CN107541503A - A kind of transmethylase GenL and its encoding gene genL and application - Google Patents

Abstract

The invention discloses a kind of transmethylase GenL and its encoding genegenLAnd application, belong to biomedicine field.The transmethylase GenL and its encoding gene of the present inventiongenLSequence respectively as shown in SEQ ID NO.1,2, GenL has methyl transferase activity.Passing through willgenLGene, which is connected in prokaryotic expression carrier, builds recombinant expression plasmid, then recombinant expression plasmid is transformed into Escherichia coli and obtains GenL through inducing, purifying.By routine operation, it can knock out or introduce in specific bacterial straingenLGene.The present invention GenL andgenLAvailable for specific gentamicin component, or synthesizing new glucoside-containing component is obtained, method therefor is easily operated, and for cost than relatively low, environmental pollution is small, is adapted to large-scale application, is with a wide range of applications in terms of research, drug field and treatment.

Description

A kind of transmethylase GenL and its encoding gene genL and application

Technical field

The present invention relates to biomedicine field, more particularly to a kind of transmethylase GenL and its encoding gene genL and should With.

Background technology

As a kind of aminoglycoside antibiotics for being once widely used in clinical treatment, gentamicin (gentamicin) is right Most Gram-negative bacterias and the microbial infection of part Gram-positive have good efficacy.Clinically conventional gentamicin For the mixture of Multiple components, its mainly include Gentamicin C1a, gentamicinC2 b, gentamicinC2 a, gentamicinC2 and Five kinds of components of gentamicinC1, each group molecular structure are as shown in Figure 1.But the drug effect and toxicity between its each component have not With the difference of degree, different pathogenic bacteria are also inconsistent to the tolerance level of each component.For exploitation high-efficiency low-toxicity, there is specific aim Gentamicin product, it is necessary to obtain corresponding gentamicin one-component.

The chemical synthesis difficulty of glucoside-containing component is larger, and is largely obtained from gentamicin mixture using HPLC Take one-component with high costs, therefore by the method for biology and metabolic engineering, be it is a kind of than chemical synthesis and molecular design more For effective, economic and environment-friendly means.In order to reach this purpose, it is necessary to use the synthetic gene and its phase of gentamicin Close albumen.

Along with the appearance of multidrug resistant pathogenic bacteria, people expect synthesizing new aminoglycoside antibiotics to be answered It is right, if reaching the purpose by the approach of biology, need the gene and enzyme of correlation.In addition, methylating on 6 '-N is repaiied Decorations are implicitly present in influence for drug resistance, if for the resistance to the action of a drug of pathogenic bacteria, this will be a decorating site for being worth attempting. In recent years, successively it has been reported that treatment of the gentamicin to part cancer and HIV shows potentiality, for the field, According to convention, different gentamicin components is likely that there are specific drug effect and distinguished, and the acquisition nothing of gentamicin one-component The progress of research for helping correlation is suspected to have, in order to realize the target for obtaining one-component, it is also desirable to understand its biosynthesis correlation Gene and albumen.

The content of the invention

The primary and foremost purpose of the present invention is to provide a kind of transmethylase GenL and its encoding gene genL.The present invention's is another One purpose is the preparation method for providing above-mentioned transmethylase GenL.The present invention also aims to provide above-mentioned methyl transfer Enzyme GenL and its encoding gene genL application.

The purpose of the present invention is achieved through the following technical solutions：

A kind of transmethylase GenL, its amino acid sequence is as shown in SEQ ID NO.1.The enzyme is with aminoglycoside chemical combination Thing is substrate, has methyl transferase activity, and Gentamicin C1a can be converted into gentamicinC2 b, also can be big mould by celebrating Plain C2 is converted into gentamicinC1, will 6 '-N positions of corresponding substrate molecule methylated.

Above-mentioned transmethylase GenL encoding gene, i.e. genL, its nucleotide sequence is as shown in SEQ ID NO.2.

Described transmethylase GenL preparation method comprises the following steps：Its encoding gene genL is connected to protokaryon Recombinant expression plasmid is built in expression vector；And constructed recombinant expression plasmid is converted to Host Strains (e. coli bl21) In, through induced expression, purifying, obtain transmethylase GenL.

Activity analysis is carried out to obtained transmethylase GenL, it is found that it has methyl transferase activity, can be to celebrating Big mycin C2 and Gentamicin C1a 6 '-N positions carry out the specific modification that methylates, big so as to obtain gentamicinC1 and celebrating Mycin C2b, show that it is with a wide range of applications in terms of the biosynthesis of related compound.

Methyl transferase activity based on GenL, the present invention also provide above-mentioned transmethylase GenL or its encoding gene Applications of the genL in the biosynthesis of glucoside-containing component, especially gentamicin.By to gentamicin producing strains In ATCC15835 transmethylase encoding gene genL (or in other glucoside-containing component producing strains genL it is same Source gene) knocked out, specifically obtain and methylate that (including but not limited to celebrating is big for the gentamicin component of modification without 6 '-N Mycin C1a and gentamicinC2).By to the genL in gentamicin producing strains ATCC15835 (or to other aminoglycosides GenL homologous gene in compound producing strains) carry out overexpression, specifically obtain containing 6 '-N methylate modification celebrating it is big Mycin component, or improve gentamicin mixture in 6 '-N methylate modification component ratio.6 '-N are modified using GenL not It is methylated the gentamicin component of modification, obtains 6 '-N and methylate the gentamicin component of modification.By exogenous add GenL is artificially imported host and carries out endogenous expression by GenL, and making it, (such as celebrating is big in glucoside-containing component molecule Mycin) 6 '-N positions or the position equivalent to 6 '-N methylate.

The invention has the advantages that：

(1) the transmethylase GenL using glucoside-containing component as substrate, have to enter-the N of gentamicin molecule 6 ' The activity of the capable modification that methylates, the enzyme and its encoding gene genL with this feature do not have been reported that.

(2) transmethylase encoding gene genL of the invention is easy to carry out molecular cloning and genetic manipulation, and methyl turns Genetic engineering bacterium of the enzyme GenL preparation based on routine is moved, the extraction of gentamicin class compound has ready-made method to abide by Follow, be adapted to large-scale culture and separation and Extraction, production technology is uncomplicated, and equipment requirement is simple, with short production cycle, and cost compares Low, environmental pollution is small.

(3) transmethylase GenL and its encoding gene genL can be used for obtaining specific gentamicin component, has and repaiies The similar substrate of decorations structure simultaneously produces the potentiality of novel amino sugar aminated compounds, has in terms of research, drug field and treatment Have wide practical use.

Brief description of the drawings

Fig. 1 is the molecular structure of gentamicin.

Fig. 2 is the restructuring transmethylase GenL prepared in embodiment 1 SDS-PAGE figures.Swimming lane M represents albumen Marker, swimming lane ni represent the protein extract of the Escherichia coli without induction, and swimming lane ip is represented to induce and carry out to have children outside the state plan and crushed The insoluble solids remained afterwards, swimming lane 60 represent and use buffer solution (60mM imidazoles, 20mM TrisHCl, 500mM NaCl, pH 7.9) elute obtained component, swimming lane 250 represent using buffer solution (250mM imidazoles, 20mM TrisHCl, 500mM NaCl, PH7.9 obtained component) is eluted.

Fig. 3 is the restructuring transmethylase GenL catalysis gentamicinC2 and Gentamicin C1a generation prepared in embodiment 2 The LC-MS figures to methylate.(A) pure substrate gentamicinC2；(B) GenL and products therefrom after gentamicinC2 reaction；(C) pure bottom Thing Gentamicin C1a；(D) GenL and products therefrom after gentamicinC2 reaction.(E) MS/MS of each component of gentamicin is tested Card figure.

Fig. 4 is the structure and proof diagram of mutant strain ATCC15835 Δs genL in embodiment 3.(A) gene knockout schematic diagram； (B) PCR the results；(C) Southern Blot the results.

Fig. 5 is the LC-MS analysis results of mutant strain ATCC15835 Δs genL tunnings in embodiment 3.(A) conduct pair According to the ATCC15835 strain fermentation product analysis of group；(B) mutant strain ATCC15835 Δs genL tunnings are analyzed.

Embodiment

With reference to embodiment, the present invention will be further described.Embodiment is intended to carry out citing description to the present invention, and It is non-to limit the invention in any form.

The routinely reagent being related in embodiments of the present invention is existing commercially produced product, such as：Plasmid extraction, The corresponding reagent box for all using " Tiangeng biochemical technology Co., Ltd " is reclaimed in DNA (PCR primer) purifying, DNA fragmentation from gel； Restriction enzyme and ligase are purchased from " New England Biolabs companies ", Britain.

Embodiment 1 recombinates transmethylase GenL preparation

(1) extraction of STb gene

By 100 μ L micromonospora ATCC15835 mycelium be fully suspended in 500 μ L SET buffer (75mM NaCl, 25mM EDTA [pH8.0], 20mM Tris-HCl [pH7.5]) in buffer solution, 10 μ L lysozymes (50mg/mL) are added after 37 DEG C water-bath 1 hour.Then add after 14 μ L Proteinase K Solutions (60mg/mL) and be sufficiently mixed uniformly to it, add 60 μ L10% SDS, it is well mixed again after 55 DEG C of water-baths 1 hour.200 μ L 5M sodium chloride solution is continuously added after incubation, it is fully mixed It is even.500 μ L chloroform is continuously added, is mixed.Centrifuge 10min in 12000rpm, and by supernatant be transferred completely into one it is new from In heart pipe.The isopropanol of 0.6 times of volume is added to it, mixes and centrifuges 1min after 12000rpm.Supernatant is removed, adds 1mL 70% ethanol fully washs precipitation twice.Supernatant is removed, after room temperature all volatilizes ethanol, DNA is filled with 200 μ L sterilized waters Divide dissolving.

(2) structure of His-GenL plasmids is recombinated

Using micromonospora ATCC15835 STb gene as template, with purchased from " New England Biolabs companies " Fusion enzymatic amplification genL fragments, its specific primer sequence are as follows：

genL-F：5 '-AAACATATGCTGAGCATCTCCGATCTACG-3 ',

genL-B：5’-AAAGAATTCACGAGGTGGTCTACGCGAT-3’.

Enter performing PCR amplification first, reaction condition is：98℃3min；98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, circulate 30 times； Last 72 DEG C of extensions 5min.Pcr amplification product is reclaimed with PCR primer gel reclaims kit, is expanded with NdeI and EcoRI digestions Product, 2-8 hours are connected in room temperature with the prokaryotic expression carrier pET28a (+) through same enzyme digestion with T4DNA ligases.Will be even Thing of practicing midwifery is imported into competent escherichia coli cell DH10B, passes through the matter of the purposeful gene of kalamycin resistance screening zone Grain, the plasmid product of extraction obtain recombinant plasmid His-GenL through sequencing detection checking.

(3) transmethylase GenL expression and purifying is recombinated

Extract the recombinant plasmid His-GenL in Escherichia coli DH10B, and with calcium chloride transformation by recombinant plasmid transformed Into e. coli bl21 (DE3), and it is coated on containing on 50 μ g/mL kanamycins 2 × TY solid mediums, filters out conversion Son, and it is seeded to 37 DEG C of overnight incubations in 2 × TY fluid nutrient mediums containing 50 μ g/mL kanamycins.Second day inoculation 10mL kind Sub- liquid is cultivated to OD for 37 DEG C into 2 × TY fluid nutrient mediums of the 1L containing 50 μ g/mL kanamycins₆₀₀=0.6 or so adds IPTG extremely Final concentration 0.1mM, 20h is induced in 16 DEG C.Induced cultures are centrifuged 10 minutes in 4 DEG C of 6000rpm, take precipitation 30mL's Binding buffer (20mM Tris-HCl pH 8.0,200mM NaCl, 5mM imidazoles) fully suspend, under condition of ice bath, With ultrasonic disruption cell, (condition is：2 seconds working times, 5 seconds intermittent times, total duration 30min).Gains are in 4 DEG C 12000rpm centrifuges 20 minutes to remove cell fragment, supernatant and Ni²⁺Affinity column mixes.Then binding is used respectively Buffer and wash buffer (20mM Tris-HCl pH 8.0,200mM NaCl, 50-100mM imidazoles) elute foreign protein Matter, target protein is eluted with elution buffer (20mM Tris-HCl pH 8.0,200mM NaCl, 200mM imidazoles). Recovery destination protein obtains the restructuring His-GenL albumen of high-purity after PD-10 desalting column desalinations (see Fig. 2).

Embodiment 2 recombinates transmethylase GenL external biochemical reaction

Reacted respectively using gentamicinC2 and Gentamicin C1a as substrate, reaction system and reaction condition are as follows：10 Restructuring His-GenL albumen, the 0.2mg substrates to be detected (gentamicinC2 or Gentamicin C1a) of μ g after purification react in 1mL In 31 DEG C of reactions in buffer solution (20mM Tris-HCl, 200mM NaCl, 1mM S-adenosylmethionine, final pH 8.0) 4h.Reaction adds isometric chloroform and mixed after terminating, take supernatant to be concentrated into 0.25mL after stratification.Sample LC- after reaction MS is detected, and testing conditions are as follows：

The high resolution mass spectrometer LTQ Obitrap XL produced using Thermo companies are detector, are used Phenomenex Luna C18 (2) reverse chromatograms post (250 × 4.6mm, 5 μm) carries out separation detection to gentamicin component.Stream Dynamic phase includes：A phases are 0.2%TFA, and pH to 2.0 Milli-Q ultra-pure waters are adjusted with ammoniacal liquor；B phases are acetonitrile.Gradient elution bar Part is：0-14min, 2%B to 6%B；14-16min, 6%B to 8%B；16-25min, 8%B to15%B；25- 26min, 15%B to 40%B；26-34min, 40%B；34-35min, 40%B to 2%B；35-45min, 2%B.Flow velocity For 400 μ L/min, sample injection volume is 20 μ L.

Mass Spectrometer Method is detected from positive ion mode.Ion gun condition is：Cation mode, capillary temperature 275 DEG C, spray voltage 3.5kv, sheath gas 50L/h, 350 DEG C of ion source temperature, secondary air speed 10L/h, collision energy 35eV。

LC-MS testing results are shown in Fig. 3, it is seen that sterling Gentamicin C1a and C2 have successfully been separately converted to celebrating by GenL Big mycin C2b and C1, complete the modification that methylates of 6 '-N positions.

Embodiment 3

GenL genes are knocked out in gentamicin producing strains micromonospora ATCC15835 by homologous recombination double crossing over method, Obtain mutant strain ATCC15835 Δs genL and fermented and product detection, obtain the gentamicinC group to be methylated without 6 '-N Point.

(1) ATCC15835 Δs genL structure

Using following four pairs of primers, the homology arm on the left of genL with right side is obtained by PCR.

genL-left-S：5 '-GCACATATGGCAGGCGTGGGTCAACAG-3 ',

genL-left-A：5’-CCGTCTAGAGTCTACTCCGTCGGCGAG-3’；

genL-right-S：5 '-GCGTCTAGAGGAGATGCTCAGCACGGT-3 ',

genL-right-A：5’-GCCAAGCTTATCACCGAGTACGGTCGC-3’.

PCR reaction conditions are：98℃3min；98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 90s are circulated 30 times；Last 72 DEG C of extensions 7min.Fragment (left side homology arm is NdeI and XbaI enzymes, and right side homology arm is HindIII and XbaI enzymes) after digestion is reclaimed, Be connected to by NdeI and HindIII cutting pYH7 carriers (structure of pYH7 carriers refers to document " Analysis of functions in plasmid pHZ1358influencing its genetic and structural stability The sequence of in Streptomyces lividanS1426 ", pYH7 carriers is as shown in SEQ ID NO.3) on, obtain plasmid pWHU2755.The plasmid is converted into Escherichia coli ET12567/pUZ8002, obtains the Escherichia coli containing target plasmid ET12567/pUZ8002, it is seeded to 2 × TY of 5mL fluid nutrient mediums (ampicillin containing 100mM, 50mM card The chloramphenicol of that mycin and 25mM) in, after 37 DEG C of 220rpm shaking table cultures 12h, transferred according to 1% inoculum concentration in 5mL fresh 2 In × TY fluid nutrient mediums (ampicillin containing 50mM, 25mM kanamycins and 12.5mM chloramphenicol), 37 DEG C 220rpm shaking table cultures are to OD₆₀₀About 0.4-0.6,5000rpm centrifuge 10min, abandon supernatant, and trained with fresh 2 × TY liquid It is standby twice to support base washing thalline.Recipient bacterium micromonospora (shaking table culture 2-3d) bacterium solution 10mL, 5000rpm centrifugation 10min is taken, Supernatant is abandoned, and it is standby twice with fresh 2 × TY fluid nutrient mediums washing thalline.100 μ L coli somatics and small are taken respectively After sporangium thalline mixes, coating ABB13 solid mediums flat board (soy peptone 5.0g, soluble starch 5.0g, CaCO₃3.0g, 3- (N- morpholinyls)-propane sulfonic acid 2.1g, FeSO₄0.012g, Thiamine HCl 0.01g, agar powder 20.0g, Milli-Q ultra-pure water 1000mL, pH to 7.0 is adjusted with KOH.Final concentration of 10mM MgCl is added during use₂), it is placed in 28 DEG C of trainings After supporting 14-16h, the sterilized water of 20 μ g/mL apramycins and 25 μ g/mL nalidixic acids is contained with 1mL, uniform fold is entirely put down Plate, after the interior drying of superclean bench covers moisture, 28 DEG C of cultures are placed in, about joint element can be observed in 5-7d.

Will on engagement transfer flat board the joint element that grow, fallen within sterile toothpick picking single bacterium and fresh contain 20 μ g/ (soluble starch 10.0g, corn flour 2.5g, yeast carry the A culture medium flat plates of mL apramycins and 25 μ g/mL nalidixic acids Take thing 3.0g, CaCO₃3.0g, FeSO₄0.012g, agar powder 20.0g, Milli-Q ultra-pure water 1000mL) on be coated with small bacteria block, And using recipient bacterium micromonospora as negative control, after 28 DEG C of culture 3-4d, the bacterial strain of growth is the mutation that single-swap occurs Strain.Single-swap mutant strain is taken after the A culture medium flat plate upper sheet bacterium colonies without antibiotic, 28 DEG C of culture 5-7d, picking single bacterium Fall respectively at being coated with small bacteria block on the A culture medium flat plates containing corresponding antibiotic and the A culture medium flat plates without antibiotic, and with Recipient bacterium micromonospora after 28 DEG C are cultivated 3-4d, is chosen at containing not grown on antibiotic flat board, only not as negative control Containing the bacterium colony grown on antibiotic flat board, its STb gene is extracted as template, entering performing PCR using following checking primer, (checking is drawn Thing -1：5 '-CGTGTCGCTGTTCTGGGTCA-3 ', verify primer -2：5 '-CTCGTGCTCGTCATCGCCTA-3 '), to investigate Whether the bacterium colony is the mutant strain that double crossing over occurs (1073bp bands are mutant strain, and 1752bp bands are wild type).If not Double crossing over mutant strain is screened, then continues to carry out the culture that relaxes on the A culture medium flat plates without antibiotic, until screening hair Untill the mutant strain of raw double crossing over, the mutant strain is ATCC15835 Δ genL, enters performing PCR immediately with same checking primer Probe is obtained, Southern Blot checkings are carried out to the mutant strain, it is correct to confirm the mutant strain.The structure of mutant strain and The result is shown in Fig. 4.

(2) ATCC15835 Δs genL fermentation and product extraction

Gentamicin producing strains ATCC15835, its mutant strain ATCC15835 Δs genL are inoculated in ATCC 172 and consolidated respectively Body culture medium (glucose 10.0g, soluble starch 20.0g, yeast extract 5.0g, casein hydrolysis (N-Z Amine Type A) 5.0g, CaCO₃1.0g, agar powder 20.0g, Milli-Q ultra-pure water 1000mL) on, 28 DEG C of culture 4d.Take about 1cm²Fungus block connects Kind after 28 DEG C of 220rpm shaking table cultures 2d, is transferred in fresh in the fluid nutrient mediums of ATCC 172 according to 2% inoculum concentration In the fluid nutrient mediums of ATCC 172,28 DEG C of 220rpm shaking table culture 8d, ATCC15835 and ATCC15835 Δs genL liquid is obtained Body fermentate.

With 6M H₂SO₄The liquid fermentate of gentamicin producing strains or its mutant strain is acidified, adjusts pH to 2.0, After shaken at room temperature acidifying 2h, 8000rpm centrifugation 10min, take supernatant and filtered with filter paper.Weigh 2.0g DOWEX 50WX8- 200 cationic ion-exchange resins (Sigma companies), the supernatant after being filtered with 10mL mix concussion 3h.It is then ultrapure with Milli-Q Water washing resin 2-3 times, and resin is filled into post.Finally eluted with the ammoniacal liquor of 10mL 2% and collect eluent.In volatilization eluent Ammonia after, be placed in freeze drier (LABCONCO companies) and sample be concentrated and dried.By the sample after freeze-drying It is dissolved in 500 μ L Milli-Q ultra-pure waters, 12000rpm centrifugation 10min, takes supernatant and with after 0.22 μm of membrane filtration, press LC-MS detections are carried out according to the method in embodiment 2, as a result see Fig. 5, it is observed that in ATCC15835 Δs genL, celebrating is big mould Plain tri- kinds of components of C1a, C2a and C2 can still be produced, but gentamicinC1 and C2b are not produced, show the methyl of 6 '-N positions Change does not occur in mutant strain.

Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (9)

1. A kind of 1. transmethylase GenL, it is characterised in that：Amino acid sequence is as shown in SEQ ID NO.1.
2. 2. the encoding gene genL of the transmethylase GenL described in claim 1, it is characterised in that：Nucleotide sequence such as SEQ Shown in ID NO.2.
3. 3. the preparation method of the transmethylase GenL described in claim 1, it is characterised in that comprise the following steps：Will by right Ask the encoding gene genL described in 2 to be connected in prokaryotic expression carrier and build recombinant expression plasmid, the recombination expression that will be formed Plasmid is transformed into Host Strains, through induced expression, purifying, obtains transmethylase GenL.
4. 4. the encoding gene genL described in transmethylase GenL or claim 2 described in claim 1 is in aminoglycoside Application in the biosynthesis of compound.
5. 5. application according to claim 4, it is characterised in that：By exogenous add transmethylase GenL or artificial Ground by genL channel genes host carry out endogenous expression, make glucoside-containing component molecule 6 '-N positions or equivalent to 6 '-N position methylates.
6. 6. the encoding gene genL described in transmethylase GenL or claim 2 described in claim 1 is in gentamicin Application in biosynthesis.
7. 7. applications of the transmethylase GenL in the preparation of gentamicin component described in claim 1, it is characterised in that：Utilize The gentamicin component that transmethylase GenL is not methylated to 6 '-N carries out the modification that methylates.
8. 8. applications of the encoding gene genL in the preparation of gentamicin component described in claim 2, it is characterised in that：By right GenL genes or its homologous gene in gentamicin producing strains carry out gene knockout, specifically obtain and are methylated without 6 '-N The gentamicin component of modification.
9. 9. applications of the encoding gene genL in the preparation of gentamicin component described in claim 2, it is characterised in that：By right GenL genes or its homologous gene in gentamicin producing strains carry out overexpression, specifically obtain to methylate containing 6 '-N and repair The gentamicin component of decorations, or improve gentamicin mixture in 6 '-N methylate modification component ratio.