CN106906238A - A kind of streptomycete antibiotic biological synthesis gene cluster multicopy amplification method and application - Google Patents
A kind of streptomycete antibiotic biological synthesis gene cluster multicopy amplification method and application Download PDFInfo
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Abstract
The present invention relates to a kind of streptomycete antibiotic biological synthesis gene cluster multicopy amplification method and application.The present invention is while some uncorrelated gene clusters are deleted, introducing multiple attB sites, by site-specific recombination system, once realizes that target gene cluster multicopy is expanded, and greatly improves target product yield.The method of the present invention is not only simple to operate effective, and the genetic stability of engineering bacteria is good.
Description
Technical field
The invention belongs to metabolic engineering field, more particularly it relates to a kind of streptomycete antibiotic biosynthesis base
Because of cluster multicopy amplification method and application.
Background technology
Microorganism produce the natural products (also known as secondary metabolite) with bioactivity, be the mankind resistance cancer,
The treasure of aging and infectious diseases etc..By taking antibiotic as an example, according to statistics, in the antibiotic of the natural origin reported
In, having 50% or so is produced by the streptomycete of Actinomy cetaceae.Antibiotic biological synthesis gene cluster contain structure, resistance,
The gene such as outer row, regulation and control and rear modification, gene cluster size is general in 20-150kb.Along with the rapid hair of metabolic engineering
Exhibition, improved by designing antibiotic yield strategy be developed with application, including Optimum Regulation network, reduce competition approach, carry
Height endurability, increase precursor supply etc..These strategies avoid required substantial amounts of time and human cost when classic mutagenesis are screened,
The biosynthesis of secondary metabolite can effectively be optimized.
Streptomyces, in the Streptomycetaceae of Actinomycetal, is a kind of prokaryotes, and it is the Typical Representative of actinomyces, is belonged to
Aerobic bacteria.It is distributed widely in that water content is relatively low, ventilation is good, in the abundant and subalkaline soil of organic matter, in fresh water, seawater
And be also distributed in mud.
Rotation streptomycete (Streptomyces pristinaespiralis) be it is a kind of under suitable condition, Ke Yisheng
Produce the streptomycete of pyostacin (Pristinamycin).
Although streptomycete has more been applied to produce antibiotic now, in view of its metabolic mechanism is complicated, production
During uncertain factor it is more, this area still need further exploitation optimize its biosynthesis antibiotic metabolism on the way
The technology of footpath, Optimum Regulation network etc., to further improve the yield of its biosynthesis antibiotic.
The content of the invention
It is an object of the invention to provide a kind of streptomycete antibiotic biological synthesis gene cluster multicopy amplification method and should
With.
In the first aspect of the present invention, there is provided one kind makes to be integrated into multicopy target gene or target in streptomyces gene group
The method of gene cluster, methods described includes：
(1) the attB sites of at least one external source are introduced in streptomyces gene group, acquisition carries external source attB sites
Streptomycete；
(2) method by engaging transfer, target gene or target gene cluster are incorporated into the streptomycete of step (1),
Selection target gene or target gene cluster are incorporated into the strepto- in the endogenous attB sites of streptomyces gene group and external source attB sites
Bacterium, so as to obtain the streptomycete for being integrated into multi-copy gene or gene cluster.
In a preference, the attB sites of described at least one external source are the 1-2 attB site of external source；Preferably
Ground is 2 attB sites of external source.
In another preference, the attB sites of 1-2 described external source are incorporated into streptomyces gene group selected from following
1-2 position：
I types polyketone biological synthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in streptomyces gene group
Position, and replace the former I types polyketone biological synthesis gene cluster of the position；It is preferred that corresponding to rotation streptomycete genome
Sequence (GenBank accession number：NZ_ABJI00000000.2), described I type polyketone biological synthesis gene cluster is located at the
The site of 4050261-4072243.
Non-ribosomal peptides biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 2,
BGC2) position, and replace the former non-ribosomal peptides biological synthesis gene cluster of the position；It is preferred that corresponding to the rotation strepto- that begins
Bacterium genome sequence (GenBank accession number：NZ_ABJI00000000.2), described non-ribosomal peptides biological synthesis gene cluster
Positioned at the site of 1106415-1130923；Or
Type III polyketone biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 3,
BGC3) position, and replace the former type III polyketone biological synthesis gene cluster of the position；It is preferred that corresponding to rotation streptomycete
Genome sequence (GenBank accession number：NZ_ABJI00000000.2), described non-ribosomal peptides biological synthesis gene cluster position
In the site of 686202-696578.
In another preference, described streptomycete includes but is not limited to：Rotation streptomycete, streptomyces coelicolor, rose
Rare spore streptomycete or streptomyces hygroscopicus.
In another preference, described streptomycete is missing from papR5 genes and comprising papR4 and papR6 genes.
In another preference, there is 1 endogenous attB site in the genome of described streptomycete.
In another preference, described target gene or target gene cluster is included but is not limited to：Pyostacin component
II (PII) synthetic gene cluster, pyostacin I (PI) synthetic gene cluster, Daptomycin synthetic gene cluster or rapamycin synthesis base
Because of cluster.
In another preference, in step (2), target gene or target gene cluster are introduced by the method for engaging transfer
Include to the method in the streptomycete of step (1)：By the plasmid of foreign gene-carrying or gene cluster to engage transfer in the way of lead
Enter in the streptomycete of step (1).
In another aspect of this invention, there is provided a kind of streptomyces gene engineering bacteria, wrapped in the genome of the genetic engineering bacterium
Include the attB sites of at least one external source, 1 endogenous endogenous attB site.
In a preference, the attB sites of described at least one external source are the 1-2 attB site of external source；Preferably
Ground is 2 attB sites of external source.
In another preference, the attB sites of 1-2 described external source are incorporated into streptomyces gene group selected from following
1-2 position：
I types polyketone biological synthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in streptomyces gene group
Position, and replace the former I types polyketone biological synthesis gene cluster of the position；
Non-ribosomal peptides biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 2,
BGC2) position, and replace the former non-ribosomal peptides biological synthesis gene cluster of the position.
Type III polyketone biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 3,
BGC3) position, and replace the former type III polyketone biological synthesis gene cluster of the position.(it is preferred that corresponding to the rotation strepto- that begins
Bacterium genome sequence (GenBank accession number：NZ_ABJI00000000.2), described non-ribosomal peptides biological synthesis gene cluster
Positioned at the site of 686202-696578.)
In another aspect of this invention, there is provided the purposes of described streptomyces gene engineering bacteria, for preparing, carrying to be copied
The streptomyces gene engineering bacteria of shellfish foreign gene or gene cluster.
In another preference, described multicopy foreign gene or gene cluster is 2-3 copies.
In another aspect of this invention, there is provided multicopy target gene or target gene cluster are integrated with a kind of genome
Streptomyces gene engineering bacteria, includes up to 3 target gene clusters of copy in the genome of the genetic engineering bacterium, and positioned at following
In position as many as 3 positions：
Endogenous attB sites position in streptomyces gene group, and replace the original attB sites in the position；It is preferred that
Corresponding to rotation streptomycete genome sequence (GenBank accession number：NZ_ABJI00000000.2), described endogenous attB
Point is located at the site of 4303498-4303477.
I types polyketone biological synthesis gene cluster (Biosynthetic gene cluster 1, BGC1) in streptomyces gene group
Position, and replace the former I types polyketone biological synthesis gene cluster of the position；Or
Non-ribosomal peptides biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 2,
BGC2) position, and replace the former non-ribosomal peptides biological synthesis gene cluster of the position；Or
Type III polyketone biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 3,
BGC3) position, and replace the former type III polyketone biological synthesis gene cluster of the position.
In another preference, described target gene or target gene cluster is included but is not limited to：Pyostacin component
II (PII) synthetic gene cluster, pyostacin I (PI) synthetic gene cluster, Daptomycin synthetic gene cluster or rapamycin synthesis base
Because of cluster etc..
In a preference, there is provided a kind of method of utilization streptomycete high efficient expression target gene or target gene cluster,
Described method includes：
A () prepares streptomycete in described method, make to be integrated into multicopy target gene or target in streptomyces gene group
Gene cluster；
B streptomycete that () incubation step (a) is obtained, so that high efficient expression target gene or target gene cluster.
In another preference, in step (b), also macroporous absorbent resin is added in the nutrient solution of streptomycete；It is preferred that
Add 1-12% (w/v)；The more preferably macroporous absorbent resin of 3-8%.
Other side of the invention, due to this disclosure, is to those skilled in the art apparent
's.
Brief description of the drawings
Fig. 1, the tactful schematic diagram of gene cluster multicopy amplification.Using CRISPR Cas9 systems knock out other non-targeted bases
During because of cluster, direct in-situ introduces an attB site；BAC-F1F15 (is contained into PII synthetic genes by engaging branch mode
Cluster) import in improved bacterial strain, can respectively obtain the engineering bacteria for increasing one, two or three copy.
The efficiency comparative that Fig. 2, difference PII synthetic genes cluster copy number are introduced.
(A) while introducing two efficiency of copy of PII synthetic genes cluster.
(B) while introducing three efficiency of copy of PII synthetic genes cluster.M representation DNAs marker, C represent control strain
Δ papR5+R4R6 (is named as SBJ1000).
PII (the Pristinamycin II of the engineering bacteria that Fig. 3, difference PII synthetic gene clusters copy number are obtained after introducingA)
Yield comparison.
Fig. 4, engineering bacteria SBJ1003 (comprising the three PII synthetic genes clusters copy being exogenously introduced) are in the macropore of addition 6%
PII yield after polymeric adsorbent HB60.
Specific embodiment
It is more clean in order to obtain in the related biosynthesis research process of target gene cluster is carried out using streptomycete
Metabolism spectrum, the present inventor deletes some and the incoherent gene cluster of target gene cluster in streptomycete, is found during being somebody's turn to do, and deletes
While some non-targeted gene clusters, attB sites directly are re-introduced into corresponding position, by carrying out engagement transfer, be capable of achieving
The disposable multicopy insertion of external source target gene cluster, the related biosynthesis of further optimization aim gene cluster.
As used herein, term " locus specificity restructuring " is that a class depends on the homologous heavy of small range homologous sequence joint conference
Prescription formula, it is necessary to by the participation of integrase (such as phiC31) and specific recombination site (such as attB/attP), most external source at last
In plasmid integration to genome.
As used herein, term " introducing " or " conversion " refers to that exogenous polynucleotide is transferred into the host cell (present invention
In be streptomycete).Alternatively, exogenous polynucleotide can be integrated into host genome.
As used herein, " external source " or " heterologous " gene or albumen refer to not to be naturally contained in primary object (this
It is streptomycete in invention) gene or albumen in genome.
The invention provides a kind of side for making to be integrated into multicopy target gene or target gene cluster in streptomyces gene group
Method, methods described includes：(1) the attB sites of at least one external source are introduced in streptomyces gene group, acquisition carries external source
The streptomycete in attB sites；(2) method by engaging transfer, step (1) is incorporated into by target gene or target gene cluster
Streptomycete in, selection target gene or target gene cluster are incorporated into the endogenous attB sites of streptomyces gene group and external source attB
The streptomycete in site, so as to obtain the streptomycete for being integrated into multi-copy gene or gene cluster.
The attB sites of at least one external source are introduced in streptomyces gene group, 2 the attB of external source is preferably introduced
Point.There is 1 attB site due to generally endogenous in streptomyces gene group, so as at least 2 external source targets of copy can be introduced
Gene or target gene cluster；Preferably introduce 3 the external source target genes or target gene cluster of copy.
The present inventor has found that the copy number of introducing is not The more the better under study for action, outside being introduced using 4 attB sites
When source target gene or target gene cluster, cause thalline dead because integrase occurs cutting in many places, mesh is obtained after introducing
Bacterial strain efficiency it is not high.
There is 1 attP site on plasmid containing target gene or gene cluster, after transfer is engaged, it is common with attB
Effect, so as to can realize that foreign gene or the fixed point of foreign gene cluster are introduced.
As preferred embodiment of the invention, while attB sites are introduced, also deleted and target on specific position
Incoherent other gene clusters of biosynthesis of gene or target gene cluster, so as to reduce precursor competition, obtain more clean
Metabolism spectrum.
Deleting specific gene cluster and introduce attB gene clusters can be using some technology well known in the art, as this
The preferred embodiment of invention, is sieved using CRISPR/Cas9 systems and subtracts incoherent gene cluster.At present, in the prior art, had
Some are used for the plasmid for realizing carrying out streptomyces gene editor based on CRISPR/Cas9 principles, such as pKCcas9dO.
Used as preferred embodiment of the invention, described streptomycete is rotation streptomycete, introduces 1-2 the attB of external source
Point, and be incorporated into streptomyces gene group selected from 1-2 following position：
4050261-4072243 sites in streptomyces gene group, and replace the former I types polyketone biosynthesis gene of the position
Cluster (BGC1)；Or
1106415-1130923 sites in streptomyces gene group, and replace the former non-ribosomal peptides biosynthesis of the position
Gene cluster (BGC2)；Or
The site of 686202-696578 in streptomyces gene group, and replace the biological conjunction of former type III polyketone of the position
Into gene cluster (BGC3).
It is further preferable that described rotation streptomycete is missing from papR5 genes and simultaneously overexpression papR4 and papR6 bases
The streptomycete of cause.Missing papR5 or overexpression papR4 and papR6 can effectively improve PII yield, and obtain regulated and control network
More lax chassis bacterium.
Used as preferred embodiment of the invention, described target gene or target gene cluster is：Pyostacin component II (PII)
Synthetic gene cluster.In order that PII can efficiently be expressed, introduce external source attB while, the present inventor also delete with
Pyostacin synthesis it is incoherent, gene cluster synthesize secondary metabolite when with pyostacin synthesis compete acetyl coenzyme A, third
The gene cluster of two acyl coenzyme A or amino acid precursor.
Present invention also offers streptomyces gene engineering bacteria prepared by application preceding method, the genome of the genetic engineering bacterium
Include the attB sites of at least one external source, 1 endogenous endogenous attB site.It is preferred that described is at least outer containing 1
The attB sites in source；The preferably 2 attB sites of external source.More preferably, the attB sites of 1-2 described external source are incorporated into
1-2 following position is selected from streptomyces gene group：
I types polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former I types polyketone of the position to give birth to
Thing synthetic gene cluster；Or
Non-ribosomal peptides biological synthesis gene cluster position in streptomyces gene group, and replace the former non-ribose of the position
Body peptide biological synthesis gene cluster；Or
Type III polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former type III of the position to gather
Ketone biological synthesis gene cluster.
Present invention also offers the streptomycete base that multicopy target gene or target gene cluster are integrated with a kind of genome
Because of engineering bacteria, 3 target gene clusters of copy are included up in the genome of the genetic engineering bacterium, and be located at：
Endogenous attB sites position in streptomyces gene group, and replace the original attB sites in the position；
I types polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former I types polyketone of the position to give birth to
Thing synthetic gene cluster；Or
Non-ribosomal peptides biological synthesis gene cluster position in streptomyces gene group, and replace the former non-ribose of the position
Body peptide biological synthesis gene cluster；Or
Type III polyketone biological synthesis gene cluster in streptomyces gene group (Biosynthetic gene cluster 3,
BGC3) position, and replace the former type III polyketone biological synthesis gene cluster of the position.
In a particular embodiment of the present invention, the present inventor introduces multiple while other non-targeted gene clusters are deleted
AttB sites, are expanded with the multicopy for realizing target gene cluster.By taking PII synthetic gene clusters as an example, in rotation streptomycete (Δ
PapR5+R4R6 external source imports a PII synthetic gene cluster in), can be by 1.5 times of PII output increaseds.Copied for detection continues increase
Whether PII yield can further improve during shellfish number, and the present inventor is deleting other and the pyostacin uncorrelated gene cluster of synthesis
While, an attB site is introduced in same position, so as to increased attB numbers in genome, to realize more PII
The insertion of synthetic gene cluster copy number.Result finds, two with three copies while the efficiency that introduce be respectively 100% and
10%, it was confirmed that multiple attB sites can utilize locus specificity recombination form effectively to mediate the amplification of target gene cluster.
As preferred embodiment of the invention, after above-mentioned streptomyces gene engineering bacteria is obtained, when fermented and cultured is carried out,
Also macroporous absorbent resin is added in the nutrient solution of streptomycete；It is preferred that adding 1-12% (w/v)；The more preferably macropore of 3-8%
Polymeric adsorbent.
In a particular embodiment of the present invention, with rotation streptomycete (Δ papR5+R4R6) (SBJ1000) for chassis bacterium, point
The PII yield of engineering bacteria SBJ1001, SBJ1002 and SBJ1003 that Zeng Jia be after one, two or three copy is respectively reached
468,508 and 720mg/L, yield lifting is notable.By adding macroporous absorbent resin to weaken pyostacin to the anti-of itself
Feedback depression effect and the poisonous effect to thalli growth, the PII yield of SBJ1003 reach 1.75g/L, and pole is lifted with respect to chassis bacterium
Its is notable.
The method of the gene cluster multicopy amplification of site-specific recombination system mediation of the invention, can be widely used for various
The genetic modification of actinomyces, for the fermentation level for improving important microbe natural products has established good basis.
The method of the present invention introduces multiple attB, by locus specificity recombination system while other gene clusters are deleted
System, once realizes that target gene cluster multicopy is expanded, and greatly improves target product yield.Due to introduce target gene cluster into from
Scattered distribution, the relative gene cluster cascade amplification realized using ZouA-RsA/B systems is not only simple to operate effective, and engineering bacteria
Genetic stability more preferably, can be widely used for other industrial actinomyces carries out genetic modification.
Into discrete distribution after being introduced due to target gene cluster, homologous recombination, the chain that the method for the present invention is obtained are not susceptible to
The genetic stability of mould genetic engineering bacterium is fine.Therefore, the method for the present invention is established as actinomyces secondary metabolites
Output optimization provides a kind of sane, pervasive Reconstruc-tion policy, and site-specific recombination system is utilized when competition approach is weakened
The amplification of gene cluster copy number is realized, so as to obtain the superior strain of inheritance stability.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Bacterial strain, plasmid and reagent that the present invention is used：
(it is named as the present invention relates to rotation streptomycete Streptomyces pristinaespiralis Δs papR5+R4R6
SBJ1000) for laboratory early stage builds, construction method is referring to Li et al., Metabolic Engineering, 2015.
E.coli S17-1 (are shifted) for engaging：Purchased from Biomedal Life Science companies.
HCCB10218 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on November 23rd, 2011
Center (CGMCC), preserving number CGMCC NO.5486.
The plasmid pKCcas9dO arrived used in the present invention, construction method is referring to Huang et al., Acta Biochim
Biophys Sin(Shanghai),2015。
The plasmid BAC-F1F15 (containing PII synthetic genes cluster) arrived used in the present invention, wherein containing Escherichia coli
Bacterial chromosome (BAC) copied cells, apramycin resistant gene aac (3) IV expression cassettes, phiC31 integrases expression cassette,
Integration site attP and oriT (RK2) site related to engagement transfer.Construction method referring to Lei Li et al.,
Metabolic Engineering,2015。
DNA glue reclaims are purified and plasmid extraction kit is purchased from Axygen companies.
E.Z.N.A.BAC/PAC DNA Kit are purchased from Omega Bio-Tek companies.
KOD plus new are purchased from TOYOBO companies.
Macroporous absorbent resin HB60 is upper extra large MinYong Industrial Co., Ltd.s purchase.
Other conventional reagents are that domestic analysis is pure or import packing.
The culture medium that the present invention is used：
1. LB liquid medium (1L)
Peptone 10g, yeast extract 5g, NaCl 10g, 121 DEG C sterilize 20 minutes.
2. solid LB media (1L)
Peptone 10g, yeast extract 5g, NaCl 10g, agar powder 20g；121 DEG C sterilize 20 minutes.
3. solid RP culture medium prescriptions (1L)
Soluble starch 20g, soybean cake powder 10g, NaCl 2g, KH2PO40.5g, MgSO4.7H2O 1g, CaCO33g, fine jade
Cosmetics 20g, adjusts 6.4,121 DEG C of pH to sterilize 20 minutes.
4th, seed culture medium (1L)
Soluble starch 15g, glucose 10g, soybean cake powder 15g, dusty yeast 5g, KNO32.5g, NaCl 2g, CaCO3
4g, adjusts 7.0~7.2,121 DEG C of pH to sterilize 20 minutes.
5th, fermentation medium (1L)
Soluble starch 40g, glucose 22.5g, cotton seed meal 32g, dusty yeast 3.5g, KH2PO40.1g, ZnSO4.7H2O
0.5g, MgSO4.7H2O 0.5g, CaCO36g, adjusts 6.0,121 DEG C of pH to sterilize 20 minutes.
The gene cluster multicopy amplification strategy of embodiment 1, site-specific recombination system mediation
1st, the selection of gene cluster and attB insertion points is deleted
Such as Fig. 1, an attB site is normally only contained in streptomyces gene group, introduce new attB sites and be conducive to introducing
The target gene cluster of more multicopy.At the same time, delete and synthesize incoherent other gene clusters with pyostacin, other can be avoided
When secondary metabolite synthesizes the precursors such as acetyl coenzyme A, malonyl coenzyme A or amino acid are competed with pyostacin synthesis.
In order to realize deleting uncorrelated gene cluster and introduce the purpose in attB sites, the present inventor by studying point repeatedly
Analysis, have chosen 3 non-targeted gene clusters in rotation streptomycete, respectively positioned at rotation streptomycete genome (GenBank accession number：
NZ_ABJI00000000.2 4050261-4072243 (BGC1)), 1106415-1130923 (BGC2) and 686202-
696578 (BCG3), may encode a kind of unknown I types polyketone, a kind of non-ribosomal peptides and a kind of type III polyketone respectively.Using
CRISPR/Cas9 systems are deleted it and are added attB sites simultaneously one by one.
2nd, it is target gene cluster with PII synthetic genes cluster, builds engineered strain
Using rotation streptomycete SBJ1000 as starting strain, using BGC1, BGC2, BGC3 as non-by what is deleted
Target gene cluster, and add attB in corresponding position of deleting.It is target gene cluster with PII biological synthesis gene clusters, builds work
Journey bacterial strain.Such as Fig. 1.
As a example by knocking out one attB site of BGC1 and introducing in situ, first using primer BGC1-up-fw/rev, BGC1-
Down-fw/rev and BGC1-sgRNA-fw/DM-sgRNA-rev, with HCCB10218 genomes as template, PCR amplifications are obtained respectively
The gRNA Expression elements BGC1-sgRNA of upstream and downstream homology arm BGC1-UP, the BGC1-DOWN that must be knocked out and targeting BGC1.Then
It is primer to use BGC1-up-fw and DM-sgRNA-rev, and above three PCR primer is template, is expanded with overlapping PCR
The mode of increasing obtains BGC1-UP-DOWN-sgRNA, then using Spe I and Hind III digestions, is connected to using identical limitation
Property endonuclease digestion carrier pKCcas9dO in, it is final to obtain BGC1 targeting knock out carriers pKCcas9-BGC1-sgRNA.Its
In, attB sites have been superimposed upon primer, and in BGC1-down-fw, (italic blackens underscore mark in being shown in Table 1 corresponding primer
Sequence).PKCcas9-BGC1-sgRNA engagements are transferred in chassis bacterium SBJ1000, the bacterium colony for growing using ID-BGC1-fw with
ID-BGC1-rev enters performing PCR identification and sequencing.The correct bacterial strain of result is cultivated 2-3 generations in non-resistant RP culture mediums and is voluntarily lost
Fall plasmid pKCcas9-BGC1-sgRNA, obtain engineering bacteria SBJ1000::attB2.In the bacterial strain, an attB for external source is replaced
Original BGC1 (i.e. 4050261-4072243 of rotation streptomycete genome, referring to GenBank step on by genome sequence
Record number：NZ_ABJI00000000.2 so that there are 2 attB sites in genome).
The knockout of BGC2 is similar with BGC1, with SBJ1000::AttB2 is the bacterium that sets out, and knockout BGC2 introduces the 3rd attB and obtains
Obtain SBJ1000::attB3.Specific method is：First using primer BGC2-up-fw/rev, BGC2-down-fw/rev with
BGC2-sgRNA-fw/DM-sgRNA-rev, with HCCB10218 genomes as template, PCR amplifications obtain the upper and lower of knockout respectively
The gRNA Expression elements BGC2-sgRNA of trip homology arm BGC2-UP, BGC2-DOWN and targeting BGC2.Then BGC2-up- is used
Fw and DM-sgRNA-rev is primer, and above three PCR primer is template, is obtained in the way of overlapping PCR are expanded
BGC2-UP-DOWN-sgRNA, then using Spe I and Hind III digestions, is connected to using identical digestion with restriction enzyme
Carrier pKCcas9dO in, it is final to obtain BGC2 targeting knock out carriers pKCcas9-BGC2-sgRNA.Wherein, attB sites have been
Added in primer to BGC2-down-fw (italic blackens the sequence that underscore is marked in being shown in Table 1 corresponding primer).
PKCcas9-BGC2-sgRNA engagements are transferred to chassis bacterium SBJ1000::In attB2, the bacterium colony for growing using ID-BGC2-fw with
ID-BGC2-rev enters performing PCR identification and sequencing.The correct bacterial strain of result is cultivated 2-3 generations in non-resistant RP culture mediums and is voluntarily lost
Fall plasmid pKCcas9-BGC2-sgRNA, obtain engineering bacteria SBJ1000::attB3.In the bacterial strain, a new external source attB is inserted
Enter original BGC2 (i.e. rotation streptomycete genomes 1106415-1130923, referring to GenBank accession number of replacement：NZ_
ABJI00000000.2) so that there are 3 attB sites in genome.
With SBJ1000::AttB3 introduces the 4th attB and obtains SBJ1000 for the bacterium that sets out knocks out BGC3::attB4.Specifically
Method is：Primer BGC3-up-fw/rev, BGC3-down-fw/rev and BGC3-sgRNA-fw/DM-sgRNA- are used first
Rev, with HCCB10218 genomes as template, PCR amplifications obtain upstream and downstream homology arm BGC3-UP, BGC3- of knockout respectively
The gRNA Expression elements BGC3-sgRNA of DOWN and targeting BGC3.Then BGC3-up-fw and DM-sgRNA-rev is used to draw
Thing, above three PCR primer is template, obtains BGC3-UP-DOWN-sgRNA in the way of overlapping PCR are expanded, so
Afterwards using Spe I and Hind III digestions, it is connected in the carrier pKCcas9dO using identical digestion with restriction enzyme, most
BGC3 targeting knock out carriers pKCcas9-BGC3-sgRNA is obtained eventually.Wherein, attB sites have been superimposed upon primer to BGC3-
In down-fw (italic blackens the sequence that underscore is marked in being shown in Table 1 corresponding primer).PKCcas9-BGC3-sgRNA engagement transfers
To chassis bacterium SBJ1000::In attB3, the bacterium colony for growing is entered performing PCR identification and is surveyed using ID-BGC3-fw and ID-BGC3-rev
Sequence.The correct bacterial strain of result cultivates 2-3 generations in non-resistant RP culture mediums and voluntarily loses plasmid pKCcas9-BGC3-sgRNA, obtains
Obtain engineering bacteria SBJ1000::attB4.In the bacterial strain, the attB of external source replaces original BGC3 (i.e. rotation streptomycete genes
686202-696578 of group, referring to GenBank accession number：NZ_ABJI00000000.2) so that have 4 in genome
Individual attB sites.The primer sequence used in above method is as shown in table 1.
Table 1, list of primers
Note：Roman underscore represents restriction enzyme site, and italic blackens underscore and is labeled as attB sites.
2nd, efficiency comparative when different copy gene clusters are introduced
Using above-mentioned strategy, the present inventor builds obtain engineering bacteria SBJ1000 respectively::attB2、SBJ1000::
AttB3 and SBJ1000::AttB4, contains 2,3 and 4 attB sites respectively in its genome.
By the plasmid (BAC-F1F15) containing PII synthetic gene clusters to engage transfer in the way of imported it is above-mentioned
SBJ1000、SBJ1000::AttB2 and SBJ1000::In tri- plants of bacterium of attB3, bacterium colony to be grown enters performing PCR amplification, and pass through
Gel electrophoresis is identified.
Result such as Fig. 2, while the efficiency of 2 copies of insertion reaches 100%, the efficiency for inserting 3 copies is only 10%, and
4 PII synthetic genes clusters of insertion do not have bacterium colony to grow when copying.Supposition be when introducing more PII synthetic genes clusters and copying, it is whole
Synthase phiC31 (being brought into by BAC-F1F15 plasmids) is cut but can not be repaired in time in genome many places attB sites, from
And cause thalline dead.The identification primer of 2 copies of insertion is ID-attB-fw/ID-BGC1-attB-fw and ID-BAC-
F1F15-rev, inserts 3 identification primers of copy in addition to above-mentioned two pairs of primers, separately there is ID-BGC2-attB-fw and ID-BAC-
F1F15-rev, its sequence is as shown in table 1.
Engineering bacteria PII yield comparisons after embodiment 2, the copy number introducing of difference PII synthetic genes cluster
By above-mentioned strategy, the present inventor is chassis bacterium with SBJ1000, constructs and contains increase PII synthetic genes cluster 1
Individual, 2 or 3 engineering bacterias for copying, are respectively designated as SBJ1001, SBJ1002 and SBJ1003.
The present inventor carries out fermenting experiment to SBJ1001, SBJ1002 and SBJ1003 respectively, with SBJ1000,
SBJ1000::attB2、SBJ1000::AttB3 is control.
(1) fermentation process and condition
Rotation streptomycete SBJ1000 and each derivative engineering bacteria line RP flat boards respectively, 30 DEG C of quiescent cultures 3-5 days,
The isometric bacterium block of picking accesses seed culture medium respectively, after 27 DEG C of 240rpm cultures 44-48h, is transferred with 8% (v/v) ratio
Continue to cultivate into fermentation medium, sampling carries out HPLC measure behind 30,48,72,96 with 120h respectively.
(2) pyostacin assay method
The μ l of nutrient solution 600 are drawn, equal-volume acetone is added, room temperature places 60min after vibration, and 12000 leave heart 5min, take
Supernatant, carries out HPLC detections, pillar model：Agilent Eclipse, filler：5 microns of XDB C18, size：4.6mm×
150mm, wavelength 206nm, flow velocity 1ml/min, mobile phase：Acetonitrile：0.03M KH2PO4Buffer solution=45：55.
(3) fermentation results
Fermenting experiment result shows, with the increase step by step of copy number, PII (Pristinamycin IIA) yield also phase
Should improve, SBJ1001, SBJ1002 and SBJ1003 are respectively 468,508 and 720mg/L, the relative bacterium (yield 323mg/L) that sets out
45%, 57% and 123% is respectively increased, such as Fig. 3.
Meanwhile, the inventors discovered that, engineering bacteria SBJ1000::AttB2 and SBJ1000::The PII yield of attB3 with set out
Bacterium is not significantly different from.
The above results show that the further increase of the PII yield of engineering bacteria SBJ1002 and SBJ1003 is due to PII synthesis
Caused by the increase of gene copy number is deleted rather than other gene clusters.
(4) genetic stability is determined
Meanwhile, engineering bacteria SBJ1003 has been carried out the continuous passage culture of 5 generations by the present inventor, fermentation results display offspring's
PII yield is not significantly different from female generation, it was demonstrated that this multi-copy gene cluster based on site-specific recombination system integrates institute
The engineering bacteria of structure is very stable in heredity, and homologous recombination is not susceptible between homologous genes cluster.
Embodiment 3, fermentation technology optimization further improves the PII yield of engineering bacteria
The present inventors have additionally discovered that, because pyostacin has stronger inhibition for itself synthesis with thalli growth,
Addition macroporous absorbent resin can effectively alleviate this poisonous effect, further improve the biosynthesis of pyostacin.
Therefore, the present inventor with the addition of the macroporous absorbent resin HB60 of 6% (w/v) in SBJ1003 fermentation process, as a result
It was found that, Pristinamycin IIAYield lifts 1.4 times again when being fermented without resin relatively, and with respect to starting strain
SBJ1000 also improves 1.3 times under same culture conditions.It is excellent with the fermentation in later stage by gene cluster multicopy amplification method
Change, most 4.4 times of PII output increaseds at last, from original 323mg/L be promoted to 1.75g/L, such as Fig. 4.
The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can
Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (14)
1. a kind of method for making to be integrated into multicopy target gene or target gene cluster in streptomyces gene group, it is characterised in that
Methods described includes：
(1) the attB sites of at least one external source are introduced in streptomyces gene group, acquisition carries the strepto- in external source attB sites
Bacterium；
(2) method by engaging transfer, target gene or target gene cluster are incorporated into the streptomycete of step (1), are selected
Target gene or target gene cluster are incorporated into the streptomycete in the endogenous attB sites of streptomyces gene group and external source attB sites, from
And obtain the streptomycete for being integrated into multi-copy gene or gene cluster.
2. the method for claim 1, it is characterised in that the attB sites of described at least one external source are 1-2 external source
AttB sites；The preferably 2 attB sites of external source.
3. method as claimed in claim 2, it is characterised in that the attB sites of 1-2 described external source are incorporated into streptomycete
1-2 following position is selected from genome：
I types polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the biological conjunction of former I types polyketone of the position
Into gene cluster；
Non-ribosomal peptides biological synthesis gene cluster position in streptomyces gene group, and replace the former non-ribosomal peptides of the position
Biological synthesis gene cluster；Or
Type III polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former type III polyketone of the position to give birth to
Thing synthetic gene cluster.
4. the method for claim 1, it is characterised in that described streptomycete includes：Rotation streptomycete, sky blue strepto-
Bacterium, Streptomyces roseosporus or streptomyces hygroscopicus.
5. the method for claim 1, it is characterised in that described target gene or target gene cluster includes：It is original mould
Plain component II synthetic gene clusters, pyostacin I synthetic gene clusters, Daptomycin synthetic gene cluster or rapamycin synthetic gene
Cluster.
6. the method for claim 1, it is characterised in that in step (2), by engaging the method for transfer by target gene
Or the method that target gene cluster is incorporated into the streptomycete of step (1) includes：By the plasmid of foreign gene-carrying or gene cluster with
The mode for engaging transfer is imported into the streptomycete of step (1).
7. a kind of streptomyces gene engineering bacteria, it is characterised in that the genome of the genetic engineering bacterium includes at least one external source
AttB sites, 1 endogenous endogenous attB site.
8. streptomyces gene engineering bacteria as claimed in claim 7, it is characterised in that attB of described at least one external source
Point is the 1-2 attB site of external source；The preferably 2 attB sites of external source.
9. streptomyces gene engineering bacteria as claimed in claim 8, it is characterised in that the attB sites of 1-2 described external source
It is incorporated into streptomyces gene group selected from 1-2 following position：
I types polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the biological conjunction of former I types polyketone of the position
Into gene cluster；
Non-ribosomal peptides biological synthesis gene cluster position in streptomyces gene group, and replace the former non-ribosomal peptides of the position
Biological synthesis gene cluster；Or
Type III polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former type III polyketone of the position to give birth to
Thing synthetic gene cluster.
10. the purposes of any described streptomyces gene engineering bacterias of claim 7-9, it is characterised in that carry many for preparing
The streptomyces gene engineering bacteria of copy foreign gene or gene cluster.
The streptomyces gene engineering bacteria of multicopy target gene or target gene cluster, its feature are integrated with a kind of 11. genomes
As many as it is that 3 target gene clusters of copy are included up in the genome of the genetic engineering bacterium, and in following position
3 positions：
Endogenous attB sites position in streptomyces gene group, and replace the original attB sites in the position；
I types polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the biological conjunction of former I types polyketone of the position
Into gene cluster；Or
Non-ribosomal peptides biological synthesis gene cluster position in streptomyces gene group, and replace the former non-ribosomal peptides of the position
Biological synthesis gene cluster；Or
Type III polyketone biological synthesis gene cluster position in streptomyces gene group, and replace the former type III polyketone of the position to give birth to
Thing synthetic gene cluster.
The streptomyces gene of multicopy target gene or target gene cluster is integrated with 12. genomes as claimed in claim 11
Engineering bacteria, it is characterised in that described target gene or target gene cluster includes：Pyostacin component II synthetic gene clusters, it is former
Beginning mycin I synthetic gene cluster, Daptomycin synthetic gene cluster or rapamycin synthetic gene cluster etc..
The method of a kind of 13. utilization streptomycete high efficient expression target genes or target gene cluster, it is characterised in that described method
Including：
A () prepares streptomycete in the method described in claim 1, make to be integrated into streptomyces gene group multicopy target gene or
Target gene cluster；
B streptomycete that () incubation step (a) is obtained, so that high efficient expression target gene or target gene cluster.
14. methods as claimed in claim 13, it is characterised in that in step (b), add big also in the nutrient solution of streptomycete
Macroporous adsorbent resin；It is preferred that adding 1-12% (w/v)；The more preferably macroporous absorbent resin of 3-8%.
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| CN113249401A (en) * | 2020-02-10 | 2021-08-13 | 中国科学院分子植物科学卓越创新中心 | Method for improving streptomyces rapamycin yield |
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| CN110951765A (en) * | 2018-09-27 | 2020-04-03 | 中国科学院上海生命科学研究院 | Multi-copy integration method for synthetic gene cluster of natural product of actinomycetes |
| CN110951765B (en) * | 2018-09-27 | 2022-03-08 | 中国科学院分子植物科学卓越创新中心 | Multi-copy integration method for synthetic gene cluster of natural product of actinomycetes |
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| CN113249401A (en) * | 2020-02-10 | 2021-08-13 | 中国科学院分子植物科学卓越创新中心 | Method for improving streptomyces rapamycin yield |
| CN113249401B (en) * | 2020-02-10 | 2022-11-04 | 中国科学院分子植物科学卓越创新中心 | Method for improving streptomyces rapamycin yield |
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