CN106906238B - Multi-copy amplification method and application of streptomycete antibiotic biosynthesis gene cluster - Google Patents
Multi-copy amplification method and application of streptomycete antibiotic biosynthesis gene cluster Download PDFInfo
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Abstract
The invention relates to a multi-copy amplification method and application of a streptomycete antibiotic biosynthesis gene cluster. The invention introduces a plurality of attB sites while deleting some irrelevant gene clusters, realizes multi-copy amplification of the target gene cluster at one time by means of a site-specific recombination system, and greatly improves the yield of target products. The method of the invention is not only simple and effective to operate, but also good in genetic stability of the engineering bacteria.
Description
Technical Field
The invention belongs to the field of metabolic engineering, and particularly relates to a streptomyces antibiotic biosynthesis gene cluster multi-copy amplification method and application.
Background
The natural products (also called secondary metabolites) produced by microorganisms and having biological activity are valuable resources for human beings to resist cancers, aging, infectious diseases and the like. Taking antibiotics as an example, it is statistical that about 50% of the antibiotics of natural origin have been reported to be produced by Streptomyces of the family Actinomycetaceae. The antibiotic biosynthesis gene cluster comprises genes of structure, resistance, efflux, regulation, post-modification and the like, and the size of the gene cluster is generally different from 20 kb to 150 kb. With the rapid development of metabolic engineering, strategies for increasing antibiotic production by design have been developed and applied, including optimizing regulatory networks, reducing competitive pathways, increasing tolerance, increasing precursor supply, and the like. These strategies avoid the large time and labor costs associated with traditional mutagenesis screens and can effectively optimize the biosynthesis of secondary metabolites.
Streptomyces belongs to the family of Streptomyces of the order Actinomycetales, is a prokaryote, a typical representative of actinomycetes, and belongs to the family of aerobic bacteria. It is widely distributed in soil with low water content, good ventilation, rich organic matter and alkalescence, and also in fresh water, seawater and sludge.
Streptomyces pristinaespiralis is a Streptomyces which produces Pristinamycin (Pristamycin) under appropriate conditions.
Although streptomyces has been widely used for producing antibiotics, in view of the complex metabolic mechanism and the large number of uncertain factors in the production process, the technology for optimizing the metabolic pathway of the biosynthetic antibiotics, optimizing the regulation network and the like is still needed to be further developed in the field so as to further improve the yield of the biosynthetic antibiotics.
Disclosure of Invention
The invention aims to provide a streptomyces antibiotic biosynthesis gene cluster multi-copy amplification method and application.
In a first aspect of the invention, there is provided a method of integrating multiple copies of a gene or cluster of genes of interest into the genome of streptomyces, the method comprising:
(1) introducing at least 1 exogenous attB site into a streptomycete genome to obtain a streptomycete carrying the exogenous attB site;
(2) introducing a target gene or a target gene cluster into the streptomycete in the step (1) by a joint transfer method, and selecting the streptomycete with the target gene or the target gene cluster integrated into endogenous attB sites and exogenous attB sites of a streptomycete genome, thereby obtaining the streptomycete integrated with multiple copies of genes or gene clusters.
In a preferred embodiment, the at least 1 exogenous attB site is 1-2 exogenous attB sites; preferably 2 exogenous attB sites.
In another preferred embodiment, said 1-2 exogenous attB sites are introduced into the genome of Streptomyces at 1-2 positions selected from the group consisting of:
the position of a type I polyketone biosynthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in a streptomycete genome is replaced by the original type I polyketone biosynthesis gene cluster at the position; preferably, said type I polyketone biosynthetic gene cluster is located at the site of positions 4050261-4072243, corresponding to the genomic sequence of Streptomyces pristinaespiralis (GenBank accession: NZ _ ABJI 00000000.2).
The position of a non-ribosomal peptide biosynthesis gene cluster (Biosynthetic gene cluster 2, BGC2) in the streptomyces genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; preferably, the non-ribosomal peptide biosynthesis gene cluster is located at the site 1106415-1130923, corresponding to the genomic sequence of Streptomyces pristinaespiralis (GenBank accession No.: NZ _ ABJI 00000000.2); or
The position of a type III polyketone biosynthesis gene cluster (Biosynthetic gene cluster 3, BGC3) in a streptomycete genome is replaced by the original type III polyketone biosynthesis gene cluster at the position; preferably, the non-ribosomal peptide biosynthesis gene cluster is located at the site 686202-69678, corresponding to the genomic sequence of Streptomyces pristinaespiralis (GenBank accession: NZ _ ABJI 00000000.2).
In another preferred embodiment, the streptomyces includes (but is not limited to): streptomyces pristinaespiralis, Streptomyces coelicolor, Streptomyces roseosporus or Streptomyces hygroscopicus.
In another preferred example, the streptomyces is deleted of the papR5 gene and comprises papR4 and papR6 genes.
In another preferred embodiment, 1 endogenous attB site is present in the genome of said Streptomyces.
In another preferred embodiment, the target gene or target gene cluster includes (but is not limited to): pristinamycin component II (PII) synthetic gene cluster, Pristinamycin I (PI) synthetic gene cluster, daptomycin synthetic gene cluster or rapamycin synthetic gene cluster.
In another preferred embodiment, in step (2), the method for introducing the target gene or the target gene cluster into the streptomyces of step (1) by the conjugative transfer method comprises: introducing a plasmid carrying a foreign gene or gene cluster into the streptomyces in the step (1) in a conjugative transfer mode.
In another aspect of the present invention, a streptomycete genetically engineered bacterium is provided, wherein the genome of the genetically engineered bacterium comprises at least 1 exogenous attB site and 1 endogenous attB site.
In a preferred embodiment, the at least 1 exogenous attB site is 1-2 exogenous attB sites; preferably 2 exogenous attB sites.
In another preferred embodiment, said 1-2 exogenous attB sites are introduced into the genome of Streptomyces at 1-2 positions selected from the group consisting of:
the position of a type I polyketone biosynthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in a streptomycete genome is replaced by the original type I polyketone biosynthesis gene cluster at the position;
the position of a non-ribosomal peptide biosynthesis gene cluster (Biosynthetic gene cluster 2, BGC2) in the genome of Streptomyces, and replaces the original non-ribosomal peptide biosynthesis gene cluster at that position.
The type III polyketone biosynthesis gene cluster (Biosynthetic gene cluster 3, BGC3) in the streptomycete genome is positioned, and the original type III polyketone biosynthesis gene cluster at the position is replaced. (preferably, the non-ribosomal peptide biosynthesis gene cluster is located at the site 686202-69678 corresponding to the genomic sequence of Streptomyces pristinaespiralis (GenBank accession: NZ _ ABJI 00000000.2))
In another aspect of the invention, the application of the streptomyces gene engineering bacteria is provided, which is used for preparing streptomyces gene engineering bacteria carrying multiple copies of exogenous genes or gene clusters.
In another preferred embodiment, the multiple copies of the foreign gene or gene cluster are 2-3 copies.
In another aspect of the present invention, there is provided a streptomyces genetically engineered bacterium having multiple copies of a target gene or a target gene cluster integrated into its genome, the genetically engineered bacterium having at most 3 copies of the target gene cluster in its genome, and being located at most 3 of the following positions:
the position of an endogenous attB site in a streptomycete genome is replaced by the original attB site; preferably, the endogenous attB site is located at position 4303498-4303477, corresponding to the genomic sequence of Streptomyces pristinaespiralis (GenBank accession: NZ _ ABJI 00000000.2).
The position of a type I polyketone biosynthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in a streptomycete genome is replaced by the original type I polyketone biosynthesis gene cluster at the position; or
The position of a non-ribosomal peptide biosynthesis gene cluster (Biosynthetic gene cluster 2, BGC2) in the streptomyces genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or
The type III polyketone biosynthesis gene cluster (Biosynthetic gene cluster 3, BGC3) in the streptomycete genome is positioned, and the original type III polyketone biosynthesis gene cluster at the position is replaced.
In another preferred embodiment, the target gene or target gene cluster includes (but is not limited to): pristinamycin component II (PII) synthetic gene cluster, Pristinamycin I (PI) synthetic gene cluster, daptomycin synthetic gene cluster or rapamycin synthetic gene cluster, etc.
In a preferred embodiment, a method for efficiently expressing a target gene or a target gene cluster by using streptomyces is provided, which comprises the following steps:
(a) preparing streptomycete by the method, and integrating a multi-copy target gene or a target gene cluster into a streptomycete genome;
(b) culturing the streptomycete obtained in the step (a) so as to efficiently express the target gene or the target gene cluster.
In another preferred example, in step (b), a macroporous adsorption resin is also added into the culture solution of streptomyces; preferably, 1-12% (w/v) is added; more preferably 3-8% of macroporous adsorption resin.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
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FIG. 1 is a schematic diagram of a gene cluster multicopy amplification strategy. When other non-target gene clusters are knocked out by using a CRISPR \ Cas9 system, an attB site is directly introduced in situ; one, two or three copies of the engineering bacteria can be respectively obtained by introducing BAC-F1F15 (containing a PII synthetic gene cluster) into the modified strain by means of conjugal transfer.
FIG. 2 comparison of efficiency of copy number introduction for different PII synthetic gene clusters.
(A) Efficiency of introducing two copies of the PII synthetic gene cluster simultaneously.
(B) Efficiency of introducing three copies of the PII synthetic gene cluster simultaneously. M stands for DNA marker and C for the control strain Δ papR5+ R4R6 (named SBJ 1000).
FIG. 3 shows the PII (Pristinamycin II) of the engineered bacteria obtained after the introduction of different PII synthetic gene cluster copy numbersA) And (5) comparing the yield.
FIG. 4, PII yield of the engineered bacterium SBJ1003 (containing three exogenously introduced copies of the PII synthetic gene cluster) after addition of 6% macroporous adsorbent resin HB 60.
Detailed Description
In the process of utilizing streptomyces to carry out biosynthesis research related to a target gene cluster, the inventor deletes some gene clusters unrelated to the target gene cluster in the streptomyces in order to obtain a cleaner metabolic profile.
As used herein, the term "site-specific recombination" is a type of homologous recombination that relies on the association of a small range of homologous sequences, requiring the eventual integration of a foreign plasmid into the genome by the participation of an integrase (e.g., phiC31) with a specific recombination site (e.g., attB/attP).
As used herein, the term "introduction" or "transformation" refers to the transfer of an exogenous polynucleotide into a host cell (Streptomyces in the present invention). Alternatively, the exogenous polynucleotide may be integrated into the host genome.
As used herein, a "foreign" or "heterologous" gene or protein refers to a gene or protein that is not naturally contained in the genome of the protozoan organism (Streptomyces in this invention).
The invention provides a method for integrating multiple copies of a target gene or target gene cluster into the genome of streptomyces, which comprises the following steps: (1) introducing at least 1 exogenous attB site into a streptomycete genome to obtain a streptomycete carrying the exogenous attB site; and (2) introducing the target gene or the target gene cluster into the streptomycete in the step (1) by a joint transfer method, and selecting the streptomycete with the target gene or the target gene cluster integrated into endogenous attB sites and exogenous attB sites of the streptomycete genome, thereby obtaining the streptomycete integrated with the multicopy gene or the gene cluster.
At least 1 exogenous attB site, preferably 2 exogenous attB sites, are introduced into the Streptomyces genome. Because 1 attB site is usually endogenous in the streptomycete genome, at least 2 copies of an exogenous target gene or target gene cluster can be introduced; preferably 3 copies of the foreign target gene or target gene cluster are introduced.
The inventor finds in research that the more the copy number is introduced, the better, and when a foreign target gene or a target gene cluster is introduced by using 4 attB sites, the bacterial body is dead due to the cutting of integrase at multiple positions, and the efficiency of obtaining a target strain after introduction is not high.
There are 1 attP site on the plasmid containing the target gene or gene cluster, and after conjugative transfer, it cooperates with attB, so that the site-directed introduction of the exogenous gene or exogenous gene cluster can be realized.
As a preferred mode of the invention, attB sites are introduced, and other gene clusters irrelevant to the biosynthesis of the target gene or the target gene cluster are deleted at specific positions, so that precursor competition is reduced, and a cleaner metabolic profile is obtained.
Deletion of a particular gene cluster and introduction of an attB gene cluster may employ some techniques known in the art, and as a preferred mode of the invention, the CRISPR/Cas9 system is employed to screen for unrelated gene clusters. Currently, in the prior art, there are some plasmids for realizing streptomycete gene editing based on CRISPR/Cas9 principle, such as pKCcas9 dO.
As a preferred mode of the invention, the Streptomyces is Streptomyces pristinaespiralis, and 1-2 exogenous attB sites are introduced and introduced into the genome of the Streptomyces at 1-2 positions selected from the group consisting of:
4050261-4072243 locus in streptomycete genome, and replaces the original I-type polyketone biosynthesis gene cluster (BGC 1); or
1106415-1130923 locus in streptomyces genome, and replacing the original non-ribosomal peptide biosynthesis gene cluster at the position (BGC 2); or
The 686202-696578 site in the streptomycete genome, and replaces the original type III polyketone biosynthesis gene cluster (BGC 3).
More preferably, the Streptomyces pristinaespiralis is Streptomyces sp.sp.deleted of the papR5 gene and overexpresses both papR4 and papR6 genes. Deletion of papR5 or overexpression of papR4 and papR6 can effectively improve PII yield, and obtain a chassis bacterium with a more relaxed regulatory network.
In a preferred embodiment of the present invention, the target gene or the target gene cluster is: pristinamycin component II (PII) the synthetic gene cluster. To enable PII to be expressed efficiently, the inventors also deleted the gene cluster unrelated to pristinamycin synthesis, competing with pristinamycin synthesis for acetyl-coa, malonyl-coa or amino acid precursors when synthesizing secondary metabolites from the gene cluster, while introducing exogenous attB.
The invention also provides the streptomycete genetic engineering bacteria prepared by the method, and the genome of the genetic engineering bacteria comprises at least 1 exogenous attB site and 1 endogenous attB site. Preferably, said at least one exogenous attB site comprises 1 exogenous attB site; preferably 2 exogenous attB sites. More preferably, said 1-2 exogenous attB sites are introduced into the genome of Streptomyces at 1-2 positions selected from the group consisting of:
the position of the I-type polyketone biosynthesis gene cluster in the streptomycete genome is replaced by the original I-type polyketone biosynthesis gene cluster at the position; or
The position of a non-ribosomal peptide biosynthesis gene cluster in the streptomycete genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or
And (3) the position of the type III polyketone biosynthesis gene cluster in the streptomycete genome, and replacing the original type III polyketone biosynthesis gene cluster at the position.
The invention also provides a streptomycete gene engineering bacterium integrated with a multi-copy target gene or a target gene cluster in a genome, wherein the genome of the gene engineering bacterium at most comprises 3 copies of the target gene cluster and is positioned in:
the position of an endogenous attB site in a streptomycete genome is replaced by the original attB site;
the position of the I-type polyketone biosynthesis gene cluster in the streptomycete genome is replaced by the original I-type polyketone biosynthesis gene cluster at the position; or
The position of a non-ribosomal peptide biosynthesis gene cluster in the streptomycete genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or
The type III polyketone biosynthesis gene cluster (Biosynthetic gene cluster 3, BGC3) in the streptomycete genome is positioned, and the original type III polyketone biosynthesis gene cluster at the position is replaced.
In a specific embodiment of the invention, the inventor introduces a plurality of attB sites while deleting other non-target gene clusters so as to realize multi-copy amplification of the target gene cluster. Taking the PII synthetic gene cluster as an example, the PII yield can be improved by 1.5 times by exogenously introducing a PII synthetic gene cluster into streptomyces pristinaespiralis (delta papR5+ R4R 6). To examine whether further improvements in PII production could be achieved by continuing to increase copy number, the inventors introduced an attB site at the same position while deleting other gene clusters not related to pristinamycin synthesis, thereby increasing the number of attB in the genome to allow for insertion of more PII synthetic gene cluster copy numbers. The results show that the efficiency of the simultaneous introduction of two copies and three copies is 100% and 10%, respectively, and the fact that a plurality of attB sites can effectively mediate the amplification of a target gene cluster by using a site-specific recombination mode is proved.
As a preferred mode of the invention, after the streptomycete genetic engineering bacteria are obtained, when fermentation culture is carried out, macroporous adsorption resin is also added into a culture solution of the streptomycete; preferably, 1-12% (w/v) is added; more preferably 3-8% of macroporous adsorption resin.
In the embodiment of the invention, the yield of PII of the engineering bacteria SBJ1001, SBJ1002 and SBJ1003 respectively reaches 468, 508 and 720mg/L after one, two or three copies of the engineering bacteria SBJ1001, SBJ1002 and SBJ1003 are respectively added by taking Streptomyces starterp (delta papR5+ R4R6) (SBJ1000) as the Chassis bacteria, and the yield is obviously improved. The feedback inhibition effect of the pristinamycin on the pristinamycin and the toxic effect on the growth of the thalli are weakened by adding the macroporous adsorption resin, the PII yield of the SBJ1003 reaches 1.75g/L, and the promotion of the yield to the Chassis bacteria is extremely obvious.
The method for the site-specific recombination system-mediated gene cluster multi-copy amplification can be widely used for genetic modification of various actinomycetes, and lays a good foundation for improving the fermentation level of important microorganism natural products.
The method introduces a plurality of attB while deleting other gene clusters, realizes multi-copy amplification of the target gene cluster at one time by means of a site-specific recombination system, and greatly improves the yield of target products. Because the introduced target genes are clustered into discrete distribution, compared with the gene cluster tandem amplification realized by using a ZouA-RsA/B system, the method has the advantages of simple and effective operation and better genetic stability of engineering bacteria, and can be widely used for genetic modification of other industrial actinomycetes.
Because the target gene cluster is discretely distributed after being introduced, homologous recombination is not easy to occur, and the genetic stability of the streptomycete genetic engineering bacteria obtained by the method is good. Therefore, the establishment of the method provides a stable and universal modification strategy for the yield optimization of the secondary metabolites of the actinomycetes, and the site-specific recombination system is utilized to realize the amplification of the copy number of the gene cluster when the competitive path is weakened, so that the high-yield strain with stable heredity is obtained.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
The strains, plasmids and reagents used in the invention:
the invention relates to a prophase construction of Streptomyces pristinaespiralis delta papR5+ R4R6 (named as SBJ1000) in laboratory, wherein the construction method is shown in Li et al, Metabolic Engineering, 2015.
Coli S17-1 (for conjugation transfer): purchased from Biomedal Life Science, inc.
HCCB10218 is preserved in China general microbiological culture Collection center (CGMCC) at 11/23.2011, and the preservation number is CGMCC NO. 5486.
Plasmid pKCcas9dO used in the present invention was constructed as described in Huang et al, Acta BiochimBiophys Sin (Shanghai), 2015.
The plasmid BAC-F1F15 (containing a PII synthetic gene cluster) used in the present invention contains a bacterial chromosomal (BAC) replication unit of E.coli, an apramycin resistance gene aac (3) IV expression cassette, a phiC31 integrase expression cassette, an integration site attP, and an oriT (RK2) site associated with conjugative transfer. See Lei Li et al, Metabolic Engineering,2015 for construction methods.
DNA gel recovery and purification and plasmid extraction kits were purchased from Axygen.
E.Z.N.A.
BAC/PAC DNA Kit was purchased from Omega Bio-Tek.
KOD plus new was purchased from TOYOBO.
The macroporous adsorption resin HB60 is purchased from Shanghai Min Yongchun industries Co.
Other conventional reagents are all made in domestic analytically pure or imported for subpackage.
The culture medium used in the present invention:
1. liquid LB medium (1L)
10g of peptone, 5g of yeast extract and 10g of NaCl, and sterilizing at 121 ℃ for 20 minutes.
2. Solid LB medium (1L)
10g of peptone, 5g of yeast extract, 10g of NaCl and 20g of agar powder; sterilizing at 121 deg.C for 20 min.
3. Solid RP Medium formulation (1L)
20g of soluble starch, 10g of soybean cake powder, 2g of NaCl and KH2PO40.5g，MgSO4.7H2O 1g，CaCO33g of agar powder and 20g of agar powder, adjusting the pH value to 6.4, and sterilizing for 20 minutes at 121 ℃.
4. Seed culture medium (1L)
15g of soluble starch, 10g of glucose, 15g of soybean cake powder, 5g of yeast powder and KNO32.5g，NaCl 2g，CaCO34g, adjusting the pH value to 7.0-7.2, and sterilizing for 20 minutes at 121 ℃.
5. Fermentation medium (1L)
40g of soluble starch, 22.5g of glucose, 32g of cottonseed powder, 3.5g of yeast powder and KH2PO40.1g，ZnSO4.7H2O0.5g，MgSO4.7H2O 0.5g，CaCO36g, adjusting pH to 6.0, and sterilizing at 121 ℃ for 20 minutes.
Example 1 site-specific recombination System-mediated Gene Cluster multicopy amplification strategy
1. Selection of deleted Gene Cluster and attB insertion site
As shown in FIG. 1, the streptomyces genome generally contains only one attB site, and the introduction of a new attB site is favorable for introducing more copies of a target gene cluster. Meanwhile, other gene clusters irrelevant to the synthesis of the pristinamycin are deleted, so that the condition that other secondary metabolites compete with the synthesis of the pristinamycin for precursors such as acetyl coenzyme A, malonyl coenzyme A or amino acid and the like during the synthesis of the pristinamycin can be avoided.
In order to achieve the purpose of deleting unrelated gene clusters and introducing attB sites, the inventors selected 3 non-target gene clusters in Streptomyces pristinaespiralis, which are respectively located at positions 4050261-4072243(BGC1), 1106415-1130923 (BGC2) and 686202-69678 (BCG3) of the genome of Streptomyces pristinaespiralis (GenBank accession: NZ _ ABJI00000000.2), and respectively may encode an unknown type I polyketide, a non-ribosomal peptide and a type III polyketide through repeated research and analysis. It was subjected to one-to-one deletion using CRISPR/Cas9 system and attB sites were added at the same time.
2. Constructing engineering strain by taking PII synthetic gene cluster as target gene cluster
The streptomyces pristinaespiralis SBJ1000 is used as a starting strain, BGC1, BGC2 and BGC3 are respectively used as non-target gene clusters to be deleted, and attB is added at the corresponding deletion position. And constructing an engineering strain by taking the PII biosynthesis gene cluster as a target gene cluster. As in fig. 1.
Taking BGC1 knockout and attB site in-situ introduction as an example, firstly, primers BGC1-UP-fw/rev, BGC1-DOWN-fw/rev and BGC1-sgRNA-fw/DM-sgRNA-rev are adopted, and HCCB10218 genome is taken as a template, PCR amplification is carried out to respectively obtain knockout upstream and downstream homologous arms BGC1-UP, BGC1-DOWN and a gRNA expression element BGC1-sgRNA of targeting BGC 1. Then BGC1-UP-fw and DM-sgRNA-rev are used as primers, the three PCR products are used as templates, BGC1-UP-DOWN-sgRNA is obtained in an overlapping PCR amplification mode, Spe I and Hind III are used for enzyme digestion and are connected into a vector pKCcas9dO digested by the same restriction enzyme, and a BGC1 targeted knockout vector pKCcas9-BGC1-sgRNA is finally obtained. Wherein attB sites have been added to the primers to BGC1-down-fw (see the sequences marked in italics and black underline in the corresponding primers in Table 1). pKCcas9-BGC1-sgRNA is transferred to the Chassis bacteria SBJ1000 in a conjugal manner, and the grown colonies are subjected to PCR identification and sequencing by adopting ID-BGC1-fw and ID-BGC 1-rev. The strain with correct result is cultured in a non-resistant RP culture medium for 2-3 generations to automatically discard the plasmid pKCcas9-BGC1-sgRNA, and the engineering bacterium SBJ1000: attB2 is obtained. In this strain, an exogenous attB replaces the original BGC1 (i.e.position 4050261-4072243 of the genome of Streptomyces pristinaespiralis, the genomic sequence is referred to GenBank accession No.: NZ _ ABJI00000000.2, so that there are 2 attB sites in the genome).
The BGC2 knockout is similar to BGC1, SBJ1000:: attB2 is used as a starting bacterium, and BGC2 knockout is carried out to introduce the 3 rd attB to obtain SBJ1000:: attB 3. The specific method comprises the following steps: firstly, primers BGC2-UP-fw/rev, BGC2-DOWN-fw/rev and BGC2-sgRNA-fw/DM-sgRNA-rev are adopted, and by taking HCCB10218 genome as a template, PCR amplification is carried out to respectively obtain knocked-out upstream and downstream homologous arms BGC2-UP, BGC2-DOWN and a gRNA expression element BGC2-sgRNA of a targeting BGC 2. Then BGC2-UP-fw and DM-sgRNA-rev are used as primers, the three PCR products are used as templates, BGC2-UP-DOWN-sgRNA is obtained in an overlapping PCR amplification mode, Spe I and Hind III are used for enzyme digestion and are connected into a vector pKCcas9dO digested by the same restriction enzyme, and a BGC2 targeted knockout vector pKCcas9-BGC2-sgRNA is finally obtained. Wherein attB sites have been added to the primers to BGC2-down-fw (see the sequences marked in italics and black underline in the corresponding primers in Table 1). pKCcas9-BGC2-sgRNA was transferred into SBJ1000, attB2, and the grown colonies were identified and sequenced by PCR using ID-BGC2-fw and ID-BGC 2-rev. The strain with correct result is cultured in a non-resistant RP culture medium for 2-3 generations to automatically discard the plasmid pKCcas9-BGC2-sgRNA, and the engineering bacterium SBJ1000: attB3 is obtained. In this strain, a new foreign attB was inserted into the original BGC2 (i.e.Streptomyces pristinaespiralis genome, position 1106415-1130923, see GenBank accession No.: NZ _ ABJI00000000.2) in place of the original BGC2, so that 3 attB sites were present in the genome.
SBJ1000: attB4 is obtained by introducing the 4 th attB into starting strain BGC3 knocked out by using SBJ1000: attB 3. The specific method comprises the following steps: firstly, primers BGC3-UP-fw/rev, BGC3-DOWN-fw/rev and BGC3-sgRNA-fw/DM-sgRNA-rev are adopted, and by taking HCCB10218 genome as a template, PCR amplification is carried out to respectively obtain knocked-out upstream and downstream homologous arms BGC3-UP, BGC3-DOWN and a gRNA expression element BGC3-sgRNA of a targeting BGC 3. Then BGC3-UP-fw and DM-sgRNA-rev are used as primers, the three PCR products are used as templates, BGC3-UP-DOWN-sgRNA is obtained in an overlapping PCR amplification mode, Spe I and Hind III are used for enzyme digestion and are connected into a vector pKCcas9dO digested by the same restriction enzyme, and a BGC3 targeted knockout vector pKCcas9-BGC3-sgRNA is finally obtained. Wherein attB sites have been added to the primers to BGC3-down-fw (see the sequences marked in italics and black underline in the corresponding primers in Table 1). pKCcas9-BGC3-sgRNA was transferred into SBJ1000, attB3, and the grown colonies were identified and sequenced by PCR using ID-BGC3-fw and ID-BGC 3-rev. The strain with correct result is cultured in a non-resistant RP culture medium for 2-3 generations to automatically discard the plasmid pKCcas9-BGC3-sgRNA, and the engineering bacterium SBJ1000: attB4 is obtained. In this strain, an exogenous attB replaces the original BGC3 (i.e.686202. sup. 696578 of the genome of Streptomyces pristinaespiralis, see GenBank accession No.: NZ _ ABJI00000000.2) so that there are 4 attB sites in the genome. The primer sequences used in the above method are shown in Table 1.
TABLE 1 primer List
Note: the restriction sites are underlined in positive and the attB sites are marked in italics and in black.
2. Comparison of efficiency when different copy gene clusters are introduced
By utilizing the strategy, the inventor respectively constructs and obtains engineering bacteria SBJ1000:: attB2, SBJ1000:: attB3 and SBJ1000:: attB4, wherein the genome respectively contains 2, 3 and 4 attB sites.
A plasmid (BAC-F1F15) containing a PII synthetic gene cluster is introduced into the SBJ1000, SBJ1000:: attB2 and SBJ1000:: attB3 three strains in a conjugal transfer mode, and a colony is grown, PCR amplification is carried out, and identification is carried out through gel electrophoresis.
The results are shown in FIG. 2, where the efficiency of inserting 2 copies at the same time was 100%, the efficiency of inserting 3 copies was only 10%, and no colonies grew when inserting 4 copies of the PII synthetic gene cluster. It is presumed that when more copies of the PII synthetic gene cluster are introduced, the integrase phiC31 (carried in by the BAC-F1F15 plasmid) cuts at multiple attB sites in the genome but cannot repair in time, resulting in bacterial cell death. The identifying primers inserted with 2 copies are ID-attB-fw/ID-BGC1-attB-fw and ID-BAC-F1F15-rev, and the identifying primers inserted with 3 copies are ID-BGC2-attB-fw and ID-BAC-F1F15-rev except for the two pairs of primers, and the sequences of the identifying primers are shown in Table 1.
Example 2 comparison of PII yields of engineered bacteria after introduction of copy numbers of different PII synthetic Gene clusters
Through the strategy, the inventor constructs engineering bacteria which contain 1, 2 or 3 copies of the PII synthetic gene cluster and are named as SBJ1001, SBJ1002 and SBJ1003 respectively by taking SBJ1000 as a substrate bacterium.
The inventor carries out fermentation experiments on SBJ1001, SBJ1002 and SBJ1003 respectively, and uses SBJ1000, attB2, SBJ1000, attB3 as a control.
(1) Fermentation process and conditions
The streptomyces pristinaespiralis SBJ1000 and the derived engineering bacteria are respectively streaked on an RP plate, are statically cultured for 3-5 days at 30 ℃, are respectively selected with the same volume and inoculated into a seed culture medium, are cultured for 44-48h at 27 ℃ and 240rpm, are transferred into a fermentation culture medium according to the proportion of 8% (v/v) for continuous culture, and are respectively sampled for HPLC determination after 30, 48, 72, 96 and 120 h.
(2) Method for measuring pristinamycin
Sucking culture medium 600 μ l, adding equal volume of acetone, shaking, standing at room temperature for 60min, centrifuging at 12000 rpm for 5min, collecting supernatant, and performing HPLC detection with a column type of Agilent Eclipse, a filler of XDB C185 μm, a size of 4.6mm × 150mm, a wavelength of 206nm, a flow rate of 1ml/min, and a mobile phase of BNitrile: 0.03M KH2PO4Buffer 45: 55.
(3) results of fermentation
The fermentation experiment results show that with the gradual increase of the copy number, the PII (Pristina mycin II)A) The yield is also correspondingly improved, SBJ1001, SBJ1002 and SBJ1003 are 468, 508 and 720mg/L respectively, and are improved by 45%, 57% and 123% respectively relative to the spawn (the yield is 323mg/L), as shown in FIG. 3.
Meanwhile, the inventor finds that the PII yield of the engineering bacteria SBJ1000:: attB2 and SBJ1000:: attB3 is not obviously different from that of the starting bacteria.
The results show that the further increase of PII yield of the engineering bacteria SBJ1002 and SBJ1003 is caused by the increase of copy number of PII synthetic genes but not deletion of other gene clusters.
(4) Genetic stability assay
Meanwhile, the engineering bacteria SBJ1003 are subjected to 5-generation continuous subculture, and the fermentation result shows that the yield of the PII of the offspring is not obviously different from that of the parent generation, so that the engineering bacteria constructed by the integration of the multi-copy gene cluster based on the site-specific recombination system is proved to be very stable in heredity, and homologous recombination is not easy to occur among the same gene clusters.
Example 3 optimization of fermentation Process to further increase the PII yield of engineering bacteria
The inventor also finds that the addition of the macroporous adsorption resin can effectively relieve the toxic effect and further improve the biosynthesis of the pristinamycin because the pristinamycin has stronger inhibition effects on self-synthesis and thallus growth.
Therefore, the inventor adds 6% (w/v) of macroporous adsorption resin HB60 in the SBJ1003 fermentation process, and as a result, found that Pristina mycin IIAThe yield is improved by 1.4 times compared with the yield obtained by fermentation without adding resin, and is also improved by 1.3 times compared with the starting strain SBJ1000 under the same culture conditions. Through a gene cluster multi-copy amplification method and later-stage fermentation optimization, the PII yield is finally improved by 4.4 times, and is increased from the original 323mg/L to 1.75g/L, as shown in FIG. 4.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (13)
1. A method for integrating multiple copies of a target gene or cluster of target genes into the genome of a streptomycete, the method comprising:
(1) introducing at least 1 exogenous attB site into a streptomycete genome to obtain a streptomycete carrying the exogenous attB site; the exogenous attB site is introduced into the streptomyces genome at 1-2 positions selected from the group consisting of: the position of the I-type polyketone biosynthesis gene cluster in the streptomycete genome is replaced by the original I-type polyketone biosynthesis gene cluster at the position; the position of a non-ribosomal peptide biosynthesis gene cluster in the streptomycete genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or the position of the type III polyketone biosynthesis gene cluster in the streptomycete genome, and replacing the original type III polyketone biosynthesis gene cluster at the position;
(2) introducing a target gene or a target gene cluster into the streptomycete in the step (1) by a joint transfer method, and selecting the streptomycete with the target gene or the target gene cluster integrated into endogenous attB sites and exogenous attB sites of a streptomycete genome, thereby obtaining the streptomycete integrated with multiple copies of genes or gene clusters;
wherein the at least 1 exogenous attB site is 1-2 exogenous attB sites.
2. The method of claim 1, wherein said at least 1 exogenous attB site is 2 exogenous attB sites.
3. The method of claim 1, wherein the streptomyces comprises: streptomyces pristinaespiralis, Streptomyces coelicolor, Streptomyces roseosporus or Streptomyces hygroscopicus.
4. The method of claim 1, wherein the target gene or target gene cluster comprises: a pristinamycin component II synthetic gene cluster, a pristinamycin I synthetic gene cluster, a daptomycin synthetic gene cluster or a rapamycin synthetic gene cluster.
5. The method of claim 1, wherein in step (2), the method for introducing the target gene or the target gene cluster into the streptomyces of step (1) by the method of conjugative transfer comprises: introducing a plasmid carrying a foreign gene or gene cluster into the streptomyces in the step (1) in a conjugative transfer mode.
6. A streptomycete genetic engineering bacterium is characterized in that the genome of the genetic engineering bacterium comprises at least 1 exogenous attB site and 1 endogenous attB site; the exogenous attB site is introduced into the streptomyces genome at 1-2 positions selected from the group consisting of: the position of the I-type polyketone biosynthesis gene cluster in the streptomycete genome is replaced by the original I-type polyketone biosynthesis gene cluster at the position; the position of a non-ribosomal peptide biosynthesis gene cluster in the streptomycete genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or the position of the type III polyketone biosynthesis gene cluster in the streptomycete genome, and replacing the original type III polyketone biosynthesis gene cluster at the position; wherein the at least 1 exogenous attB site is 1-2 exogenous attB sites.
7. The streptomyces genetically engineered bacterium of claim 6, wherein the at least 1 exogenous attB site is 2 exogenous attB sites.
8. The use of the streptomyces genetically engineered bacterium as claimed in any one of claims 6 to 7, which is used for preparing streptomyces genetically engineered bacterium carrying multiple copies of foreign genes or gene clusters.
9. A streptomycete genetic engineering bacterium integrated with multiple copies of a target gene or a target gene cluster in a genome is characterized in that the genome of the genetic engineering bacterium comprises 2-3 copies of the target gene cluster and is positioned at 2-3 of the following positions:
the position of an endogenous attB site in a streptomycete genome is replaced by the original attB site;
the position of the I-type polyketone biosynthesis gene cluster in the streptomycete genome is replaced by the original I-type polyketone biosynthesis gene cluster at the position; or
The position of a non-ribosomal peptide biosynthesis gene cluster in the streptomycete genome is replaced by the original non-ribosomal peptide biosynthesis gene cluster at the position; or
And (3) the position of the type III polyketone biosynthesis gene cluster in the streptomycete genome, and replacing the original type III polyketone biosynthesis gene cluster at the position.
10. The streptomyces genetically engineered bacterium having multiple copies of a target gene or target gene cluster integrated into its genome of claim 9, wherein the target gene or target gene cluster comprises: a pristinamycin component II synthetic gene cluster, a pristinamycin I synthetic gene cluster, a daptomycin synthetic gene cluster or a rapamycin synthetic gene cluster.
11. A method for efficiently expressing a target gene or a target gene cluster by using streptomyces, which is characterized by comprising the following steps:
(a) preparing streptomycete by the method of claim 1, wherein a plurality of copies of a target gene or a target gene cluster are integrated into the streptomycete genome;
(b) culturing the streptomycete obtained in the step (a) so as to efficiently express the target gene or the target gene cluster.
12. The method of claim 11, wherein in step (b), a macroporous adsorbent resin is also added to the culture broth of streptomyces.
13. The method of claim 12, wherein 1-12% (w/v) macroporous adsorbent resin is added.
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