CN113774005A

CN113774005A - High-yield engineering strain of chlorosulicin and construction method thereof

Info

Publication number: CN113774005A
Application number: CN202010526235.XA
Authority: CN
Inventors: 谭华荣; 李月; 张集慧; 郑家珍
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-10
Anticipated expiration: 2040-06-10
Also published as: CN113774005B

Abstract

The invention provides a high-yield engineering strain of chloramphenicol and a construction method thereof. The invention constructs recombinant engineering strains F2OE and YL04 by utilizing the concept that a strong promoter drives a positive control gene of the chloramphenicol. Compared with the wild type, the level of the chloramphenicol produced by the recombinant engineering strains F2OE and YL04 constructed in two different ways is greatly increased, and the recombinant engineering strains are proved to be streptomyces antibioticus engineering strains with high yield of the chloramphenicol.

Description

High-yield engineering strain of chlorosulicin and construction method thereof

Technical Field

The invention belongs to the field of antibiotic genetic engineering, and particularly relates to a high-yield engineering strain of chloramphenicol and a construction method thereof.

Background

Streptomyces is a gram-positive filamentous bacterium with high GC content, belongs to Streptomyces of actinomycetemcomitans, exists in natural soil and rotten plants, and inhabits in fresh water, seawater, marine sediments, animals and plants, and even marine organisms. The streptomyces can produce abundant and various secondary metabolites, and the secondary metabolites have various activities of resisting fungi, bacteria, malaria, tumors, plant viruses, insects and the like, and have important application values in the industries of clinical medicine, agriculture, animal husbandry and the like.

Since the engineering strains require time in the steps of culturing and fermenting, and use a large amount of media, large culture tanks, and the like, and a large amount of consumables are required for purification treatment for recovering a target component from a bacterial solution, it is desired to increase the concentration of the target component per cell as much as possible and to improve the ability of the engineering strains to produce secondary metabolites beneficial to humans. Therefore, the bacterial strain with high secondary metabolite yield is beneficial to improving the working efficiency in scientific research and reducing the industrial production cost.

The biosynthesis of antibiotics is responsible for a series of structural genes arranged in clusters and is controlled by regulators encoded by pathway-specific regulatory genes within the clusters. These pathway-specific regulators are different from global regulators and pleiotropic regulators, and belong to the most basic and bottom-most regulation. The regulation of the biosynthesis of secondary metabolites involves a very complex regulatory network which can respond to the environment or to nutritional factors, signaling molecules and even to antibiotics themselves or to intermediate metabolites of their synthetic pathways.

Chlorofusicins, a class of spirolactone polyketide antibiotics, were produced by Streptomyces antibioticus (Streptomyces antibioticus Tu 99, DSMZ culture Collection number DSM40725, hereinafter also referred to as Streptomyces antibioticus DSM 40725) and were first isolated in 1969. It has anti-infective, anti-tumor, anti-malarial and cholesterol synthesis inhibitory activities (Lacoske, M.H., and Theodorakis, E.A. spirotetron polyketides as leaves in drug delivery. J Nat Prod, 2015, 78: 562-575). However, there is a problem that the yield of chloramphenicol is low in the existing wild-type Streptomyces strain capable of producing chloramphenicol with respect to its utilization.

The biosynthesis gene cluster of chlorosis was cloned in 2006 by Liu research team of Shanghai institute of Chinese academy of sciences, and the DNA sequence of the cluster was uploaded (NCBI website https:// www.ncbi.nlm.nih.gov/nuccore/DQ116941.2, GenBank accession number: DQ116941.2), and the biosynthesis pathway thereof was analyzed (non-patent document 1). This cluster is known to contain 35 genes involved in biosynthesis, constituting a DNA sequence of 101.8 kb.

Specific regulators of ChlF1, ChlF2 are known to exist in the chloramphenicol biosynthetic pathway. Previous work by the inventors shows that ChlF1 is an important activator for the synthesis of chloramphenicol, directly activates the transcription of the acetyl-CoA carboxytransferase encoding gene chlJ, which is closely related to the synthesis of chloramphenicol, and negatively regulates the transcription of the self-encoding gene chlF1, the type II thioesterase encoding gene chlK, and the active synergistic protein encoding gene chlG.

However, the inventors also found that only glycosylated chloramphenicol and its intermediate responded as a signal molecule to the modulator ChlF1, suggesting that there was some correlation between the biological activity of the signal molecule and the response of the signal molecule to ChlF1 and the glycosylation of chloramphenicol (non-patent document 2). In the reported high expression strain of chlF1, the yield of chloramphenicol was improved, but the improvement was not large (less than one-fold).

Another pathway-specific regulator, ChlF2, belongs to the SARP family of regulatory proteins (Streptomyces antigenic regulatory proteins, SARPs). SARPs are a large family of transcriptional activators, and generally contain an N-terminal OmpR-type DNA binding domain and a bacterial transcriptional activation domain (non-patent document 3). For example, ActII-ORF4 in Streptomyces coelicolor, RedD and DnrI in Streptomyces bauseyi belong to the classical SARP family, which regulate the biosynthesis of actinorhodin, undecylprodigiosin and daunomycin, respectively (non-patent documents 4 and 5). However, the regulatory mechanism of ChlF2 in the biosynthesis pathway of chloramphenicol is still unclear.

Therefore, the inventor takes streptomyces S.antibioticus DSM40725 as a research material, researches a molecular mechanism of ChlF2 for regulating and controlling the biosynthesis of the chloramphenicol, establishes a method for improving the yield of the chloramphenicol by a genetic engineering means, and obtains a high-yield engineering strain of the chloramphenicol.

Reference to the literature

Non-patent document

Non-patent document 1: jia, x.y., Tian, z.h., Shao, l., et al, genetic characterization of the chlorothricin gene cluster as a model for spirotetron anti-biotic biosynthes chem Biol, 2006, 13: 575-585)

Non-patent document 2: li, Y, Li, J, Tian, Z, et al (2016) coding Modulation of a Biosynthesis by Binding of the Glycosylated Intermediates and End Product to a Responsive Regulator ChlF1.J Biol Chem 291, 5406-

Non-patent document 3: tanaka, a., Takano, y., ohnisi, y., and horiouchi, s.afsr recurxits RNA polymerase to the afsS s promoter: a model for translational actuation by SARPs.J. Mol Biol, 2007, 369: 322-333.

Non-patent document 4: arias, p, Fernandez-Moreno, m.a., and maprotida, f.characteristics of the path-specific positive transcriptional regulator for expression of biochemical in Streptomyces coelicolor a3(2) as a DNA-binding protein.j Bacteriol, 1999, 181: 6958-6968.

Non-patent document 5: tang, l., Grimm, a., Zhang, y.x., and Hutchinson, c.r. purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of a systemic biotin in Streptomyces bacterium, 1996, 5: 801-13.

Disclosure of Invention

The invention relates to streptomyces antibioticus with high expression of chloramphenicol and a construction method thereof.

In one embodiment of the invention, P is inserted tracelessly into a chromosome in the genome of Streptomyces antibioticus expressing chloramphenicol by the homologous double crossover method_kanReplacement of the coding region of the chlK gene (SEQ ID NO: 6) by kan (SEQ ID NO: 5) so that expression of kan and chlF2 are both in the strong promoter P_kanDrives the high expression of the chlF2, and finally realizes the high expression of the chloramphenicol by the streptomyces antibioticus.

In another embodiment of the invention, an integrative plasmid is introduced into Streptomyces antibioticus expressing chloramphenicol, in which case P is present on the chromosome of Streptomyces antibioticus_chlKSequence-unchanged from chlK-chlF 2(SEQ ID NO: 8), by means of the integrative plasmid using a strong promoter P_kanThe expression of the genes of chlK and chlF2 is driven, and finally the streptomyces antibioticus can highly express the chloramphenicol.

Specifically, the present invention includes the following:

1. a method of constructing a strain expressing chloramphenicol, comprising:

replacing the chlK gene in the genome of streptomyces chlorosis producing with a strong promoter sequence, preferably said strong promoter being additionally linked to a kanamycin resistance selection gene kan, more preferably said kan having a sequence as shown in SEQ ID NO:4 is shown in the specification;

alternatively, the method comprises transforming a recombinant plasmid comprising a strong promoter sequence, an expression cassette for the chlK and chlF2 genes in the plasmid backbone into a streptomyces for the production of chloramphenicol, wherein preferably the chlK gene sequence is as set forth in SEQ ID NO:6, the sequence of the chlF2 gene is shown as SEQ ID NO:2, respectively.

2. The method of item 1, wherein the strong promoter is selected from the group consisting of P_kanPromoter, P_ermE*Promoter, or P_kasO*Promoter, preferably P_kanPromoter, more preferably P_kanThe sequence is shown as SEQ ID NO:3, respectively.

3. The method of item 1, wherein the replacement is performed by means of traceless knock-in, recombinant plasmid introduction, recombinant plasmid integration.

4. The method of item 1, wherein the strong promoter sequence is set forth in SEQ ID NO:3, the sequences of the expression cassettes of the genes chlK and chlF2 are shown as SEQ ID NO: shown at 7.

5. The method of item 1, wherein the plasmid backbone is selected from pSET152, pKC1139 or pIJ10500, preferably pSET 152.

6. The process of item 1, wherein the Streptomyces chlorothricin-producing Streptomyces is Streptomyces antibioticus Tu 99, Streptomyces antibioticus DSM 40725.

7. A strain expressing chloramphenicol produced by the method of any one of items 1-6.

8. Use of the strain expressing chloramphenicol described in item 7 for the production of chloramphenicol.

In order to clarify the regulatory mechanism of chloramphenicol, the present inventors first constructed a gene-disrupted mutant Δ chlF2 and a gene-complemented strain Δ chlF2/pSET152 of chlF2, respectively: : p_chlkThe positive regulation of chlF2 in the biosynthesis of chloramphenicol was determined by analysis of the yields of chloramphenicol from chlK-chlF2 (both using the S.antibioticus DSM40725 strain, the starting plasmid pKC1139 and the starting plasmid pSET 152).

Then, a semi-quantitative PCR experiment proves that the genes chlK and chlF2 are in a co-transcription relation, and on the basis, the following strains (figure 3A) are respectively constructed by using a genetic engineering means and a method for preparing streptomyces antibioticus with high expression of the chloramphenicol is established:

traceless insertion of P on the chromosome of S.chloramphenicol antibiotic (e.g., S.antibioticus DSM40725 strain)_kan-kan instead of the coding region of the chlK gene (SEQ ID NO: 6), thus obtaining the recombinant Streptomyces antibioticus (F2OE) highly expressing chloramphenicol of the present invention.

Alternatively, a strong promoter P was constructed_kanVectors driving the genes chlK and chlF2 (integration plasmid, pSET 152:: P)_kanThe chlK-chlF2 plasmid) and introduced into a chloramphenicol-expressing Streptomyces antibioticus strain (e.g., S.antibioticus DSM40725 strain, hereinafter sometimes abbreviated as WT) to thereby obtain a highly expressed chloramphenicol recombinant Streptomyces antibioticus WT/pSET152 of the present invention: : p_kan-chlK-chlF2(YL04)。

Dechlorinated-chlorosis is an intermediate in a biosynthesis pathway of the chlorosis, a side chain 6-methyl salicylic acid and a main skeleton spirolactone ring are synthesized through a type I PKS, wherein the 6-methyl salicylic acid needs to be methylated and halogenated to form a modified 2-methoxy-5 chloro-6 methyl salicylic acid, the modified 2-methoxy-5 chloro-6 methyl salicylic acid and two deoxyolivine sugars and a spirolactone structure form a structure of the chlorosis, the chlorosis and the dechlorinated-chlorosis structurally different by one chlorine atom, and the activity of the chlorosis is stronger than that of the dechlorinated-chlorosis on partial positive gram bacteria, so that the obtained higher-activity chlorosis has important significance.

The inventors found that the ratio of the recombinant Streptomyces antibioticus (F2OE) and the recombinant Streptomyces antibioticus WT/pSET 152: : p_kanBoth yields of chloramphenicol and dechlorinated-chlorosis were significantly higher in-chlK-chlF 2(YL04) than in wild type.

Drawings

In order to make the object, technical scheme and product effect of the invention more clear, the invention provides the following figures and tables and carries out detailed description.

FIG. 1 and FIG. 1A are schematic diagrams of a gene cluster for biosynthesis of chloramphenicol. Fig. 1B shows on the left side the expression of the wild type strain WT (s. amylotics DSM40725, the same applies below), the chlF2 knockout strain Δ chlF2 and the gene complementation strain Δ chlF2/pSET 152: : p_chlkHPLC analysis of-chlK-chlF 2, the chemical structure of chloramphenicol is shown at the top left, with the chlorine atom marked with a box. CHL represents Chlorothricin (Chlorothricin), and Des-CHL represents dechlorinated-Chlorothricin (Desschloro-Chlorothricin). The right side of fig. 1B shows the results of the biological activity tests performed on the HPLC effluents of the three strains with staphylococcus aureus and bacillus subtilis as indicator bacteria, respectively.

FIG. 2 and FIG. 2 are schematic diagrams of the positions of primer designs and the results of semi-quantitative PCR experiments in the co-transcription analysis of the genes chlK and chlF 2. Wherein "+" is a positive control and indicates a PCR amplification product using genomic DNA as a template; "-" is a negative control, and indicates a product of PCR amplification using RNA that has not been reverse-transcribed into cDNA as a template; c represents PCR amplification product using cDNA as template.

FIGS. 3 and 3A are schematic diagrams showing the construction of the chlF2 driven in different ways in two strains, the expression "derived from pSET152 vector" represents that the gene is constructed on pSET152 (the pSET152 vector is an integrative vector, i.e., the plasmid is not episomal after introduction but is inserted in an integrated form into the attB site on the Streptomyces genome, and the DNA sequence on the plasmid is replicated along with the replication of the chromosome), and the expression "derived from the cluster of chlorosilacins gene" represents the arrangement of the gene on the genomic DNA of each strain; FIG. 3B is a comparison of yields of chloramphenicol and dechlorinated-chloramphenicol for each strain.

Detailed Description

The invention is further illustrated by the following examples.

In the present invention, Streptomyces antibioticus Tu 99 (a non-model strain, which is given by Liu-Wen researchers of Shanghai institute of organic science, deposited at DSMZ culture Collection, Germany, with the deposit number DSM40725, see non-patent document 1, e.g., available from the Chinese microbial species query network Http:// www.biobw.org) is used as the Streptomyces antibioticus. However, it will be understood by those skilled in the art that other strains that produce chloramphenicol as well as strains that achieve increased production of chloramphenicol using the methods of the present specification are also within the scope of the present invention.

Wherein, the gene of the specific regulatory gene chlF2 of the biosynthesis pathway of the chloramphenicol involved in the present invention is derived from a gene cluster of the biosynthesis gene cluster of the chloramphenicol of Streptomyces antibioticus DSM40725 (GenBank accession number: DQ116941.2) shown in sequence No.1(SEQ ID NO: 1) of the sequence Listing. The amino acid sequence of the regulatory protein ChlF2 coded by the protein is shown in the sequence No.2(SEQ ID NO: 2) of the sequence table.

Those skilled in the art will understand that for SEQ ID NO:2, such proteins are still suitable for use in the present invention, as they have the function of positively regulating chloramphenicol or even have enhanced activity, by way of mutation or modification of one or more amino acids, such as substitution, addition or deletion of a portion of the amino acids. And the gene encoding the polypeptide or the protein can also be used for the invention to construct a recombinant bacterium capable of improving the yield of the chloramphenicol.

The promoter used in the present invention is not limited to P_kanPromoters (shown in SEQ ID NO: 3), but also other promoters commonly used in the genetic manipulation of Streptomyces, such as P_ermE*Promoter (Bibb, M.J., White, J., Ward, J.M., and Janssen, G.R.The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site.Mol Microbiol，1994，14：533-545.)、P_kasO*Promoters (Wang, W., Li, X., Wang, J., Xiaoing, S., Feng, X., and Yang, K.an engineered strong promoter for Streptomyces. appl Environ Microbiol, 2013, 79: 4484-.

The plasmid for high expression of chlF2 in the present invention is not limited to pSET152 (available from Novegen) (Bierman, M., Logan, R., O' Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E.plasmid cloning vectors for the conjugate vector of DNA from Escherichia coli to Streptomyces sp.Gene, 1992, 116: 43-49), but may be an episomal high copy plasmid such as pKC1139(Kieser, T., Bibby, M.J., Butter, Chater, K.F., and Hopwood, D.A.practical plasmids, 2000, John Innes, Normal, Kinectic, molecular, and genome, D.A.practical plasmids, 2000, John Innes, university, integrity, and integrity, or other plasmids such as plasmid, integrity, clone, 2003, integrity, clone, 53185, and integrity, etc. (available from Bacillus).

The plasmid of the present invention can be integrated into the genome of Streptomyces by conjugative transfer technology, protoplast fusion technology, electroporation transformation, chemical transformation, and the like. The present invention prefers the bond transfer technique but is still effective with other techniques.

The recombination technique used in the present invention may be any suitable technique used in the art, for example, by knocking the target gene into the genome of the strain at any position, introducing a recombinant plasmid containing the target gene (for example, episomal high-copy plasmid pKC 1139) into the strain, and integrating the recombinant plasmid containing the target gene into the genome of the bacterial cells.

Analysis of yields of Chlorosuccinin and dechlorinated Chlorosuccinins

Unless otherwise specified, the following materials, conditions and HPLC parameters were used in the present invention for the production analysis of both chloramphenicol and dechlorinated-chloramphenicol.

Fermentation and culture materials of the strains:

seed culture medium: adopting YEME: peptone 0.5% by BD, yeast extract 0.3% by Oxoid, malt extract 0.3% by Oxoid, glucose 1%, sucrose 20%, MgCl₂0.1 percent, prepared by distilled water and sterilized for 30 minutes at 115 ℃.

The fermentation medium adopts the following formula: soybean cake powder (cold pressing, Haimingwei corporation) 2%, mannitol (Hai Ke hong Chuang corporation) 2%, CaCO₃(national drug group) 0.2%, distilled water, and sterilizing at 115 deg.C for 30 min.

Common culture and conjugation transfer were performed using MS: 2 percent of soybean cake powder (common), 2 percent of mannitol, 2 percent of agar (stringh is more than 1300), and 2 percent of distilled water, and sterilizing for 30 minutes at 115 ℃.

The fermentation process comprises the following steps:

s.antibioticus DSM40725, or a recombinant strain based on s.antibioticus DS M40725 constructed in the following step, was cultured on an MS plate for about 6 days, and after spores were matured (in gray black), a spore suspension was prepared using 20% glycerol.

Each strain was inoculated as a spore suspension (30. mu.L) into seed medium YEME (50mL) (250mL Erlenmeyer flask). After culturing at 28 ℃ and 220rpm for 48 hours, the cells were inoculated into a fermentation medium in an amount of 6% and cultured at 220rpm and 28 ℃ for 7 days. After fermentation, the fermentation broth (100mL) was centrifuged at 10000rpm for 10min to collect mycelia, the supernatant was discarded, and the mycelia were removed by freeze-drying. After extraction with 30mL of methanol for 6h, the methanol was removed by rotary evaporation, and the entire extract obtained was dissolved in 1mL of anhydrous methanol, filtered and analyzed by HPLC.

Conditions for HPLC analysis:

agilent ZORBAX SB-C18 column (5 μm, 4.6X 250mm) was analyzed by Agilent liquid chromatography.

Mobile phase A: deionized water (0.05% trifluoroacetic acid)

Mobile phase B: chromatographic grade acetonitrile (0.05% trifluoroacetic acid)

Detection wavelength: 222nm

Flow rate: 1ml/min

Sample loading amount: 40 μ l

Time(min)	0.00	5.00	20.0	25.0	30.0
						Mobile phase B%	40％	40％	85％	85％	40％

Antibacterial activity

The antibacterial activity of the HPLC effluent was examined by a conventional method in the art using Bacillus subtilis and Staphylococcus aureus (both available from Beijing Baiopaownwei Biotechnology Co., Ltd.) as indicator bacteria.

Other main materials involved in the embodiments of the present invention.

Table 1 plasmids used in the study

Table 2 strains used in the study

TABLE 3 primer sequences

Example 1 Positive Regulation of Chlorofusin biosynthesis by ChlF2

1. Blocking mutant Δ chlF2 and gene complementation strain Δ chlF 2/: : p_chlKConstruction of-chlK-chlF 2

Blocking mutant Δ chlF 2: the upstream and downstream sequences of the coding region of the chlF2 gene in the chloramphenicol gene cluster (gene cluster Genbank No: DQ116941.2) of the S.antibioticus DSM40725 strain were each selected to be about 1.6kb, respectively, as upstream and downstream homology arms for performing homologous double crossover. The coding region of the chlF2 gene is shown in SEQ ID NO: 1, the ChlF2 protein sequence is shown in SEQ ID NO: 2.

PCR was carried out using the extracted genomic DNA of S.antibioticus DSM40725 as a template and primers F2DML-F/R (5 '-3') and F2DMR-F/R (5 '-3') (sequences shown in SEQ ID NOS: 10, 11, 12 and 13, all primers were synthesized by Invitrogen, the same applies hereinafter) to obtain upstream and downstream fragments (1.6 kb each).

At the same time, the plasmid pUC 119: : neo as a template, and PCR was performed using primers Bkan-F/R (shown in SEQ ID NOS: 14, 15) to obtain a fragment (P) including the kanamycin resistance gene and its promoter_kan-kan)。P_kanThe sequence is shown in SEQ ID NO:3, kan sequence shown in SEQ ID NO:4, P_kan-kan sequence shown in SEQ ID NO: 5.

for the upstream and downstream fragments as PCR products, and P_kanThe kan sequence fragments were digested with HindIII/BglII, BglII/XbaI and BamHI/BglII, respectively, and recovered and purified with an agarose gel recovery kit. The recovered fragment was ligated with the temperature-sensitive plasmid pKC1139 which had been digested with HindIII/XbaI to obtain the chlF2 knock-out plasmid pLY201 (Table 1).

The chlF2 knockout plasmid pLY201 was introduced into the WT strain by a homologous double crossover strategy, whereby the kanamycin resistance selection gene and its promoter were inserted and the chlF2 gene was deleted from the chromosome to obtain the chlF2 knockout Streptomyces antibioticus strain Δ chlF 2.

Gene complementation strain Δ chlF 2/: : p_chlK-chlK-chlF2：

Similarly, PCR was performed using the extracted S.antibioticus DSM40725 genomic DNA as a template and using primers F2zspg-F/F2zspg-R (SEQ ID NOS: 16, 17) to obtain amplification products of chlF2, chlK and their promoter regions, amounting to about 3.2kb (5 '-3'), i.e., P_chlK-chlK-chlF2 sequence, shown in SEQ ID NO: 8.

the resulting fragment was digested with XbaI/BamHI, and ligated with pSET152 cleaved with the same cleavage site using T4 DNA ligase to construct a gene complementing plasmid pSET 152: : p_chlK-chlK-chlF2, i.e. pLY 202. This plasmid was introduced into a knockout strain Δ chlF2 by conjugative transfer (e.coli ET12567/pUZ8002) to obtain a complementation strain Δ chlF2/pSET 152: : p_chlK-chlK-chlF2, sometimes also denoted as Δ chlF2/pLY 202.

2. Comparison of Chlorosuccinin yields in wild-type, blocked mutant and Gene-complemented strains

Comparison of yields of chloramphenicol by three strains was performed using HPLC, and the fermentation, HPLC conditions and parameters of the strains were the same as those described in the detailed description.

As shown in FIG. 1B, no peak ascribed to chloramphenicol was observed in the blocking mutant strain Δ chlF2, indicating that chloramphenicol was not normally synthesized in the strain after the chlF2 knockout. And in the gene complementation strain Δ chlF 2/: : p_chlKA characteristic peak of chloramphenicol was observed in-chlK-chlF 2, indicating thatThe synthesis capacity of the chloramphenicol is recovered through the complementation of the chlF2 gene.

From the above results, it was demonstrated that chlF2 is a gene that plays a positive regulatory role in the biosynthesis pathway of chloramphenicol.

The inventors further examined the antibacterial activity of the effluent at 24.9min when the wild type, the disrupted mutant and the gene complementation strain were analyzed by HPLC using Bacillus subtilis and Staphylococcus aureus (both from Beijing Baiohobowei Biotech Co., Ltd.) as indicator bacteria. The results show that: as with the results of the HPLC analysis, the effluent of the blocking mutant Δ chlF2 no longer had activity against gram-positive bacteria and staphylococcus aureus, whereas the gene replenisher Δ chlF 2/: : p_chlKThe effluent of the-chlK-chlF 2 has bacteriostatic activity. The results are shown in FIG. 1B. Further proves that the chlF2 has an indispensable important role in the biosynthesis of the chloramphenicol.

In conclusion, the inventors found that the knockout of chlF2 by genetic engineering techniques resulted in the failure of the synthesis of chloramphenicol, but that the yield of chloramphenicol was restored after the complementation of chlF2 gene, confirming that chlF2 exerts a positive regulatory effect on the biosynthesis of chloramphenicol.

Example 2 Co-transcriptional analysis of chlK and chlF2

The inventors speculate that the possibility of co-transcription of the two genes exists, based on the fact that the orientation of the ORFs of the chlK and chlF2 genes is the same and they are arranged in close proximity. To determine whether co-transcriptional relationships exist, semi-quantitative PCR experiments were performed.

First, s.antibioticus DSM40725, which is a wild strain, was extracted and reverse-transcribed to obtain cDNA. PCR was performed using the cDNAs (test group), genomic DNAs extracted from the strains (positive control), and RNAs not reverse-transcribed into cDNAs (negative control) as templates, respectively, using internal reference primers B16S-F/R (SEQ ID NOS: 18, 19, which can amplify the internal sequence of the 16S rRNA-encoding gene of S.antibioticus DSM 40725), and primers PF2-F/PK-R (SEQ ID NOS: 20, 21) for testing the co-transcription of chlK and chlF 2.

As a result, in PCR amplification using genomic DNA (positive control) and cDNA (test group) as templates, a band of about 427bp (16S rRNA) was obtained using each of the primers B16S-F/R, and no band was amplified in the negative control group. When genomic DNA (positive control) and cDNA (test group) were used as templates, a band of approximately 1397bp was obtained using the primer PF2-F/PK-R, whereas no band was amplified in the negative control group. These results demonstrate that the gene chlK and chlF2 are in a co-transcriptional relationship (FIG. 2).

Example 3 increasing the expression of Chlorosuccinin by high expression of the chlF2 Gene

1. Construction of strain F2 OE:

first, using the extracted genomic DNA of S.antibioticus DSM40725 as a template, PCR amplification reactions were carried out using F2H1-F/R and F2H2-F/R (SEQ ID NOS: 22, 23, 24, 25) to obtain upstream and downstream fragments (about 2.5kb each) of the coding region of the chlK gene as upstream and downstream homology arms for homologous double crossover, respectively.

The contents of pUC 119: : neo (Table 1) as a template, and Nkan-F/R (SEQ ID NOS: 26 and 27) as a primer were subjected to PCR amplification to obtain a fragment (P) including the kanamycin-resistant gene and its promoter_kan-kan)。P_kan-kan sequence shown in SEQ ID NO: 5.

for upstream and downstream fragments and P as PCR products_kanThe kan fragment was digested with HindIII/NdeI, NdeI/XbaI, NdeI/NdeI, respectively, and recovered and purified with an agarose gel recovery kit. The recovered fragment was ligated with the HindIII/XbaI-digested temperature-sensitive plasmid pKC1139 (Table 1) to obtain plasmid pLY202 (Table 1).

Plasmid pLY202 was introduced into S.antibioticus DSM40725 strain by conjugal transfer (Streptomyces-Escherichia coli ET12567/pUZ8002) for homologous double crossover to obtain recombinant strain F2OE strain (FIG. 3A).

The fermentation process and the culture medium used were the same as those used in example 1. At this time, in F2OE, the coding region of the chlK gene (SEQ ID NO: 6) on the chromosome belonging to S.antibioticus DSM40725 was replaced with P_kan-kan。

It should be noted that, in the construction of F2OE strain, the foreign plasmid was not integrated, but the gene encoding chlK on the chromosome was deleted andin situ replacement by P_kan-kan。

2. Construction of strain YL 04:

first, the contents of pUC 119: : the DNA of neo plasmid (Table 1) and S.antibioticus DSM40725 genomic DNA were used as templates, and PCR amplification reaction was carried out using primers Gkan-F/R (SEQ ID NOS: 28, 29) and KP-F/F2P-R (SEQ ID NOS: 30, 31) to obtain a fragment of about 0.19kb and a fragment of about 1.78kb, respectively. These two fragments were digested with SpeI and EcoRI, respectively, recovered and purified using an agarose gel recovery kit, and the recovered fragments were ligated with the plasmid pSET152 (Table 1) purified by digestion with XbaI/EcoRI to obtain plasmid pSET 152: : p_kan-chlK-chlF2 (Table 1).

P contained in the plasmid_kan-chlK-chlF2 is shown in SEQ ID NO: 9.

the pSET152 constructed above: : p_kanThe plasmid chlK-chlF2 was introduced into strain S.antibioticus DSM40725 by means of conjugative transfer (Streptomyces-Escherichia coli ET12567/pUZ8002) to obtain recombinant strain YL04 (FIG. 3A), the fermentation process and the media used were the same as those used in example 1.

Comparison of yields of dechlorinated-and-chlorosilotins from F2OE and YL04

Changes in yields of dechlorinated-chlorosis-CHL (des-CHL) and chlorosis-CHL (CHL) were measured in three strains of WT, F20E and YL04 using HPLC, wherein the conditions and parameters used were as in the detailed description section. The experimental data are from three independent experimental results and are expressed by a standard deviation +/-variance method.

The results showed that dechlorinated-and-54-fold increases in F2OE strain compared to wild-type (WT) were 6.3-fold and 54-fold, respectively (FIG. 3B). In particular, dechlorinated-and-4-fold increases in YL04 strain were 11-fold and 8.4-fold, respectively, compared to Wild Type (WT) (fig. 3B).

Thus, it was found that P was used in the F2OE strain_kan-kan replaces the majority of the chlK coding region in the chromosome, with pSET 152: : p_kanthe-chlK-chlF 2 plasmid increased production of dechlorinated and chlorosulicin and in both strainsIn addition, the yield-increasing effect of chloramphenicol was superior to that of dechlorinated-chlorosis (FIG. 3B).

Taken together, the above results demonstrate that both strategies can greatly improve the production of dechlorinated-chloroses (des-CHL) and Chloroses (CHL) in chloramphenicol-producing Streptomyces antibioticus, regardless of the use of a strong promoter in place of the chlK coding region in the chromosome, or the introduction of a plasmid containing the strong promoter-chlK-chlF 2 sequence.

Therefore, the inventor successfully obtains the recombinant streptomyces antibioticus with high expression of the chloramphenicol and provides a method for establishing an engineering strain with high expression of the chloramphenicol by a genetic engineering means.

Industrial applicability

The recombinant streptomyces antibioticus and the establishment method thereof provided by the invention can be widely applied to industries related to the application of the chloramphenicol, such as scientific research work, clinical medical treatment, pharmacy, agriculture, aquatic product animal husbandry and the like, and have important application value.

Claims

1. A method of constructing a strain expressing chloramphenicol, comprising:

replacing the chlK gene in the genome of streptomyces for producing the chloramphenicol with a strong promoter sequence, preferably the strong promoter is additionally connected with a kanamycin resistance screening gene kan, and more preferably the sequence of the kan is shown as SEQ ID NO. 4;

alternatively, the method comprises transforming a recombinant plasmid into streptomyces for producing chlorosulicin, the recombinant plasmid comprising a strong promoter sequence, an expression cassette for chlK and chlF2 genes in the plasmid backbone, wherein preferably the chlK gene sequence is shown as SEQ ID NO. 6 and the chlF2 gene sequence is shown as SEQ ID NO. 2.

2. The method of claim 1, wherein the strong promoter is selected from the group consisting of P_kanPromoter, P_ermE*Promoter, or P_kasO*Promoter, preferably P_kanPromoter, more preferably P_kanThe sequence is shown as SEQ ID NO. 3.

3. The method of claim 1, wherein the replacement is performed by way of traceless knock-in, recombinant plasmid introduction, recombinant plasmid integration.

4. The method of claim 1, wherein the strong promoter sequence is shown in SEQ ID NO 3 and the sequences of the expression cassettes for the genes chlK and chlF2 are shown in SEQ ID NO 7.

5. The method of claim 1, wherein the plasmid backbone is selected from pSET152, pKC1139 or pIJ10500, preferably pSET 152.

6. The process of claim 1, wherein the chloramphenicol-producing Streptomyces is Streptomyces antibioticus Tu 99, Streptomyces antibioticus DSM 40725.

7. A strain expressing a chloramphenicol produced by the method of any one of claims 1-6.

8. Use of the strain expressing chloramphenicol according to claim 7 for the production of chloramphenicol.