Method for increasing yield of milbemycins A3/A4 or derivatives thereof
阅读说明：本技术 提高米尔贝霉素a3/a4或其衍生物产量的方法 (Method for increasing yield of milbemycins A3/A4 or derivatives thereof ) 是由 陈少欣 芦银华 姜卫红 卫科科 李雷 吴远杰 于 2018-08-23 设计创作，主要内容包括：本发明涉及提高米尔贝霉素A3/A4或其衍生物产量的方法。本发明中,确定了以其基因组中A1和/或A2-R两段基因簇作为重组克隆的靶标,在菌株中重组表达该两段基因簇,使得米尔贝霉素A3/A4或其衍生物的产量呈现极为显著的提高。本发明还提供了遗传工程化的米尔贝霉素A3/A4或其衍生物的生产菌及其应用。(The present invention relates to a method for increasing the yield of milbemycins A3/A4 or derivatives thereof. In the invention, two gene clusters of A1 and/or A2-R in the genome are determined as targets of recombinant cloning, and the two gene clusters are expressed in a strain in a recombinant mode, so that the yield of the milbemycins A3/A4 or derivatives thereof is remarkably improved. The invention also provides a genetically engineered milbemycins A3/A4 or a production strain of the derivatives of the milbemycins A3/A4 and application of the genetically engineered milbemycins A3/A4.)
1. A method of increasing the yield of milbemycin A3/a4 or a derivative thereof, comprising: in a producing bacterium of milbemycins A3/A4 or derivatives thereof, there are introduced:
(1) a1 gene cluster, and/or
(2) The a2-R gene cluster or a variant thereof in which the milF gene is down-regulated.
2. The method of claim 1, wherein the derivative of milbemycins A3/a4 is 5-keto-milbemycins A3/a 4.
3. The method of claim 1, wherein said a1 gene cluster comprises the milA1 gene; or
The A2-R gene cluster comprises the genes of mil A2, mil C, mil E, mil A4, sbi _00730, mil F, sbi _00732, mil A3 and mil R.
4. The method of claim 1, wherein the a1 gene cluster is:
(a) 1 or a degenerate sequence thereof;
(b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 1 or a degenerate sequence thereof;
(c) polynucleotide whose nucleotide sequence has homology of greater than or equal to 85% with the nucleotide sequence shown in SEQ ID NO. 1 or its degenerated sequence.
5. The method of claim 1, wherein the a2-R gene cluster is:
(a) 2 or a degenerate sequence thereof;
(b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 2 or a degenerate sequence thereof; or
(c) Polynucleotide whose nucleotide sequence has homology of more than 85% with the nucleotide sequence shown in SEQ ID NO. 2 or its degenerated sequence.
6. The method according to claim 1, wherein the two gene clusters of (1) and (2) are obtained separately and introduced separately into a strain producing milbemycin A3/A4 or a derivative thereof; preferably, the (1) and the (2) are simultaneously transferred into a producing strain of the milbemycin A3/A4 or the derivatives thereof, or two gene clusters of the (1) and the (2) are respectively obtained and are transferred into a producing strain of the milbemycin A3/A4 or the derivatives thereof after splicing; more preferably, the two gene clusters obtained in (1) and (2) are transferred into a producing strain of milbemycin A3/A4 or a derivative thereof, and then the producing strain carrying the two gene clusters of (1) and (2) is obtained by means of conjugative transfer.
7. The method of claim 1, wherein the strain producing milbemycins A3/a4 or derivatives thereof comprises: streptomyces (Streptomyces); preferably Streptomyces hygroscopicus.
8. The method according to claim 1, wherein the (1) and/or (2) is integrated into the Φ C31 attB or Φ BT1 attB site of a production bacterium of milbemycins A3/a4 or a derivative thereof.
9. A genetically engineered milbemycins A3/A4 or derivatives thereof producing strain, wherein:
(1) a1 gene cluster, and/or
(2) The a2-R gene cluster or a variant thereof in which the milF gene is down-regulated.
10. The genetically engineered milbemycin A3/a4 producing strain as claimed in claim 9, characterized in that the a1 gene cluster comprises the milA1 gene; or
The A2-R gene cluster comprises the genes of mil A2, mil C, mil E, mil A4, sbi _00730, mil F, sbi _00732, mil A3 and mil R.
11. The genetically engineered milbemycin A3/a4 producing bacterium of claim 9, characterised in that the a1 gene cluster is:
(a) 1 or a degenerate sequence thereof;
(b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 1 or a degenerate sequence thereof;
(c) polynucleotide whose nucleotide sequence has homology more than 85% with the nucleotide sequence shown in SEQ ID NO. 1 or its degenerated sequence.
12. The genetically engineered milbemycin A3/a4 producing bacterium of claim 9, in which the a2-R gene cluster is:
(a) 2 or a degenerate sequence thereof;
(b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 2 or a degenerate sequence thereof; or
(c) Polynucleotide whose nucleotide sequence has homology of more than 85% with the nucleotide sequence shown in SEQ ID NO. 2 or its degenerated sequence.
13. The genetically engineered milbemycin A3/a4 or a derivative thereof producer of claim 9, wherein the milbemycin A3/a4 or derivative thereof producer comprises: streptomyces (Streptomyces); preferably Streptomyces hygroscopicus.
14. Use of the genetically engineered milbemycins A3/A4 or derivatives thereof of any of claims 9 to 13 for the production of milbemycins A3/A4 or derivatives thereof.
15. A kit for producing milbemycins A3/a4 or derivatives thereof, comprising the genetically engineered milbemycins A3/a4 or derivatives thereof producing bacteria of any one of claims 9 to 13.
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a method for improving the yield of milbemycins A3/A4 or derivatives thereof.
Background
The milbemycins A3/A4 are structural analogues of abamectin, belong to polyketone compounds of sixteen-membered ring macrolides and have a molecular formula of C31H44O7/C32H46O7The chemical structural formula is as follows:
the milbemycins A3/A4 have high activity, low toxic and side effect, safety, reliability and easy degradation, and have excellent effects of expelling nematodes and killing parasites inside and outside the body, so the milbemycins A3/A4 are widely applied to anti-parasitic drugs, and the application value and the research prospect of the milbemycins A3/A4 are more and more concerned in the world.
5-keto-milbemycins A3/A4 is precursor compound for chemically synthesizing milbemycin A3/A4, and its molecular formula is C31H42O7/C32H44O7The chemical structural formula is shown as the left compound of the formula:
the milbemycin oxime (the chemical structural formula is shown as the right compound) is a derivative (the chemical reaction formula is shown as the above formula) of 5-ketone-milbemycin A3/A4 after oximation reaction with hydroxylamine, and the molecular formula is C31H43NO7/C32H45NO7. The milbemycin oxime is mainly used for preventing and treating parasites in vivo and in vitro of pet cats and dogs, and has high insecticidal activity and low toxicity.
At present, milbemycins are mainly obtained by microbial fermentation and then separation and extraction from fermentation liquor, but the cost effectiveness ratio is low, so the market price of the antibiotics is high, and the large-scale application of the antibiotics is not facilitated. Although the traditional mutagenesis screening method can improve the titer to a certain extent, the mechanism is not clear, and a rational idea cannot be provided for the next strain transformation. The synthesis of milbemycin oxime relies mainly on a two-step chemical synthesis:
the two-step chemical synthesis method comprises the following steps: first using CrO3Milbemycins A3/A4 were oxidized to 5-keto-milbemycins A3/A4, which were then reacted with hydroxylamine hydrochloride to produce milbeximes. The synthesis process has low conversion efficiency, is not environment-friendly and has heavy metal (Cr) pollution. Therefore, the 5-keto-milbemycins A3/A4 are used as chemical intermediates, and the significance of improving the potency is great.
In conclusion, there is a need in the art to develop and improve methods for increasing the yield of milbemycins A3/A4 or derivatives thereof, such as 5-keto-milbemycins A3/A4, in order to change the current conditions of low yield and high cost.
Disclosure of Invention
The invention aims to provide a method for improving the yield of milbemycins A3/A4 or derivatives thereof.
In a first aspect of the invention, there is provided a method of increasing the yield of milbemycin A3/a4 or a derivative thereof, said method comprising: in a producing bacterium of milbemycins A3/A4 or derivatives thereof, there are introduced: (1) the A1 gene cluster, and/or (2) the A2-R gene cluster or a variant thereof in which the milF gene is down-regulated.
In a preferred embodiment, the derivative of milbemycins A3/A4 is 5-keto-milbemycins A3/A4.
In another preferred example, the A1 gene cluster comprises the milA1 gene; or the A2-R gene cluster comprises the genes of mil A2, mil C, mil E, mil A4, sbi _00730, mil F, sbi _00732, mil A3 and mil R.
In another preferred embodiment, the a1 gene cluster is: (a) 1 or a degenerate sequence thereof; (b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 1 or a degenerate sequence thereof; (c) polynucleotide (preferably, it is derived from Streptomyces) having a homology of 85% (preferably 90% or more, more preferably 95% or more, e.g., 98% or more, 99%) or more with the nucleotide sequence shown in SEQ ID NO. 1 or a degenerate sequence thereof.
In another preferred embodiment, the A2-R gene cluster is: (a) 2 or a degenerate sequence thereof; (b) a polynucleotide whose nucleotide sequence is complementary to the nucleotide sequence shown in SEQ ID NO. 2 or a degenerate sequence thereof; or (c) a polynucleotide (preferably, derived from Streptomyces) having a nucleotide sequence homology of 85% or more (preferably 90% or more, more preferably 95% or more; e.g., 98% or more or 99% or more) with the nucleotide sequence shown in SEQ ID NO. 2 or a degenerate sequence thereof.
In another preferred example, the two gene clusters of (1) and (2) are obtained separately and-introduced into a strain producing milbemycin A3/A4 or a derivative thereof, respectively; preferably, the genes (1) and (2) are simultaneously transferred into a producing strain of the milbemycin A3/A4 or the derivatives thereof, or the two gene clusters of the genes (1) and (2) are respectively obtained and are transferred into a producing strain of the milbemycin A3/A4 or the derivatives thereof after splicing.
In another preferred embodiment, the producing strain of milbemycins A3/A4 or derivatives thereof comprises: streptomyces (Streptomyces); preferably Streptomyces hygroscopicus.
In another preferred example, the above-mentioned (1) and/or (2) is integrated into the site Φ C31 attB or Φ BT1 attB of a producing bacterium of milbemycins A3/a4 or a derivative thereof.
In another aspect of the present invention, there is provided a genetically engineered milbemycin A3/A4 or a derivative thereof-producing strain into which: (1) the A1 gene cluster, and/or (2) the A2-R gene cluster or a variant thereof in which the milF gene is down-regulated.
In a preferred embodiment, the A1 gene cluster comprises the milA1 gene; or the A2-R gene cluster comprises the genes of mil A2, mil C, mil E, mil A4, sbi _00730, mil F, sbi _00732, mil A3 and mil R.
In another aspect of the invention, the use of said genetically engineered milbemycins A3/A4 or derivatives thereof producing bacteria for the production of milbemycins A3/A4 or derivatives thereof is provided.
In another aspect of the present invention, there is provided a kit for producing milbemycins A3/A4 or derivatives thereof, the kit comprising a genetically engineered milbemycins A3/A4 or derivatives thereof-producing strain as described in any of the preceding.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 is a schematic diagram of the gene cluster for carrying out the biosynthesis of milbemycins (A) and 5-keto-milbemycins (B) according to the invention.
FIG. 2 is a schematic diagram of integration of plasmids containing gene clusters, which is exemplified by integration of pCAP-milA1 plasmid.
FIG. 3, map of plasmid pCAP-milA 1.
FIG. 4, map of plasmid pCAP-milA 2-R.
FIG. 5, map of plasmid pCAP-milA2- Δ F-R.
FIG. 6, map of plasmid pBTK-mil A1.
FIG. 7, map of plasmid BAC-mb.
FIG. 8, map of plasmid BAC-05.
Detailed Description
Aiming at the defect of low yield of the milbemycin A3/A4 production strain in the prior art, the inventor determines that two gene clusters of A1 and/or A2-R in the genome of the milbemycin production strain are used as targets of recombinant cloning through deep analysis of the genome of the milbemycin production strain, and the two gene clusters are expressed in the strain in a recombinant mode, so that the yield of the milbemycin A3/A4 or derivatives thereof is remarkably improved.
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" gene or protein refers to a gene or protein that is not naturally contained in the genome of the protozoan organism (Streptomyces in the present invention). Typically, a "foreign" gene or protein is introduced into an organism by recombinant techniques of genetic engineering.
As used herein, the term "derivative of milbemycins A3/A4" refers to a compound having a parent nucleus structure identical to that of milbemycins A3/A4 and which can also be produced by the recombinant producer constructed according to the present invention, such as 5-keto-milbemycins A3/A4.
As used herein, the expression "the mil gene is down-regulated" includes the expression "the mil gene is suppressed", "the mil gene is knocked out" and "the mil gene is disrupted".
Gene cluster
In the invention, the A1 and A2-R two segments of gene clusters are used as targets of recombinant cloning, and the two segments of gene clusters are expressed in a strain in a recombinant mode, so that the yield of the milbemycins A3/A4 or derivatives thereof is remarkably improved. At the beginning of the research, the inventor finds that the relative number of genes for producing various milbemycins A3/A4 is relatively large, and the cloning into a production strain is very difficult, so that the burden of the strain is greatly increased, and the strain is difficult to accept. In order to overcome the problem, the inventor conducts repeated research and experiments to divide the related genes into two sections of A1 and A2-R, clones the genes respectively, and then combines the genes by a conjugal transfer method to construct an ideal strain. This elegant genetic partitioning allows the construction of high producing strains to be successful.
In the present invention, the A1 gene cluster may have the nucleotide sequence shown in SEQ ID NO. 1 or a degenerate sequence thereof.
The A2-R gene cluster can have the nucleotide sequence shown in SEQ ID NO. 2 or the degenerate sequence thereof. The gene cluster of the present invention is preferably provided in an isolated form, and more preferably purified to homogeneity.
The A2-R gene cluster can be a modified nucleotide sequence based on SEQ ID NO. 2 or a degenerate sequence thereof, the modification can be carried out on the milF gene of the gene cluster, and after the gene is down-regulated, the product of the producing strain can be 5-keto-milbemycin A3/A4. As a preferred mode of the invention, the gene cluster subjected to the modification is named A2-delta F-R; preferably, it has the nucleotide sequence shown in SEQ ID NO. 3 or a degenerate sequence thereof.
The gene cluster may also contain one or more gene variants, including substitution variants, deletion variants and insertion variants. However, the variants do not substantially alter the function of the encoded polypeptide and still achieve the technical effects of the present invention.
The genes of the gene cluster may be naturally occurring, e.g., they may be isolated or purified from a microorganism. In addition, one or more of the genes may also be artificially prepared, for example, the relevant genes may be amplified according to conventional genetic engineering techniques, or the relevant genes may be prepared by artificial synthesis and then linked to form a gene cluster.
The gene cluster or the genes or fragments thereof can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
After understanding the novel findings of the present invention, those skilled in the art can also make various known modifications to the gene cluster or the genes or fragments thereof, such as codon optimization or site-directed mutagenesis, to further improve the expression efficiency.
After the A1 and/or A2-R and/or A2- Δ F-R gene clusters have been obtained, they are ligated into a suitable expression construct (e.g., an expression vector) and introduced into a suitable host cell. Finally, the metabolite milbemycin A3/A4 of the host cell or the derivative thereof is obtained by culturing the transformed host cell.
The expression construct may include clusters of the A1 and/or A2-R and/or A2- Δ F-R genes, and may further include additional functional sequences operably linked to the sequences of the genes to facilitate entry into the host cell and to achieve site-directed integration of the genes. In the functional sequence, an inducible or constitutive promoter can be applied according to different requirements, and the inducible promoter can realize more controllable expression and metabolite production, thereby being beneficial to industrial application. For prokaryotic cells or eukaryotic cells, suitable expression vectors are known in the art, so one can select a suitable expression vector as a backbone vector for cloning the encoding gene, and suitable expression vectors commonly used in streptomyces are pSET152, pIB139, pRT802, BAC-F15, and the like.
Milbemycin producing strain
The invention also includes genetically engineered production bacteria of milbemycins A3/A4 or derivatives thereof, into which: the A1 gene cluster, and/or the A2-R gene cluster, and/or the A2-DeltaF-R gene cluster.
The modified starting strain is Streptomyces (Streptomyces); preferably Streptomyces hygroscopicus.
The streptomyces or the streptomyces hygroscopicus can be natural strains, and can also be strains mutated or modified on the basis of the natural strains. In a preferred embodiment of the invention, the strain Streptomyces hygroscopicus NRRL5739 and 5-keto-milbemycins A3/A4 producing strain Streptomyces hygroscopicus SIPI-KF are used as milbemycins A3/A4 producing strain.
In a preferred embodiment of the present invention, a plurality of plasmids relating to the production of milbemycins (5-keto-milbemycins) A3/A4 are provided, and a plurality of microbial strains efficiently producing milbemycins A3/A4 and a plurality of microbial strains efficiently producing 5-keto-milbemycins A3/A4 are developed.
In a particularly preferred embodiment of the present invention, the present invention utilizes molecular biology to construct a plurality of plasmids pCAP-mil A1, pCAP-mil A2-R, pBTK-mil A1, pCAP-mil A2-. DELTA.F-R, BAC-mb and BAC-05, and introduces these plasmids into Streptomyces hygroscopicus NRRL5739 and Streptomyces hygroscopicus SIPI-KF strains, respectively.
In a particularly preferred embodiment of the invention, plasmids pCAP-mil A1, pCAP-mil A2-R and pCAP-mil A2-. DELTA.F-R are constructed by TAR using the pCAP01a plasmid, and then the Φ C31int/attP and aac (3) IV in pCAP-mil A1 are replaced with Φ BT1int/attP and aph (3) II elements to give plasmid pBTK-mil A1. Meanwhile, the plasmid BAC-F15 is utilized to splice A1 and A2-R and A1 and A2-delta F-R respectively to obtain the plasmid BAC-mb containing the complete gene cluster and the plasmid BAC-05 containing the complete 5-keto-milbemycin biosynthesis gene cluster.
In a particularly preferred embodiment of the invention, pCAP-mil A1 and pCAP-mil A2-R are respectively introduced into the phi C31 attB site of Streptomyces hygroscopicus NRRL5739 by a conjugal transfer method to obtain engineering strains SIPI-054-001 and SIPI-054-002; introducing pBTK-milA1 into phi BT1 attB site of SIPI-054-one 002 to obtain engineering strain SIPI-054-one 003; BAC-mb is introduced into Streptomyces hygroscopicus NRRL5739 to obtain the engineering strain SIPI-054-004. Similarly, pCAP-mil A1 and pCAP-mil A2-delta F-R are respectively introduced into a phi C31 attB site of Streptomyces hygroscopicus SIPI-KF to obtain engineering strains SIPI-KF101 and SIPI-KF102, and pBTK-mil A1 is introduced into a phi BT1 attB site of SIPI-KF102 to obtain an engineering strain SIPI-KF 103; the BAC-05 is introduced into Streptomyces hygroscopicus SIPI-KF to obtain an engineering strain SIPI-KF 201. A schematic diagram of the integration of the gene cluster into the genomic DNA of Streptomyces hygroscopicus strain is shown in FIG. 2.
Method for producing milbemycins
The present inventors have made extensive studies in an effort to improve the production efficiency of a milbemycin producing strain. In the early work, the fact that more genes and large gene clusters are related to the production of the milbemycins A3/A4 is found, cloning and recombinant expression are not facilitated, and effective improvement cannot be achieved. Further, through repeated adjustment and optimization, the A1 and/or A2-R two gene clusters in the genome are determined to be used as targets of recombinant cloning, the two gene clusters are respectively expressed in the strain in a recombinant mode, or are cloned respectively and then are expressed in the same strain in a co-expression mode, the yield improvement of the milbemycins A3/A4 or derivatives thereof is effectively achieved, and the improvement is very obvious.
Accordingly, the present invention provides a method for increasing the yield of milbemycin A3/a4 or a derivative thereof, said method comprising: in a producing bacterium of milbemycins A3/A4 or derivatives thereof, there are introduced: (1) the A1 gene cluster, and/or (2) the A2-R gene cluster or a variant thereof in which the milF gene is down-regulated.
As a preferred embodiment of the present invention, the above-mentioned (1) and/or (2) is integrated into the site of Φ C31 attB or Φ BT1 attB of a producing bacterium of milbemycins A3/A4 or a derivative thereof.
In a specific embodiment of the invention, the total production of milbemycins A3/A4 is increased by about 42.1% and 48.3%, respectively, by expressing the milbemycins A1 or A2-R in Streptomyces hygroscopicus NRRL5739, respectively; the simultaneous expression of A1 and A2-R can improve the total yield by about 98.4 percent compared with the original strain NRRL 5739; after the plasmid BAC-mb is expressed in NRRL5739, the total yield is improved by about 102.3 percent compared with the original strain. Similarly, respectively expressing A1 and A2-delta F-R in a 5-keto-milbemycin A3/A4 producing strain streptomyces hygroscopicus SIPI-KF can improve the total yield of 5-keto-milbemycin A3/A4 by about 69.0 percent and 78.5 percent respectively; the simultaneous expression of A1 and A2-delta F-R can improve the total yield of 5-keto-milbemycins A3/A4 by about 104.9 percent; after expression of plasmid BAC-05 carrying the complete 5-keto-milbemycin biosynthesis gene cluster in SIPI-KF, the total yield of 5-keto-milbemycin A3/A4 was increased by about 98.2%. The above results show that the method of the present invention can significantly improve the yield of the target product.
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.
Strains, plasmids, reagents and apparatus
Streptomyces hygroscopicus NRRL 5739: purchased from NRRL.
The 5-keto-milbemycins A3/A4 producing strain Streptomyces hygroscopicus SIPI-KF is obtained by knocking out the milF gene of Streptomyces hygroscopicus NRRL5739, and the construction method refers to Appl Microbiol Biotechnol (2014)98: 9703-9712.
S. cerevisiae VL6-48 for TAR was from: ATCC MYA-366.
Escherichia coli S17-1 for conjugation transfer: commercial E.coli cells (purchased from Gibco-BRL).
EPI300 competent cells: purchased from Epicentre.
The pCAP01a for TAR in the present invention was constructed: obtained after modification on pCAP01 plasmid, and the specific construction steps are as follows: using pSET152 plasmid as a template, and using 152-fw/rv primer to amplify a DNA fragment C31-acc (4605bp) containing phi C31int/attP and acc (3) IV elements by PCR; then, pKCas 9(tipAp) plasmid is used as a template, two pairs of primers, namely gRNA-up-fw/gRNA-rv and gRNA-dn-fw/gRNA-rv, are used for carrying out PCR amplification respectively to obtain DNA templates of gRNA-up and gRNA-dn, then the gRNA-up and the gRNA-dn are transcribed into corresponding RNA respectively by using an in vitro transcription kit, then the pCAP01 plasmid is cut by using a Cas9 in vitro enzyme digestion kit, and the DNA templates are subjected to alcohol precipitation recovery; finally, the C31-acc fragment is connected with the pCAP01 plasmid cut by Cas9 by a recombination kit, and the plasmid pCA01a can be obtained.
Wherein the construction of the pSET152 plasmid is described in the references: BIERMAN M, LOGAN R, O' BRIEN K, ethyl. plasmid cloning vectors for the conjugate transfer of DNA from Escherichia coli to Streptomyces spp [ J ] Gene,1992,116(1):43-9.
Construction of the pCAP01 plasmid references: YAMANAKA K, REYNOLDS K A, KERSTEN R D, et al.direct cloning and degrading of a silicone lipid biosynthesis genetic elements the antisense taromycen A [ J ]. Proc Natl Acad Sci U S A,2014,111(5):1957-62.
The primer sequences used were:
152-fw：GACGAGATCCTCGCCGTCGGGCATCCGCGCCTTGAACGCTGTAGGTATCTCAGTTCGGTGT(SEQ ID NO:4)；
152-rv：AGATCAGGCTTCCCGGGTGTCTCGCTACGCCGCTACAGGCTTCCCGGGTGTCTCGCTA(SEQID NO:5)；
gRNA-up-fw：GACTGACACTGATAATACGACTCACTATAGGGCATCCGCGCCTTGAGCCGTTTTAGAGCTAGAAATA(SEQ ID NO:6)；
gRNA-dn-fw：GACTGACACTGATAATACGACTCACTATAGGACGGCACGGAAGACGTAGGTTTTAGAGCTAGAAATA(SEQ ID NO:7)；
BAC-F15 plasmid for splicing: reference documents: LI L et al, Metabolic engineering,2015,29: 12-25).
pRT802 reference for PCR templates: gregory MA et al, J Bacteriol 2003; 185:5320-5323.
pKCas 9(tipAp) plasmid reference for PCR template: HUANG H et al, Acta biochimiciet bipysica Sinica,2015,47(4): 231-43.
The DNA gel recovery and purification kit is purchased from Axygen company, the plasmid extraction kit is purchased from Omega Bio-Tek company, all restriction enzymes are purchased from Thermo Fisher Scientific company, the in vitro transcription kit is purchased from Thermo Fisher Scientific company, the Cas9 in vitro digestion kit is purchased from TOLOBIO company, the recombinant ligation kit is purchased from Vazyme company, the chromatographically pure acetonitrile is purchased from Amerhyst Chemicals company, and other conventional reagents are all domestic analytically pure or imported and subpackaged.
The constant temperature fermentation shaker was purchased from Shanghai Heishi laboratory facilities, Inc., and the model 1200 high performance liquid chromatograph was purchased from Agilent Technologies, Inc.
Culture medium
1. Liquid LB medium (1L)
10g of peptone, 5g of yeast extract, 10g of NaCl and 1L of distilled water; sterilizing at 121 deg.C for 20 min.
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 M-Isp4 medium formula (1L)
5g of soybean cake powder, 5g of mannitol, 5g of starch, 2g of peptone and fermented soybean cake powder1g of mother powder, 1g of NaCl, (NH)4)2SO42g,K2HPO31g,CaCO32g of agar powder, 20g of agar powder and 1mL of inorganic salt trace element solution, and adjusting the pH value to 7.0; sterilizing at 121 deg.C for 20 min.
4. Liquid 2 XYT medium formula (1L)
16g of peptone, 10g of yeast powder and 5g of NaCl; sterilizing at 121 deg.C for 20 min.
MB solid Medium formulation (1L)
4g of sucrose, 2g of yeast extract powder, 1g of skimmed milk powder and 20g of agar powder, and adjusting the pH to 7.2; sterilizing at 121 deg.C for 20 min.
6. Seed culture medium formula (1L)
20g of cane sugar, 5g of yeast extract powder, 1g of skim milk powder and K2HPO41g, adjusting the pH value to 7.2; sterilizing at 121 deg.C for 20 min.
7. Fermentation medium formula (1L)
120g of cane sugar, 10g of skim milk powder, 10g of cottonseed cake powder and K2HPO41g，FeSO4·7H2O 0.1g，CaCO33g, adjusting the pH value to 7.2; sterilizing at 121 deg.C for 20 min.
8. Yeast selection medium (1L)
Yeast Nitrogen Base with amino acids 1.7g, Yeast synthetic drop-out additives 1.9g, sorbitol 182g, D-glucose 20g, ammonium sulfate 5g, agar powder 2g, 115 ℃ sterilization for 15 min.
YPD Medium (1L)
20g of D-glucose, 20g of tryptone, 10g of yeast extract and 0.8g of adenine sulfate, and sterilizing at 115 ℃ for 15 min.
Primer and method for producing the same
The primers used in the present invention are shown in Table 1.
TABLE 1
