CN105176899B - Method for constructing recombinant bacteria for producing or highly producing target gene products, constructed recombinant bacteria and application - Google Patents

Abstract

The invention relates to a method for constructing recombinant bacteria for producing or highly producing target gene products, which comprises the following steps: (1) selecting a starting strain and a target gene; (2) constructing an expression vector containing the target gene; (3) inserting an insulator functional sequence before a ribosome binding site of the target gene in the step (2), and inserting a heterologous or artificially synthesized promoter sequence before the insulator functional sequence; (4) and (4) introducing the expression vector constructed in the step (3) into a growth bacterium. Also relates to a recombinant bacterium constructed according to the method and a method for producing lycopene by fermenting the recombinant bacterium.

Description

Method for constructing recombinant bacteria for producing or highly producing target gene products, constructed recombinant bacteria and application

Technical Field

The invention relates to the field of genetic engineering, in particular to a method for constructing recombinant bacteria for producing or highly producing target gene products, the constructed recombinant bacteria and application.

Background

Animals, plants and microorganisms in nature can synthesize many organic small molecular compounds with structural and functional diversity. Compared with biological macromolecules such as proteins, nucleic acids, lipids and the like, the biological macromolecules do not directly participate in the growth and development of organisms, but have important biological activity and physiological functions. Such active small molecule compounds are referred to as natural products. Many natural products have important uses in medicine or in industrial and agricultural production.

In the traditional methods for developing and mining active natural products, such as high throughput screening and OSMAC (one strain management Compounds) means, it is difficult to find novel drug lead compounds (Zhu H, Sandiford SK, van Wezel GP (2014) triggerers and customers't active anti-infection products, J Ind Microbiol Biotechnol 41(2): 371-386.), compared with the number of the discovered compound molecules, a large number of secondary metabolic genes are still not expressed in the sequenced microbial genome, and the related genes are clustered in the genome and are called as gene clusters. These unexpressed gene clusters are called "silent" or "latent" gene clusters, and these silent gene clusters suggest that a library of natural products (Rebets Y,E,Tokovenko B,Luzhetskyy A(2014)Actinomycetes biosynthetic potential:How to bridge in silico and in vivo？J Ind Microbiol Biotechnol.41(2):387–402.)。

one of the important causes of silencing of these gene clusters is: in the genome of a microorganism, a large number of transcriptional and translational regulatory genes are contained, and these regulatory genes form a negative regulatory network, inhibiting the expression of gene clusters. However, it is difficult to remove these tight controls or activate silent gene clusters by using the controls, and it is difficult to find active natural products. In order to activate these silent gene clusters and obtain active natural products, researchers developed many methods of mining, roughly divided into physiological stimulation and synthetic or genetic biological manipulations against bacterial cells, and both (Aigle B, core C (2012) learning up microorganisms secondary expression of expression. methods enzyme. 517: 343-366. Luo Y, et al. (2013) Activation and transformation of a secretory polycystic mammalian expression vector synthesis enzyme microorganism. Nat. mu.4 (4):2894.Zhuo Y, et al. (2010) transformation of biological expression of protein 1125. expression of protein of expression of protein of biological expression of protein of 1125. expressing expression of protein of expression of protein of expression of protein of expression of biological of expression of. The operation of synthesis or genetic biology adopts an exogenous expression element to bypass the thallus negative control network aiming at the target gene, so as to promote the target gene to express. In the work of overexpression of a target gene using exogenous expression elements, there is some interference between the expression elements, especially between strong expression elements, or during transcription and translation of each element, so that the effect of overexpression is greatly reduced (Lou C, Stanton B, Chen Y-J, Munsky B, Voigt CA (2012) Ribozyme-based expression parts from genetic context. Nat Biotechnology 30(11): 1137-1142.).

Streptomyces gram-positive actinomycetes, which are phylogenetically classified as Streptomyces (Streptomyces), have a complex life cycle and secondary metabolic pathways. Many members of the genus produce abundant biologically active secondary metabolites. Over 70% of the currently known tens of thousands of antibiotics are produced by streptomyces. In addition, it can produce immunosuppressants, antitumor agents, insecticides, and various extracellular hydrolases such as cellulase, amylase, pectinase, protease, etc. (Zhu F, et al (2011) Clustered patterns of species orientations of nature-derived polynucleotides and clones for future bioproscopy. Proc Natl Acad Sci USA.108(31): 12943-12948.) these properties suggest that Streptomyces possesses the precursor metabolic pathways of these classes of natural products and is one of the ideal hosts for the heterologous synthesis of natural products. At present, some streptomyces have completed whole genome sequencing and established standard genetic operation procedures, and provide convenience for heterologous synthesis of natural products, such as streptomyces avermitilis.

Lycopene is a diterpene compound with antioxidant capacity, and is widely applied to the pharmaceutical health care, Food chemical industry and cosmetic industry (Namitha KK, Negi PS (2010) Chemistry and biotechnology of cardiac oil. crit Rev Food Sci 50:728-760), and commercial lycopene is mainly prepared from tomatoes or tomato products by adopting multistage chemical synthesis and chemical extraction (Lin Y, Jain R, Yan Y (2014) microbiological production of antioxidant Food ingredients vitamin a metabolic engineering.Currin Biotechnology.26: 71-78). However, the raw materials are affected by seasons and geography, the complexity of chemical synthesis and the pollution of organic waste limit the industrial application of lycopene. In recent years, with the rise of metabolic engineering and synthetic biology, researchers hope to utilize microorganisms for the fermentative production of lycopene.

Disclosure of Invention

The recombinant bacterium replaces the original regulation and control element of the target gene, eliminates the interference between a promoter and expression elements such as RBS (RBS), and the like, improves the expression of the target gene, thereby increasing the yield of the target gene product.

In one aspect of the present invention, a method for constructing a recombinant bacterium that produces or highly yields a target gene product is provided, comprising the steps of:

(1) selecting a starting strain and a target gene;

(2) constructing an expression vector containing the target gene;

(3) inserting an insulator functional sequence before a ribosome binding site of the target gene in the step (2), and inserting a heterologous or artificially synthesized promoter sequence before the insulator functional sequence;

(4) and (4) introducing the expression vector constructed in the step (3) into a growth bacterium.

In the above construction method, the initiating bacterium comprises a wild-type or genetically engineered prokaryotic microorganism; preferably, Escherichia coli, Streptomyces and Actinomycetes are included.

In the above construction method, the streptomyces comprises wild streptomyces or streptomyces obtained by modification, mutation, mutagenesis or gene recombination; preferably, the Streptomyces is Streptomyces avermitilis MA 4680.

In the above construction method, the target gene includes a gene encoded by a prokaryotic genome and a gene encoded by a eukaryotic cDNA.

In the above construction method, the genes encoded by the genome of the prokaryote include genes encoded in Streptomyces; preferably, the genes include Geranylgeranyl diphosphite synthase (crtE), Phytoene desaturase (crtI) and Phytoene synthse (crtB).

In the above construction method, the heterologous or artificially synthesized promoter includes a modified and modified original promoter sequence, a promoter sequence of another gene in the outbreak bacterium, a promoter sequence in a heterologous strain or an artificially synthesized promoter sequence; preferably, SP12, SP18, SP23, SP26, kasOp, SP43 or SP44 promoters are included.

In the above construction method, the sequence with insulator function is a sequence which has a 5' end-cutting ribozyme function after being transcribed to form RNA, and reduces interference on a ribosome binding sequence and forms an RNA hairpin structure after being transcribed by cutting the sequence before the sequence with insulator function; preferably, LtsvJ, SccJ RiboJ, SarJ, PlmJ, VtmoJ, ChmJ, ScvmJ, SltJ or PlmvJ sequences are included.

In the above construction method, the integrated plasmid vector is used to express the target gene in step (2).

In the construction method, the integrated expression vector is an escherichia coli-streptomyces shuttle vector pIJ 8660.

In another aspect of the present invention, there is provided a recombinant bacterium having a high-yield target gene, wherein the recombinant bacterium has an expression vector comprising the target gene, a sequence having an insulator function is inserted before a ribosome binding site of the target gene, and a heterologous or artificially synthesized promoter is inserted before the sequence having the insulator function.

In the recombinant bacterium, the target gene is a gene cluster comprising a crtE gene, a crtI gene and a crtB gene.

The recombinant strain, wherein the heterologous or artificial promoter comprises a modified and modified original promoter sequence, a promoter sequence of other genes in an original strain, a promoter sequence in a heterologous strain or an artificially synthesized promoter sequence; preferably, SP12, SP18, SP23, SP26, kasOp, SP43 or SP44 promoters are included.

In the recombinant bacterium, the sequence with the insulator function has a ribozyme function sheared at the 5' end after being transcribed to form RNA, and the interference of a ribosome binding sequence is weakened by cutting the sequence in front of the sequence with the insulator function, and the sequence forms an RNA hairpin structure after being transcribed; preferably, LtsvJ, SccJ RiboJ, SarJ, PlmJ, VtmoJ, ChmJ, ScvmJ, SltJ or PlmvJ sequences are included.

The recombinant strain comprises the expression of the target gene as an expression vector of an integrative plasmid vector.

In the recombinant bacteria, the integrative plasmid expression vector is an escherichia coli-streptomycete shuttle vector pIJ 8660.

The starting bacterium for constructing the recombinant bacterium comprises a wild type or prokaryotic microorganism modified by genetic engineering; preferably, Escherichia coli, Actinomycetes and Streptomyces are included.

The recombinant strain, wherein the streptomyces comprises wild streptomyces or streptomyces obtained by modification, mutation, mutagenesis or gene recombination; preferably, the Streptomyces is Streptomyces avermitilis MA 4680.

In still another aspect of the present invention, a method for producing lycopene by fermentation is provided, wherein the recombinant bacterium is any one of the above bacteria.

The invention replaces the original regulation element of the target gene (lycopene gene cluster), utilizes an artificially synthesized promoter to bypass the tight regulation of thallus, adds an insulating element between a promoter region and a Ribosome Binding Site (RBS) region, the insulating element has a 5' end-cut ribozyme function after forming RNA after transcription, reduces the interference on a ribosome binding sequence by cutting off the sequence in front of the sequence, forms an RNA hairpin structure after the transcription of the sequence, better exposes the ribosome binding sequence, well eliminates the interference between the promoter and expression elements such as RBS and the like, constructs a predictable expression system, enables the target gene or gene cluster which cannot be expressed to be expressed, obtains a corresponding product, and achieves the effect of high yield of the target compound by using the artificially synthesized strong promoter.

In terms of host selection, the streptomyces is selected, and is a preferred host for expressing secondary metabolites due to the fact that the streptomyces has a large number of secondary metabolic gene clusters, the olefin structure of the lycopene determines that the lycopene is embedded into cell membranes during microbial fermentation production, and the streptomyces avermitilis is superior to conventional hosts, namely escherichia coli and saccharomyces cerevisiae, the filamentous form of the streptomyces avermitilis endows the streptomyces avermitilis with abundant cell membrane space, so that more space is provided for accumulation of products, and the synthetic gene clusters for encoding the lycopene in the genome of the streptomyces avermitilis are found through analysis of the streptomyces avermitilis genome. However, this gene cluster is silent, and applicants successfully activated this gene cluster by using the above activation method using artificial initiation elements and insulator elements, and obtained lycopene in high yield.

Experiments prove that the yield of lycopene in the recombinant Streptomyces avermitilis SAV-SP44-RiboJ-crtEIB obtained by taking Streptomyces avermitilis MA4680 as an initial strain can reach 82.02 +/-8.69 mg/g DCW after 120 hours of shake flask culture, and is 1.62 times of the reported yield of escherichia coli (50.6mg/g DCW) (Tao S, Miao L, Li Q, et al. (2014) Production of lycopene by metabisubality-engineere Escherichia coli.Bi otech letters.36(7): 1515-one strain 1522.).

The recombinant streptomyces avermitilis gene engineering bacteria constructed by the invention can be directly used for fermentation production of lycopene, so that the yield of lycopene is improved, and the production cost is reduced.

Drawings

FIG. 1 is a schematic structural diagram of an expression vector containing a lycopene gene cluster of a high-yield lycopene recombinant bacterium provided by an embodiment of the invention;

FIG. 2 is a schematic structural diagram of pIJ8660-crtEIB plasmid provided by the embodiment of the invention;

FIG. 3 is a schematic structural diagram of pIJ8660-RiboJ-crtEIB plasmid provided in the embodiments of the present invention;

FIG. 4 is a graph showing the yield of over-expressed lycopene in Streptomyces avermitilis recombinant strains containing different promoters and insulating elements according to the embodiment of the present invention.

Wherein aac (3) IV in FIGS. 2 and 3 is an apramycin resistance gene; oriT is the origin of replication from plasmid RK 2; int is the integrase encoded by phage Φ C31; attP is an attachment sequence corresponding to int integrase; tfd is the transcription terminator Tfd of phage fd; to is the transcription terminator of bacteriophage lambda.

Detailed Description

The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of pIJ8660-Promoters-RiboJ-crtEIB expression plasmid vector

1. cloning of the crtE, crtI and crtB genes

Because of the requirement of the later multi-fragment insertion of the plasmid, three BsaI restriction site sequences need to be synonymously mutated in the crtI and crtB genes, and the three sites are designed and used for amplifying the BsaI restriction site sequences located in Streptomyces avermitilis MA4680(ATCC31267) and can be obtained through ATCC. The genes crtE (SEQ ID NO: 1) [ BA000030.3(1289024 … 1290265) ], crtI (SEQ ID NO: 2) [ BA000030.3(1290262 … 1291803) ] and crtB (SEQ ID NO: 3) [ BA000030.3(1291800 … 1292828) ] on the chromosome.

Fragment E was amplified using primer crtF (SEQ ID NO: 4) and primer crtM1R (SEQ ID NO: 5), fragment I was amplified using primer crtM1F (SEQ ID NO: 6) and primer crtM2R (SEQ ID NO: 7), fragment B1 was amplified using primer crtM2F (SEQ ID NO: 8) and primer crtM3R (SEQ ID NO: 9), and fragment B2 was amplified using primer crtM3F (SEQ ID NO: 10) and primer crtR (SEQ ID NO: 11). The total DNA of Streptomyces avermitilis MA4680 is used as a template, Q5Hot Start High-Fidelity DNA Polymerase of New England Biolabs is adopted for PCR amplification, and the fragment is recovered and purified after agarose gel electrophoresis identification. The pIJ8660 plasmid was digested with BamHI and NotI restriction enzymes from New England Biolabs, purified and recovered, and the pIJ8660 fragment, E fragment, I fragment, B1 fragment and B2 fragment were ligated together using Gibsonassambery kit (New England Biolabs), to obtain plasmid pIJ8660-crtEIB, as shown in FIG. 2.

2. Obtaining pIJ8660-RiboJ-crtEIB plasmid

RiboJ (SEQ ID NO: 30) sequence two pairs of primers, namely crt-RiobJ-Ls (SEQ ID NO: 12) and crt-RiobJ-La (SEQ ID NO: 13), crt-RiobJ-Rs (SEQ ID NO: 14) and crt-RiobJ-Ra (SEQ ID NO: 15), are synthesized, subjected to high-temperature treatment and reannealing to form an oligonucleotide double strand, subjected to T4 polynucleotide kinase of New England Biolabs and then connected with plasmid pIJ8660-crtEIB treated by restriction enzyme BsaI, and the ligase is T4 ligase of New England Biolabs to construct plasmid pIJ8660-RiboJ-crtEIB (see figure 3).

3. Construction of pIJ8660-Promoters-crtEIB expression plasmid vector

The pair primers of 7 promoters, SPL12(crt-SPL12-F/crt-SPL12-R) (SEQ ID NO: 16/SEQ ID NO: 17), SPL18(crt-SPL 4-F/crt-SPL 18-R) (SEQ ID NO: 18/SEQ ID NO: 19), SPL23(crt-SPL23-F/crt-SPL23-R) (SEQ ID NO: 20/SEQ ID NO: 21), SPL26(crt-SPL26-F/crt-SPL26-R) (SEQ ID NO: 22/SEQ ID NO: 23), kasOp (crt-kasOp-F/crt-kasOp-R) (SEQ ID NO: 24/SEQ ID NO: 25), SPL 3(crt-SPL 43-F4642-crt-SPL 43/SEQ ID NO: 737) (SEQ ID NO: 26/SEQ ID NO: 26), SPL44(crt-SPL44-F/crt-SPL44-R) (SEQ ID NO: 28/SEQ ID NO: 29). Each pair of primers was treated at high temperature and then annealed to form oligonucleotide chains, treated with T4 polynucleotide kinase, and then ligated to plasmid pIJ8660-crtEIB treated with restriction enzyme BsaI, using T4 ligase to construct plasmid pIJ8660-Promoters-crtEIB of seven-strength Promoters, see Table 2.

4. Construction of pIJ8660-Promoters-RiboJ-crtEIB expression plasmid vector

Paired primers of 7 promoters, SPL12(crt-SPL12-F/crt-SPL12-R), SPL18(crt-SPL18-F/crt-SPL18-R), SPL23(crt-SPL23-F/crt-SPL23-R), SPL26(crt-SPL26-F/crt-SPL26-R), KasOp (crt-KasOp-F/crt-KasOp-R), SPL43(crt-SPL43-F/crt-SPL43-R), SPL44(crt-SPL44-F/crt-SPL44-R) were synthesized, respectively. Each pair of primers was treated at high temperature and then annealed to form oligonucleotide strands, treated with T4 polynucleotide kinase, and ligated to plasmid pIJ8660-RiboJ-crtEIB treated with restriction enzyme BsaI, using T4 ligase to construct plasmid pIJ8660-Promoters-RiboJ-crtEIB of seven-strength Promoters, see Table 2.

Example 2 transformation of recombinant plasmid

Because of the strong restriction modification effect in the streptomyces avermitilis, the Escherichia coli DH5 alpha is directly combined with the streptomyces avermitilis for transfer, the conversion efficiency is extremely low, and sometimes even transformants cannot be obtained. And the combination transfer is carried out by using E.coli ET12567(PUZ8002) without restriction modification, so that the transformation efficiency is obviously improved. Thus, The constructed recombinant plasmid was transformed into E.coli ET12567(PUZ8002) (Kieser T, Bibb M J, Buttner M J, oral.practical Streptomyces Genetics,2000, Norwich: The John Innes Foundation.) to obtain unmethylated DNA, followed by conjugation transfer.

In the example, Streptomyces avermitilis MA4680 was selected as the starting strain, and the strain produced gray white spores on a plate.

Combining and transferring the negative control plasmid pIJ8660-crtEIB, the negative control plasmid pIJ8660-RiboJ-crtEIB, the lycopene expression plasmid pIJ8660-Promoters-crtEIB of seven Promoters with different strengths and the E.coliET12567(PUZ8002) of the lycopene expression plasmid pIJ8660-Promoters-RiboJ-crtEIB of seven Promoters with different strengths with the streptomyces avermitilis respectively, coating the mixture on an MS (soybean meal powder 2%, mannitol 2% and agar 2%) plate containing 10mM MgCl2, culturing for 16-20 h at 28 ℃, uniformly covering the plate with 1mL of sterile water containing 1000 mu g of nalidixic acid and 1000 mu g of apramycin, culturing for 5-7 days at 28 ℃, growing colonies which are transformants, and carrying out next-step fermentation research after the transformants are subjected to resistance culture and PCR verification.

Example 3 fermentation study of recombinant strains

1. Shake flask fermentation of streptomyces avermitilis

After the Streptomyces avermitilis MA4680 as the starting strain of Streptomyces avermitilis and the recombinant bacteria successfully screened for resistance grow rich spores on a slant culture medium, the lycopene yield level of the Streptomyces avermitilis transformant is tested, three times of experiments are set, the recombinant Streptomyces avermitilis SAV-RiboJ-crtEIB of the transformation plasmid pIJ8660-RiboJ-crtEIB is used as the negative control of the recombinant Streptomyces avermitilis SAV-Promoters-RiboJ-crtEIB of the transformation plasmid pIJ8660-Promoters-RiboJ-crtEIB, and the recombinant Streptomyces avermitilis SAV-Promoters-RiboJ-crtEIB of the transformation plasmid pIJ8660-Promoters-crtEIB is used as the negative control of the recombinant Streptomyces avermitilis SAV-Promoters-crtEIB of the transformation plasmid pIJ8660-Promoters-crtEIB, which is shown in Table 2. Digging inclined surface lawn 1cm²Inoculating a strain to be detected into a seed bottle filled with 25mL of sterilized seed culture medium, and performing shake cultivation at 30 ℃ for 44-48 hours at a rotating speed of 220rpm and a rotating radius of 50mm to obtain a seed culture solution. Inoculating the seed culture solution into a triangular flask containing 50mL of sterilized fermentation medium according to the inoculation amount of 2% (volume percentage), performing shake culture at 28 ℃ for 5 days, centrifuging 2 parts of 5mL fermentation liquor from each sample, collecting thallus, cleaning two sides with sterile water, lyophilizing one part, calculating dry weight, treating one part with 10mg/mL lysozyme for 3hAdding 100 mu L of dimethyl sulfoxide and 400 mu L of acetone, fully shaking and suspending thalli, adding 1mL of dichloromethane, fully shaking, performing ultrasonic treatment for 10min, centrifuging to obtain a supernatant, repeating the steps for several times if the extraction is not complete until the thalli do not have visible red, quantifying an extract, filtering with a 0.2 mu m microporous filter membrane to obtain a filtrate, performing HPLC (Agilent 1200 high performance liquid chromatograph) analysis, and determining the concentration of a fermentation product of the lycopene, wherein the specific HPLC analysis is as described in the following step 2.

In this experiment, the solid medium (soybean meal powder 2%, mannitol 2%, agar 2%), the seed medium (glucose 0.5%, soybean cake powder 1.5%, yeast extract 0.5%, pH 7.2), the fermentation medium components: glucose 60g/L, (NH)₄)₂SO₄2g/L,MgSO₄·7H₂O 0.1g/L,K₂HPO₄ 0.5g/L,NaCl 2g/L,FeSO₄·7H₂O 0.05g/L,ZnSO₄·7H₂O 0.05g/L,MnSO₄·4H₂O 0.05g/L,CaCO₃5g/L, yeast extract 2g/L, pH 7.0.

2. HPLC analysis of fermentation products

HPLC analysis was carried out using an Agilent 1200 series HPLC system (Agilent Technologies Sales & Services GmbH & Co., KG, Waldbronn), lycopene detection wavelength was 280nm, and the sample was eluted isocratically using a ZORBAX SB-C18 column (150 mm. times.4.6 mm, Agilent). The eluent was 30% (vol/vol) acetonitrile/methanol, the elution time was 35 minutes, the sample size was 50. mu.L, the flow rate was 1mL/min, and a lycopene standard (aladdin, Shanghai, China) was used for the measurement of the standard curve.

The fermentation results of the series of strains of Streptomyces avermitilis MA4680 are shown in Table 1, wherein Control is SAV-crtEIB in SAV-microorganisms-crtEIB series recombinant bacteria, and SAV-RiboJ-crtEIB in SAV-microorganisms-RiboJ-crtEIB series recombinant bacteria, and the yield cannot be observed by HPLC, which also proves that the lycopene gene is silenced in Streptomyces avermitilis MA 4680. Wherein the promoter strength level is based on the theoretical strength level of the promoter studied and verified by forensics in the laboratory, measured as kasOp as 100% strength.

From the fermentation results in FIG. 4, it can be seen that lycopene production was gradually increased with the use of different synthetic promoters, but in the absence of the addition of the insulating element RiboJ, a decrease in the level of the promoter was observed above 50% of the level of kasOp, lycopene production was not observed when the promoter reached the theoretical strength of kasOp, which may be due to interference between the expression elements, and that lycopene production gradually increased with the increase in the theoretical strength of the promoter when the insulating element RiboJ was added to the promoter and RBS regions, suggesting that this artificial expression element system has better predictability after the addition of RiboJ. The promoter SP44 and the insulating element RiboJ are used jointly, streptomyces avermitilis MA4680 is used as an initial strain, the recombinant streptomyces avermitilis SAV-SP44-RiboJ-crtEIB is obtained, and after 120 hours of shake flask culture, the yield of lycopene can reach 82.02 +/-8.69 mg/g DCW.

Table 1: overexpression of lycopene yield in streptomyces avermitilis recombinant strain by different promoters and insulating elements

Table 2: strains and plasmid materials for use in the present invention

Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (7)

1. A method for constructing recombinant bacteria for producing or highly producing target gene products is characterized by comprising the following steps:

(1) selecting an initial strain and a target gene, wherein the initial strain is Streptomyces avermitilis MA4680, and the target gene is crtE gene, crtI gene and crtB gene;

(2) constructing an expression vector containing the target gene;

(3) inserting an insulator-functional sequence before the ribosome binding site of the target gene in the step (2), and inserting a heterologous or artificially synthesized promoter sequence before the insulator-functional sequence, wherein the heterologous or artificially synthesized promoter is an SP12, SP18, SP23, SP26, kasOp, SP43 or SP44 promoter, and the insulator-functional sequence is a RiboJ sequence;

(4) and (4) introducing the expression vector constructed in the step (3) into a growth bacterium.

2. The method of claim 1, wherein the desired gene is expressed in step (2) using an integrative plasmid vector.

3. The method of claim 2, wherein the integrative expression vector is the E.coli-Streptomyces shuttle vector pIJ 8660.

4. The recombinant bacterium is characterized in that the recombinant bacterium is provided with an expression vector containing a target gene, a sequence with an insulator function is inserted in front of a ribosome binding site of the target gene, a heterologous or artificial promoter is inserted in front of the sequence with the insulator function, the target gene is a gene cluster containing a crtE gene, a crtI gene and a crtB gene, the heterologous or artificial promoter is SP12, SP18, SP23, SP26, kasOp, SP43 or SP44 promoter, the sequence with the insulator function is a RiboJ sequence, and a growth bacterium used by the recombinant bacterium is Streptomyces avermitilis MA 4680.

5. The recombinant bacterium according to claim 4, wherein the expression vector containing the target gene is an integrative plasmid vector expression vector.

6. The recombinant bacterium of claim 5, wherein the integrative plasmid expression vector is an E.coli-Streptomyces shuttle vector pIJ 8660.

7. A method for producing lycopene by fermentation, characterized in that the recombinant bacterium of any one of claims 4 to 6 is used for fermentation.