.176, LOCS-20R-GFP-R shown in SEQ ID NO.177, PRE 112-LOCS-20L-F shown in SEQ ID NO.178, PRE 112-LOCS-20L-R shown in SEQ ID NO.179, PRE 112-LOCS-20R-F shown in SEQ ID NO.180, PRE 112-LOCS-20R-R shown in SEQ ID NO.181;

The construction primer of the insertion site LOCS-21 plasmid is LOCS-21L-GFP-F shown in SEQ ID NO.182, LOCS-21L-GFP-R shown in SEQ ID NO.183, LOCS-21R-GFP-F shown in SEQ ID NO.184, LOCS-21R-GFP-R shown in SEQ ID NO.185, PRE 112-LOCS-21L-F shown in SEQ ID NO.186, PRE 112-LOCS-21L-R shown in SEQ ID NO.187, PRE 112-LOCS-21R-F shown in SEQ ID NO.188, PRE 112-LOCS-21R-R shown in SEQ ID NO.189.

2. Gene integration

(1) The integrated plasmids constructed by the 21 insertion site fragments are respectively subjected to expansion culture, 12-16 h and then are jointed with Halomonas LY 03 in a 20LB plate, 8-h and a small amount of jointed thalli are picked and coated on a 60LB plate with corresponding resistance. 36-48 h and then verifying the monoclonal colony again;

(2) The plate can carry out the de-antibiotics verification after a single colony grows out;

(3) Colonies that were successfully de-antigen were selected for double verification (universal F1/R versus specific F2/R), as shown in FIG. 2, when the target band of 3000 bp appeared, i.e., 21 insertion sites on the genome were confirmed to have successfully inserted GFP, respectively. And (5) selecting the PCR reaction liquid with double verification success, and carrying out measurement, and obtaining 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,YG-21 strain after sequencing success.

3. Fluorometry assay

(1) 21 Preparation strains containing plasmids expressing fluorescent proteins were streaked on 60LB solid plates, respectively, and subjected to activation culture, and the incubator at 37℃was inverted for 24 h.

(2) On an ultra-clean bench, single colonies were picked up and planted in a1 mL deep well plate containing 60LB, cultured at 37℃and 220 rpm for 12 h to obtain seed liquid.

(3) 1. Mu.L of each of the seed solutions was placed in a 1mL deep well plate containing 60LB and three parallel plates were used to continue culturing 12: 12 h.

(4) 20 Mu L of bacterial liquid is taken in 180 mu L of PBS solution, the solution is evenly mixed and placed in a 96-well plate with a cover, 60LB is used as a blank control, the fluorescence intensity is measured by a Thermo Varioskan Lux enzyme-labeled instrument under the conditions that the excitation wavelength is 488 nm and the emission wavelength is 520 nm, and the fluorescence intensities of 21 insertion sites are shown in table 5:

TABLE 5 fluorescence intensity at 21 insertion sites

As shown in FIG. 3, the fluorescence expression intensities of the plasmids constructed by 21 insertion sites, the average fluorescence expression intensity of GFP inserted into the LOCUS1-5 sites is about 200, and the average fluorescence intensity of GFP inserted into LOCUS-7, LOCUS-9, LOCUS-10, LOCUS-14, LOCUS-15, etc. sites is substantially more than 500. Wherein the fluorescence expression intensity at loci LOCUS-9, LOCUS-10 and LOCUS-15 is strong, and the fluorescence average values are 771.7, 791.7 and 796.7 respectively.

Example 3 comparative analysis of genomic site expression intensity and plasmid expression intensity

The data obtained in example 2, which was compared with the fluorescence intensity obtained for plasmids constructed using porin-GFP as a template, demonstrates the nature of the expression intensity at different insertion sites on the genome and the plasmid expression intensity.

Construction of GFP Gene expression plasmid

Plasmid implementation the plasmid pSEVA321 porin-GFP was constructed as a GFP fragment as in example 2-1, and the constructed plasmid is shown in FIG. 4.

The amplification primer sequences (5 '-3') are shown in SEQ ID NO.190 for GFP-R, SEQ ID NO.191 for PORIN-F, SEQ ID NO.192 for PORIN-GFP-F, and SEQ ID NO.193 for PORIN-R.

2. Comparison of plasmid fluorescence assay with genomic fluorescence expression

The fluorescence measurement operation is similar to that of examples 2-3, the fluorescence expression intensity of porin-GFP is monitored, the fluorescence average value of porin-GFP in the expression of pSEVA321 porin-GFP plasmid is 391.7, the fluorescence expression intensities of porin-GFP inserted into 21 sites are compared with the fluorescence expression intensity of the plasmid which is obtained originally, as shown in figure 5, the fluorescence intensities expressed by different sites have a certain proportion relation with the fluorescence intensity expressed by the plasmid itself, the plasmid expression of porin-GFP is basically 4 times of the genome expression of the plasmid which is positioned at the site of LOCUS-13, the fluorescence expression of porin-GFP is about 2 times of the genome expression, the loci of LOCUS-1, LOCUS-4 and LOCUS-11 are basically equivalent to the fluorescence expression intensity of the plasmid which is expressed at the insertion sites which are obtained originally, the fluorescence intensities expressed by the plasmids which are 2 times of the genome expression are also shown in the figure 5, the loci which are LOCUS-9, LOCUS-10 and LOCUS-15 are also present, the genome expression intensities can be changed by the genome expression sites which are different from the genome expression sites, and the genome expression intensities can be further clarified by different proportions of the plasmids, and the genome expression intensities can be further different than the genome expression sites can be set by changing the genome expression intensities.

Example 4 stability analysis of genomic expression

According to the data obtained in example 2, three insertion sites (LOCUS-9, LOCUS-10, LOCUS-15) with stronger fluorescence expression intensity were selected to replace templates (porin-GFP, porin140-GFP, porin 141-GFP) with different expression intensities, and the fluorescence expression intensity was monitored to verify the stability of the screening site.

1. Construction of GFP Gene expression plasmids of different expression intensities

Plasmid construction specific examples referring to example 2-1, GFP fragments of different expression intensities were amplified with plasmids pSEVA321 porin-GFP, pSEVA321 porin-GFP, pSEVA321 porin-GFP template, respectively, and plasmid information is shown in FIG. 6.

The amplified primer sequences (5 '-3') are shown in SEQ ID NO.194, PORIN-F in SEQ ID NO.195, PORIN-GFP-F in SEQ ID NO.196, PORIN-R in SEQ ID NO.197, PORIN-F in SEQ ID NO.198, PORIN-GFP-F in SEQ ID NO.199, PORIN140-R in SEQ ID NO.200, PORIN-141-F in SEQ ID NO.201, PORIN-141-GFP-F in SEQ ID NO.202, PORIN-R in SEQ ID NO.203.

2. Gene integration

Gene integration specific procedures were described in example 2-2, and finally strains with templates (porin-GFP, porin140-GFP, porin-GFP) of different expression intensities at three different sites (LOCUS-9, LOCUS-10, LOCUS-15) were obtained, respectively （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）.

The gene integration primer sequences (5 '-3') are as follows:

The insertion site LOCS-9 gene integrating primer is LOCS-9L-GFP-F, LOCS-9L-GFP-R, LOCS-9R-GFP-F, LOCS-9R-GFP-R, PRE 112-LOCS-9L-F, PRE 112-LOCS-9L-R, and PRE 112-LOCS-9R-R, respectively, is shown in SEQ ID NO.204, SEQ ID NO.205, SEQ ID NO.210, and SEQ ID NO.211, respectively;

The insertion site LOCS-10 gene integrating primer is LOCS-10L-GFP-F, LOCS-10L-GFP-R, LOCS-10R-GFP-F, LOCS-10R-GFP-R, PRE 112-LOCS-10L-F, PRE 112-LOCS-10L-R, PRE 112-LOCS-10R, and PRE 112-LOCS-10R-R, respectively, is shown in SEQ ID NO.212, 213, 214, 215, and 219 respectively;

The insertion site LOCS-15 gene integrating primer is LOCS-15L-GFP-F, LOCS-15L-GFP-R, LOCS-15R-GFP-F, LOCS-15R-GFP-R, PRE 112-LOCS-15L-F, SEQ ID NO.224, PRE 112-LOCS-15L-R, SEQ ID NO.225, PRE 112-LOCS-15R-F, SEQ ID NO.226, and PRE 112-LOCS-15R-R, SEQ ID NO.227.

PCR was performed to verify whether the loci LOCS-9, LOCS-10 and LOCS-15 were successfully inserted into GFP of different expression intensities, and the result is shown in FIG. 7, when the target fragment was 3000 bp, it was confirmed that loci LOCS-9, LOCS-10 and LOCS-15 on the genome were successfully inserted into GFP of different expression intensities, respectively.

3. Fluorometry assay

The GFP plasmids (pSEVA 321 porin-GFP, pSEVA321 porin-GFP, pSEVA321 porin-GFP) of example 4-1, which had different expression levels, were passed through examples 2-3, their fluorescence expression levels were measured, and the fluorescence expression levels of GFP inserted at three different sites with different expression levels were analyzed in comparison with the fluorescence expression levels of the plasmids, as shown in FIG. 8, for the porin-GFP, porin140-GFP, and porin-GFP, and the fluorescence intensity ratios of GFP expressed by the promoters of the three plasmids of different expression levels were about 1:4:8. As compared with the insertion of promoters of different intensities at the same site in the genome, three GFP of different expression intensities, such as porin-GFP, porin140-GFP and porin-GFP, were inserted in LOCUS-9, respectively, the ratio of the expressed fluorescence intensities was also about 1:4:8, and the same results were obtained in LOCUS-10 and LOCUS-15. This demonstrates that promoters of different intensities at a certain site on the genome maintain the ratio of their expression intensities in agreement with the ratio of intensities at the time of plasmid expression, thus demonstrating that the genome expression has stability. However, from the viewpoint of the overall expression intensity, the fluorescence intensities of the expressed GFP at the loci LOCUS-9, LOCUS-10 and LOCUS-15 were all substantially 2 times the plasmid-expressed GFP intensity for the porin-intensity promoters, and the fluorescence intensities of the expressed GFP at the loci LOCUS-9, LOCUS-10 and LOCUS-15 were also substantially 2 times the plasmid-expressed GFP intensity at these three loci by substituting the promoter intensities with porin-140-GFP and porin-GFP, LOCUS-9, LOCUS-10 and LOCUS-15.

EXAMPLE 5 validation of P34HB synthetic Gene Cluster genome expression applications

As is clear from examples 3 and 4, the three sites LOCUS-9, LOCUS-10 and LOCUS-15 are relatively stable in fluorescence expression, and thus promoter-induced gene clusters (porin-aldD-dhaT-orfz, porin140-aldD-dhaT-orfz and porin 141-aldD-dhaT-orfz) with different expression intensities are inserted into the three sites, respectively, and the yields of poly 3-hydroxybutyrate-co-4-hydroxybutyrate (P34 HB) obtained from the three genome expression gene clusters are compared with the plasmid expression, respectively, to compare the stability of the genome expression and the plasmid expression gene clusters.

Construction of expression plasmid for P34HB synthetic Gene Cluster

Specific examples of plasmid construction referring to example 2-1, the P34HB synthase gene clusters were amplified using plasmids pSEVA321 porin-P34 HB, pSEVA321 porin-P34 HB, pSEVA321 porin141-P34HB, respectively, as templates, and the plasmid information thereof is shown in FIG. 9.

The amplified primer sequences (5 '-3') were shown as GFP-R in SEQ ID NO.228, PORIN-F in SEQ ID NO.229, PORIN-GFP-F in SEQ ID NO.230, PORIN-R in SEQ ID NO.231, PORIN-F in SEQ ID NO.232, PORIN-GFP-F in SEQ ID NO.233, PORIN140-R in SEQ ID NO.234, PORIN-141-F in SEQ ID NO.235, PORIN-141-GFP-F in SEQ ID NO.236, PORIN-R in SEQ ID NO.237.

P34HB synthetic Gene Cluster genome integration

Specific procedure for Gene integration referring to example 2-2, strain （LOCUS-9-porin58-P34HB,LOCUS-9-porin140-P34HB,LOCUS-9-porin141-P34HB,LOCUS-10-porin58-P34HB,LOCUS-10-porin140-P34HB,LOCUS-10-porin141-P34HB,LOCUS-15-porin58-P34HB,LOCUS-15-porin140-P34HB,LOCUS-15-porin141-P34HB）, expressing templates of different intensities (porin-P34 HB, porin-P34 HB, porin141-P34 HB) at three different genomic insertion sites (LOCUS-9, LOCUS-10, LOCUS-15), respectively, will eventually give strains designated P9-58, P9-140, P9-141, P10-58, P10-140, P10-141, P15-58, P15-1410, P15-141, respectively.

The genome integration primer sequence (5 '-3') is shown in SEQ ID NO.238 as 9L-58-P34 HB-F; 9L-58-P34HB-R is shown in SEQ ID NO.239;9R-58-P34HB-F is shown in SEQ ID NO.240;9R-58-P34HB-R is shown in SEQ ID NO.241, PRE112-LOCUS-9L-F is shown in SEQ ID NO.242, PRE112-LOCUS-9L-R is shown in SEQ ID NO.243, PRE112-LOCUS-9R-F is shown in SEQ ID NO.244, PRE112-LOCUS-9R-R is shown in SEQ ID NO.245, 9L-140-P34HB-F is shown in SEQ ID NO.246, 9L-140-P34HB-R is shown in SEQ ID NO.247, 9L-141-P34HB-F is shown in SEQ ID NO.248, 9L-141-P34HB-R is shown in SEQ ID NO.249, 10L-58-P34HB-F is shown in SEQ ID NO.250, 10L-58-P34HB-R is shown in SEQ ID NO.252, 10R-58-P34 HB-R253, 10L-9L-140-P34 HB-F is shown in SEQ ID NO.25, 9L-141-P34HB-F is shown in SEQ ID NO.255, 9L-141-P34HB-F is shown in SEQ ID NO. 35, 10L-9-HB-L-9R is shown in SEQ ID NO. 9, 10L-9-R is shown in SEQ ID NO.250, 10L-HB-9 is shown in SEQ ID NO. 10-P34, 10L-HB-P34-HB-P34 is shown in SEQ ID NO. 9, 10-P34, 10-P34, 10-P-34, 10P-34P 34P 34; 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.271, 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.

PCR verifies whether loci LOCS-9, LOCS-10 and LOCS-15 are successfully inserted into P34HB synthetic gene clusters with different expression intensities, wherein the synthetic gene cluster is aldD-dhaT-orfz, the nucleotide sequence of aldD-dhaT-orfz is shown as SEQ ID NO.274, and when the result is shown as figure 10, the target fragment is 5000 bp, namely, the loci LOCS-9, LOCS-10 and LOCS-15 on the genome are respectively successfully inserted into P34HB synthetic gene clusters with different expression intensities.

Fermentation test of P34HB synthetic Gene Cluster expression plasmid

(1) Culture medium:

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

The component I comprises 0.2 g/L of magnesium sulfate, 1.0 g/L of urea, 10 g/L of magnesium sulfate and 3 g/L of urea, which are mixed with 50 times of concentrated mother solution;

Component II, namely potassium dihydrogen phosphate (5.2 g/L) and 50 times of mother solution 260 g/L, and glucose solution (30 g/L) and glucose mother solution 500 g/L;

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

The fermentation medium (50 MM) comprises 30 g/L of glucose, 10 g/L of 1, 4-butanediol, 50 g/L of sodium chloride, 1.2 g/L of yeast powder, 0.2-3 g/L of urea, 0.2 g/L of anhydrous magnesium sulfate, 1.5-5.5 g/L of potassium dihydrogen phosphate, and component III of Fe (III) -NH ₄-Citrate 5 g/L、CaCl₂•2H₂ O2 g/L, HCl, mol/L of component Ⅳ：ZnSO₄•7H₂O 0.1 g/L,MnCl₂•4H₂O 0.03 g/L,H₂BO₃0.3 g/L,CoCl₂•6H₂O 0.2 g/L,CuSO₄•5H₂O 0.01 g/L,NiCl₂•6H₂O 0.02 g/L.

(2) Strain activation:

the strains were taken in a laboratory-80℃refrigerator, streaked with a lance tip and inoculated onto a plate solid medium (yeast powder 5 g/L; tryptone 10 g/L; sodium chloride 60 g/L, pH 8.5), and cultured at 37℃for 24. 24 h.

(3) Primary seed culture:

Single colonies were picked up and inoculated into 12 mL shaking tubes (5 mL 60LB medium: yeast powder 5 g/L; tryptone 10 g/L; sodium chloride 60 g/L; pH 8.5), and the culture broth was placed in a shaking table 37℃and 220 rpm for cultivation of 12 h.

(4) Fermentation medium preparation:

The fermentation medium (50 MM) comprises 30: 30 g/L glucose, 10: 10 g/L1, 4-butanediol, 50: 50 g/L sodium chloride, 1.2: 1.2 g/L yeast powder, 0.2-3 g/L urea, 0.2: 0.2 g/L anhydrous magnesium sulfate and 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.

(4) Fermentation culture:

Seed solution was inoculated (2.5 mL) in 500 ml Erlenmeyer flasks and incubated 48 h on a shaker at 37℃and 220 rpm.

(5) Determination of cell dry weight, PHA content and 4HB mole fraction:

Cell Dry Weight (CDW) is prepared through fermenting 30-35 mL, loading the fermented liquid in 50 mL centrifugal tube, centrifugal at room temp for 6 min at 8000 rpm, pouring supernatant, adding deionized water to restore original volume, resuspension, centrifugal under same condition, pouring supernatant, sealing centrifugal tube in-80 deg.C refrigerator for 2h, drying in vacuum freeze drier for 12-16 hr, weighing, and calculating cell dry weight (g/L).

The PHA content and 4HB mole fraction are determined by weighing 0.05 g dry bacterial cells obtained by fermentation after grinding, placing in an esterification pipe with good sealing property, adding 2 mL chloroform, 1700 mu L methanol and 300 mu L concentrated sulfuric acid, reacting under 100 ℃ oil bath for 1h, cooling at room temperature, adding 1mL volumes of ddH ₂ O, fully shaking, mixing uniformly, standing and layering. After the aqueous and organic phases were completely separated, the chloroform layer (typically the lower layer) was filtered into a liquid phase bottle using a 0.22 μm organic filter, and GC was performed using a GC-7800 gas chromatograph, a capillary column (Rtx-5 type, length 30 m, inner diameter 0.25 mm and stationary phase 0.25 μm) and hydrogen Flame Ion Detection (FID). The carrier gas is high purity nitrogen.

The temperature programming settings are shown in table 6:

TABLE 6 program temperature settings

The sample volume was 1. Mu.L, and the PHA content and 4HB mole fraction were calculated from the peak area by quantitative analysis using an external standard method.

The fermentation results are shown in Table 7:

TABLE 7 fermentation results of plasmid strains

Fermentation test of P34HB synthetic Gene Cluster genome integration Strain

For specific procedures, reference is made to examples 5-3 and the fermentation results are shown in Table 8:

TABLE 8 genome integration Strain fermentation results

According to the data analysis of tables 7 and 8, the P34HB gene cluster is expressed in plasmids or at different sites in a genome, promoters with different intensities basically do not influence the dry weight and PHA content of cells, the dry weight of the promoters 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 plasmids, so that according to the fermentation data of the P34HB gene cluster in the strain and the genome integration strain, the growth and PHA accumulation of the gene cluster in the genome are hardly influenced, and the stability of the genome site expressed gene cluster is reflected. However, the proportion of 4HB in the genomic integration strain was about 5 mol% higher than that fermented in the plasmid, so these data indicate that expression by genomic integration is more stable than plasmid expression and that the expression intensity at different sites in the genome may be higher.

Example 6 testing and characterization of the screening methods of the invention in other microorganisms (E.coli Nissel, E.coli BL21, halophila LY01, halophila LY 03)

The method comprises the steps of sorting genome annotation of LY01, calculating positions and lengths of all inter-gene spacing regions (IGRs) on the LY01 genome to obtain 3756 inter-gene spacing regions, screening out the inter-gene spacing regions (IGRs) with the lengths smaller than 200 bp and IGRs containing a coding tRNA, rRNA, sRNA sequence and a tandem repeat sequence (TRF) to obtain 511 inter-gene spacing regions in order to avoid influence on regulation, comparing LY01 genome sequences with transcription factor binding site sequences (TF bindings) of E.coli, marking sequences with the similarity of more than 85% on LY01 genome as transcription factor binding sites of LY01, screening out the inter-gene spacing regions containing the transcription factor binding sites to obtain 123 inter-gene spacing regions, screening out the inter-gene spacing regions containing a promoter sequence by a local and online promoter prediction combined method to obtain 120 inter-gene spacing regions, and finally comparing transcriptome data of LY01 to obtain the whole inter-gene spacing regions with the adjacent gene expression intensities of 50% before the whole gene spacing regions are expressed in 9h and 22 h.

The nucleotide sequence of the LY01 inter-gene interval 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、SEQ ID NO.285, and is named LY01_1-LY01_11.

The genome annotation of LY03 is arranged, the positions and the lengths of all inter-gene spacing regions (IGRs) on the LY03 genome are calculated to obtain 3903 inter-gene spacing regions, in order to avoid the influence on regulation, firstly, the inter-gene spacing regions (IGRs) with the lengths smaller than 200 bp and the IGRs containing the coding tRNA, rRNA, sRNA sequence and the tandem repeat sequence (TRF) are screened out to obtain 428 inter-gene spacing regions, then the LY03 genome sequence and the transcription factor binding site sequence (TF bindings) of E.coli are compared, the sequences with the similarity exceeding 85% on the LY03 genome are marked as transcription factor binding sites of LY03, the inter-gene spacing regions containing the transcription factor binding sites are screened out to obtain 9 inter-gene spacing regions, and finally, in order to avoid the influence on regulation by the promoter, the inter-gene spacing regions containing the promoter sequence are screened out by a local and online promoter prediction combined method, and the 9 inter-gene spacing regions are still obtained.

The nucleotide sequence of the LY03 inter-gene interval 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、SEQ ID NO.294, and is named LY03_1-LY03_9.

The genome annotation of Nissel is arranged, the positions and the lengths of all inter-gene spacing regions (IGRs) on the genome of Nissel are calculated to obtain 9713 inter-gene spacing regions, in order to avoid the influence on regulation, firstly, the inter-gene spacing regions (IGRs) with the lengths smaller than 200 bp and IGRs containing a coding tRNA, rRNA, sRNA sequence and a Tandem Repeat (TRF) are screened out to obtain 945 inter-gene spacing regions, then the genome sequence of Nissel and a transcription factor binding site sequence (TF bindings) of E.coli are compared, the sequence with the similarity of more than 85% on the genome of Nissel is marked as a transcription factor binding site of Nissel, the inter-gene spacing regions containing the transcription factor binding site are screened out to obtain 6 inter-gene spacing regions, and finally, in order to avoid the influence of a promoter on regulation, the inter-gene spacing regions containing a promoter sequence are screened out by a method combining local and online promoter prediction, and the 6 inter-gene spacing regions are still obtained.

The nucleotide sequence of the interval region between the Nissel genes 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 as Nissel_1-Nissel_6.

The method comprises the steps of sorting genome annotation of BL21, calculating the positions and lengths of all inter-gene interval regions (IGRs) on the BL21 genome to obtain 8834 inter-gene interval regions, screening out the inter-gene interval regions (IGRs) with the length smaller than 200 bp and IGRs containing a coding tRNA, rRNA, sRNA sequence and a tandem repeat sequence (TRF) to obtain 823 inter-gene interval regions in order to avoid influence on regulation, comparing a BL21 genome sequence with a transcription factor binding site sequence (TF bindings) of E.coli, marking sequences with the similarity of more than 85% on the BL21 genome as transcription factor binding sites of BL21, screening out the inter-gene interval regions containing the transcription factor binding sites to obtain 11 inter-gene interval regions, and finally screening out the inter-gene interval regions containing a promoter sequence by a local and online promoter prediction combined method to avoid influence on regulation to obtain 4 inter-gene interval regions.

The nucleotide sequence of the BL21 gene interval 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 BL 21_1-BL21_4.

TABLE 9 fluorescence intensity at 28 insertion sites

According to the data analysis of Table 9, fluorescence intensities of 28 insertion sites ranged from 123.0 to 711.0, median 380.5, mean 468.6, where 22 sites (FI/OD 600) had a mean value of more than 300, and 6 sites (FI/OD 600) had a mean value of more than 600, indicating that the expression intensity of the introduced gene was higher on the basis of reducing the influence on cell proliferation. The overall screened sites have good effect and a small part of the sites are more prominent. These data thus demonstrate the applicability and effectiveness of the screening method for the genomic insertion site.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. The microbial genome insertion site is characterized in that the insertion site is an intergenic interval region, and the nucleotide sequence of the microbial genome insertion site is shown as any one of SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 15.

2. Use of the insertion site according to claim 1 for the stable synthesis of P34HB in halophila, comprising the steps of:

(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 the plasmid to obtain an integrated plasmid containing the P34HB synthetic gene cluster;

The P34HB synthetic gene cluster is aldD-dhaT-orfz;

(2) And (3) ligating the integrated plasmid containing the P34HB synthetic gene cluster obtained in the step (1) with halophiles to obtain recombinant halophiles containing the P34HB synthetic gene cluster, and culturing and fermenting the recombinant halophiles.