CN112430608B - Method for constructing high-yield engineering bacteria of oritavancin precursor and application - Google Patents

Abstract

The invention provides a method for constructing high-yield engineering bacteria of an oritavancin precursor, and application thereof, wherein the high-yield bacteria of the oritavancin precursor is Chromemomycin, and a positive regulatory gene strR biologically synthesized from the oritavancin precursor is introduced into amycolatopsis orientalis producing the Chromemomycin, so that the high-yield bacteria of the oritavancin precursor, namely the Chromemomycin, is obtained, and the collection number is CGMCC NO. 21140. The method provided by the invention avoids the defects of long period and manpower and material consumption caused by the traditional mutation breeding technical means. The yield is improved by 40 percent compared with the original strain and reaches 140mg/L, the generation cost of the oritavancin precursor is reduced, the biosynthesis of the oritavancin precursor Chromemomycin can be improved, the method has important application value in industrial production of the oritavancin precursor, and an effective technical means is provided for developing glycopeptide antibiotics.

Description

Method for constructing high-yield engineering bacteria of oritavancin precursor and application

Technical Field

The invention belongs to the technical field of medicinal biology, and relates to a method for constructing oritavancin high-yield engineering bacteria and application thereof.

Background

Vancomycin is an important glycopeptide antibiotic for treating drug-resistant gram-positive bacterial infection, but the problem of drug resistance of gram-positive pathogenic bacteria is becoming serious, and vancomycin-resistant staphylococcus aureus (VISA and VRSA) and vancomycin-resistant enterococcus (VRE) appear in succession around the world. Therefore, vancomycin has not been able to fully satisfy clinical needs, and there is a need to develop a new generation of glycopeptide antibiotic drugs.

In 2014, the american FDA approved Oritavancin (oritavacin) developed by medicins corporation for marketing under the trade name orabativ, which is a new generation glycopeptide antibiotic after vancomycin. Oritavancin is the first and only single dose treatment regimen approved by the FDA in the united states for the treatment of acute bacterial skin and skin structure infections (abssssis). The oritavancin injection is used for treating adult ABSSSIS infection caused by gram-positive bacteria.

Amycolatopsis orientalis is a producing strain of Cloroeremomycin (also known as A82846B) which is a precursor of Oritavancin, and the fermentation unit is low at present. Therefore, the synthetic route is reasonably reconstructed by utilizing the synthetic biotechnology to improve the fermentation level of the Chroroeremomycin, and the method has important application value for industrialization of oritavancin.

Disclosure of Invention

The invention aims to provide a construction method of high-yield Oritavancin precursor engineering bacteria, wherein the high-yield Oritavancin precursor engineering bacteria are Chromelomycin, Amycolatopsis orientalis (Amycolatopsis orientalis) with the preservation number of ACCC40175 is taken and becomes Chromemomycin producing bacteria through laboratory genetic breeding, and a positive regulatory gene strR for biosynthesis of the Oritavancin precursor Chromemomycin is introduced to obtain the high-yield Chromemomycin bacteria, and the yield of the obtained Chromemomycin is increased by about 40 percent and reaches 140 mg/L. The strain is preserved in the China general microbiological culture Collection center, and is classified and named as: amycolatopsis orientalis (Amycolatopsis orientalis) L80, accession number: CGMCC NO.21140, preservation date: 2020.11.9.

the construction method comprises the following specific steps:

(1) the specific primers were purchased from Amycolatopsis orientalis (Amycolatopsis orientalis) with the accession number of ACCC40175 under the classification name of Amycolatopsis orientalis and the genome of the primers was used as a template to amplify the sequence of SEQ ID NO.1 and recover:

the upstream primer SEQ ID NO. 3: aaaggaggcggacatcatatggtggatccgacgagagtagacc

The downstream primer is SEQ ID NO. 4: catgattacgaattcgatatcttacgcgatcgctcccgtgg

(2) Inserting the fragment recovered in the step (1) into an expression vector psoK804 to obtain a plasmid psoK 804-ermE-strR, and verifying;

(3) transducing the plasmid obtained in step (2) into E.coli ET12567/pUZ8002 by chemolysis;

(4) transferring the plasmid in the step (2) into amycolatopsis orientalis of a Chroroeremomycin producing bacterium through conjugation transduction;

(5) through fermentation and high performance liquid chromatography detection, the yield of original bacteria Chloroeremycin is improved by about 40 percent and reaches 140 mg/L.

In the above method, the sequence of SEQ ID NO.1 is inserted into the plasmid pSOK804 in step (1), to obtain the recombinant plasmid pSOK 804-ermE-strR. The SEQ ID NO.1 of the step (1) consists of 975 nucleotides, and the SEQ ID NO.2 is an amino acid sequence of a positive control gene for biosynthesis of Chroermycin.

In the method, the expression vector pSOK804 is subjected to NdeI-EcoRV double enzyme digestion and then is inserted into a sequence shown in SEQ ID NO.1 in the step (2), and a promoter is a promoter erm × p containing an erythromycin resistance gene;

in the above method, the plasmid pSOK804-ermE x-strR is introduced into E.coli ET12567/pUZ8002 by heat shock in step (3); the strain of the chloroteremomycin precursor, i.e., the amycolatopsis orientalis, is introduced into the chloroteremomycin producing strain by conjugative transfer, so that the strain of the high-yield orlistatin precursor, i.e., the chloroteremomycin, is obtained.

Through fermentation and high performance liquid chromatography detection, the yield of the genetically engineered bacteria obtained through conjugation transduction is improved by about 40 percent compared with that of the wild type bacterium Chloroeromemycin, and the yield reaches 140 mg/L.

The invention also aims to provide application of the method in high-yield origanin precursor Chloroeromemycin engineering bacteria. The yield of the produced Chromemomycin is improved by about 40 percent and reaches 140 mg/L.

The construction method of the high-yield origanin precursor Chloroeremomycin disclosed by the invention can improve the fermentation level of the origanin precursor Chloroeremomycin by about 40% and reach 140mg/L compared with that of a starting bacterium, and has a good industrial application value.

The invention has the advantages that: (1) the invention obtains the high-yield strain of the oritavancin precursor Chloroeremomycin by introducing the positive regulatory gene of the biosynthesis of the oritavancin precursor Chloroeremomycin based on a genetic engineering means, has definite target and avoids the defects of long period and manpower and material resource consumption caused by the traditional mutation breeding technical means.

(2) The yield of the high-yield origanin precursor Chloroeremomycin genetic engineering bacteria constructed by the invention is improved by 40 percent compared with that of original bacteria and reaches 140mg/L, the generation cost of the origanin precursor Chloroeremomycin is greatly reduced, and the method has important application value in industrial production of the origanin precursor Chloroeremomycin.

The invention constructs the high-yield bacterium of the Chromelomycin by a genetic engineering means, can improve the biosynthesis of the Chromelomycin precursor of the oritavancin, and provides an effective technical means for developing glycopeptide antibiotics.

Drawings

FIG. 1 is a map of expression plasmid psok804-erm star-strR.

FIG. 2 is a graph showing the time-dependent change of fermentation yield of gene engineering bacteria CGMCC NO.21140 of Chloroermycin of Amycolatopsis orientalis after the production of Chloroermycin and the growth of bacteria and gene recombination.

Detailed Description

The invention is further explained by the accompanying drawings and examples. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Media used in the examples:

YMG solid medium: yeast extract 4.0g, malt extract 10.0g, glucose 4.0g, agar 20.0g, distilled water 1000ml, pH 7.3.

LB liquid medium: 10.0g of peptone, 5.0g of yeast extract, 10g of NaCl, 20.0g of agar and 1000ml of distilled water, pH 7.3.

2 × YT liquid medium: peptone 16.0g, yeast extract 10.0g, NaCl5g, agar 20.0g, distilled water 1000ml, pH 7.3.

MS solid culture medium: 20g of mannitol, 20g of soybean meal, 20g of agarose and 1000ml of distilled water, and the pH value is 7.3.

Seed culture medium: peptone 20g, NaCl5g, glucose 2.5g, K2HPO42.5g, distilled water 1000ml, pH7.3.

Fermentation medium: 5g of peptone, 20g of glucose, NaCl1g, KCl0.5g, MgSO40.8g, KH2PO40.1g, 1000ml of distilled water and pH 7.3.

Example 1: construction method of Chloroeremomycin high-yielding strain

(1) Construction of expression vector pSOK 804-ermE-strR

The expression vector of the positive control gene for biosynthesis of Chloroeremomycin constructed in this example is named pSOK 804-ermE-strR, and the vector contains the positive control gene strR for biosynthesis of Chloroeremomycin. The sequence of the positive control gene strR for biosynthesis of Chromemomycin is shown as SEQ ID NO. 1. SEQ ID NO.1 consists of 975 nucleotides.

pSOK 804-ermE-strR was constructed as follows:

designing a specific primer, amplifying a SEQ ID NO.1 sequence by using an amycolatopsis orientalis ACCC40175 (Amycolatopsis solientalis) genome as a template, and recovering:

the upstream primer SEQ ID NO. 3: aaaggaggcggacatcatatggtggatccgacgagagtagacc

The downstream primer is SEQ ID NO. 4: catgattacgaattcgatatcttacgcgatcgctcccgtgg

And (2) connecting the SEQ ID NO.1 fragment recovered in the step (1) with an NdeI-EcoRV double-enzyme-digested expression vector pSOK804 to obtain a recombinant expression vector pSOK 804-ermE-strR, wherein the plasmid map is shown in figure 1.

(2) The specific method for obtaining the genetically engineered bacterium with high yield of Chroeremomycin by introducing the pSOK 804-ermE-strR expression vector into the starting bacterium Amycolatopsis orientalis (Amycolatopsis orientalis).

pSOK804-ermE × strR vector was transformed into demethylated E.coli ET1256/pUZ8002 by heat shock 90s at 42 ℃.

(3) The plasmid of interest pSOK804-ermE × strR was transformed into E.coli ET12567/pUZ8002 plated LB plates containing the corresponding antibiotics (Apra, Kana and Chl), picked single clones were cultured in 5mLLB at 37 ℃ and 220rpm overnight. Then transferred to 15mL LB containing the corresponding antibiotic at a ratio of 2% (scale-up culture) and cultured to OD600 of about 0.4. The cells were collected by centrifugation at 6000rpm for 5min, washed 3 times with 2ml of LB medium, and suspended in 500. mu.L of LB medium (500. mu.L of large intestine per Streptomyces tuberidus) as a donor for conjugative transduction. The mycelium is used as the acceptor bacterium, and is cultured for about 48-60h at the temperature of 28-30 ℃ and the rpm of 220 and TSB. 250 μ L of mycelia was collected by centrifugation at 6000rpm for 5min, washed 3 times with LB medium, and resuspended in 500 μ L of 2 XYT medium as recipient bacteria. Mixing the acceptor and donor bacteria: mu.L of donor bacteria and 500. mu.L of recipient bacteria were mixed well, (centrifuged at 6000rpm, the remaining 500. mu.L of liquid suspension of the cells) was spread on MS medium (containing no antibiotics) containing 10mM magnesium ions, dried on an ultra-clean bench and cultured in a 30 ℃ incubator. After culturing for 16-20h, covering the flat plate with apramycin (the final concentration is 200 mug/mL) and nalidixic acid (the final concentration is 25 mug/mL), continuously culturing for 4-5 days at 30 ℃, picking out a transformant passage resistance YMG plate (the final concentration of apramycin is 200 mug/mL), and picking out a single clone to extract a genome for identification.

Example 2: fermentation verification of producing Chloroeremomycin by using germination bacteria and genetic engineering bacteria CGMCC NO.21140

Genetically engineered bacteria CGMCC NO.21140 of the outgoing strain producing Chroroeremomycin and the Amycolatopsis orientalis are cultured on a YMG solid culture medium for 5 days. Scraping a fungus block of about 1cm multiplied by 1cm, inoculating the fungus block into a seed culture medium, and culturing at 30 ℃ for 48 hours at the rotating speed of 220 rpm; the mycelium in the seed culture medium is inoculated to the fermentation culture medium with the inoculation amount of 8 percent, the culture is carried out for 168 hours at the temperature of 30 ℃, and samples are respectively taken for 24, 48, 72, 96, 120, 144 and 168 hours of culture to determine the yield of the Chloroeremomycin.

Example 3: comparison and verification of yield of wild bacteria and high-yield bacteria Chloroeromemycin

(1) HPLC conditions: a chromatographic column: c18 column (agent, Eclipse Plus XDB, 5um, 4.6mm 250 mm); detection wavelength: 280 nm; flow rate: 1.00 mL/min; sample introduction amount: 20 ul; experimental mobile phase: the mobile phase A is water containing 0.1% formic acid, and the mobile phase B is 100% acetonitrile; HPLC procedure: 0-15min, 5% -15% of phase B; 15-20min, 15% -100% of phase B; phase B20-23 min, 100%; 23-30min, phase B5%.

(2) Analysis of Chloroeremomycin production: adding 1mL of methanol into 1mL of fermentation broth obtained by fermentation, fully oscillating, centrifuging at 12000rpm/min for 10min to settle mycelium and solid, filtering the supernatant with a 0.45um sterile microporous membrane, collecting the filtrate, and using the obtained sample for HPLC detection. FIG. 2 is a plot showing the change of yield of Chloroeremomycin synthesized by Chloroeremomycin producing strain CGMCC NO. 21140.

(3) The yield of the Chloroeremomycin in fermentation of the high-yield Chloroeremomycin amycolata (Amycolatopsis orientalis) CGMCC NO.21140 is improved by 40 percent compared with that of the original bacteria by 140mg/L, and the oritavancin can well treat skin infection caused by gram-positive bacteria, so the method for constructing the high-yield Chloroeremomycin has important application value.

Sequence listing

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Claims (4)

1. A method for constructing high-yield engineering bacteria of Oritavancin precursor is characterized in that Amycolatopsis orientalis (A) with the preservation number of ACCC40175 is takenAmycolatopsis orientalis) After genetic breedingPositive control genes of biosynthesis of Chromelomycin, a precursor of oritavancin, for a Chromelomycin producing bacteriumstrRThe strain is constructed on pSOK804 plasmid, introduced with thalli and integrated to a VWB site, so as to obtain high-yield Chloroeremomycin bacteria, the yield of the obtained Chloroeremomycin is improved by 40 percent and reaches 140mg/L, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the classification is named as: amycolatopsis orientalis(Amycolatopsis orientalis) L80, accession number: CGMCC NO.21140, preservation date: 2020.11.9, respectively; the regulatory genestrRThe sequence of (A) is as described in SEQ ID NO. 1;

the method is realized by the following steps:

(1) design of specific primers with Amycolatopsis orientalis (Amycolatopsis orientalis) Amplifying the SEQ ID NO.1 sequence by taking the genome as a template and recovering:

the upstream primer SEQ ID NO. 3: aaaggaggcggacatcatatggtggatccgacgagagtagacc the flow of the air in the air conditioner,

the downstream primer is SEQ ID NO. 4: catgattacgaattcgatatcttacgcgatcgctcccgtgg, respectively;

(2) inserting the fragment recovered in step (1) into an expression vector psoK804 to obtain a plasmid psoK 804-ermE-strRAnd verifying;

(3) the plasmid obtained in step (2) is transduced into Escherichia coli by chemolysisE.coilE.coli was obtained from ET12567/pUZ8002E.coil ET12567/pUZ8002/psoK804-ermE*-strR；

(4) By means of E.coliE.coil ET12567/pUZ8002/psoK804-ermE*-strRPerforming conjugation transduction to transfer the plasmid in the step (2) into amycolatopsis orientalis of a Chloreremomycin producing strain;

(5) through fermentation and high performance liquid chromatography detection, the yield of original bacteria Chloroeremycin is improved by 40 percent and reaches 140 mg/L.

2. The method as claimed in claim 1, wherein the plasmid pSOK 804-ermE-strRIntroduction into Chromemom by conjugation transductionin producing bacteria Amycolatopsis orientalis.

3. The method for constructing high-yielding engineering bacteria of oritavancin precursors according to claim 1, wherein SEQ ID No.1 consists of 975 nucleotides, SEQ ID No.2 is an amino acid sequence of a positive regulatory gene for biosynthesis of Chromemomycin, and the expression vector pSOK804 is subjected toNdeI-EcoRV is inserted into SEQ ID NO.1 sequence after double enzyme digestion, and the promoter is a promoter containing erythromycin resistance geneerme*p(ii) a The expression vector plasmid pSOK 804-ermE-strRTransferring into the strain producing Chloroermomycin Amycolatopsis orientalis by conjugation to obtain the high yield of Chloroermomycin precursor.

4. The method of claim 1, wherein the yield of the produced Chromelomycin is 140 mg/L.

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