CN108330113B - The Actinosynnema bacterium of one plant height Substratspezifitaet acyltransferase and its application - Google Patents

The Actinosynnema bacterium of one plant height Substratspezifitaet acyltransferase and its application Download PDF

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CN108330113B
CN108330113B CN201810191854.0A CN201810191854A CN108330113B CN 108330113 B CN108330113 B CN 108330113B CN 201810191854 A CN201810191854 A CN 201810191854A CN 108330113 B CN108330113 B CN 108330113B
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CN108330113A (en
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曹广祥
宗工理
钟传青
付加芳
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Shandong Medicinal Biotechnology Center (shandong Institute Of Virology)
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Abstract

The invention discloses the Actinosynnema bacterium of a plant height Substratspezifitaet acyltransferase; for Actinosynnema bacterium (Actinosynnema pretiosum) X4732; Guangdong Province's Culture Collection was preserved on 2 5th, 2018, deposit number is GDMCC NO.60327.The present invention is mutated the gene of acyltransferase using continuous error-prone PCR method in vitro; it is bacterium germination with acyltransferase deletion mycopremna; the mutant library containing acyl transferase gene is established by engagement transfer techniques; screening obtains positive mutating strain, and the Actinosynnema bacterium X4732 of stabilization characteristics of genetics is obtained by fermentation verifying, secondary culture.The ansamitocin AP-3 synthesis of bacterial strain of the present invention is horizontal 2.7 times higher than original strain X47, can be applied to industrial fermentation production.

Description

The Actinosynnema bacterium of one plant height Substratspezifitaet acyltransferase and its application
Technical field
The invention belongs to Microbial Breeding and field of biotechnology, and in particular to the Actinosynnema of a plant height organized enzyme Bacterium and its application.
Background technique
Ansamitocin (ansamitocin) is generated by Actinosynnema bacterium (Actinosynnema pretiosum) Ansamycin.Ansamitocin is a kind of efficient anti-tumor drug, research confirm ansamitocin can by with micro-pipe In conjunction with the proliferation for inhibiting 60 kinds of tumour cells, clinical test shows that ansamitocin drug is loose to adenocarcinoma of lung, lung squamous cancer and lung thin Born of the same parents' cancer etc. is effective in cure.Since ansamitocin has very strong cytotoxicity, the general targeting using antibody divides drug Sub- target tumor, to reduce the non-specific general toxicity of drug in clinical chemotherapy.The anticancer drug of Roche Holding Ag Trastuzumab-DM1 (T-DM1) is that (DM1 is ansamitocin to first successful ansamitocin antibody coupling drug listed Derivative), ratified within 2013 Metastasis in Breast Cancer and the end-stage patients for the treatment of HER2 receptor positive by FDA.In addition, there are also multiple peaces The antibody coupling drug of silk streptozotocin derivative is carrying out clinical test, such as: SAR-408701 is by carcinomebryonic antigen relevant cell Adhesion molecule 5 and ansamitocin derivative DM1 composition, for treating entity tumor；BT-062 is resisted by the monoclonal for targeting CD138 Body and ansamitocin derivative DM1 and DM4 composition, for treating Huppert's disease；SAR3419 is mono- by the anti-CD19 of humanization Clonal antibody and ansamitocin derivative DM4 composition, for treating the B- cell malignancies including non-Hodgkin lymphoma.
What Actinosynnema bacterium fermentation method produced ansamitocin acquisition is a kind of ansamitocin mixture, is mainly contained AP-2, AP-3 and AP-4 component, the difference between AP-2, AP-3 and AP-4 are that the acyl side-chain of C-3 hydroxyl connections is different, Such as AP-2 is propiono, AP-3 is isobutyryl, and AP-4 is isovaleryl (as shown in Figure 1).Wherein AP-3 component is to be used for AP-3, can be converted to the derivatives such as DM1 and DM4 by chemical synthesis and be used for by the intermediate for developing ansamitocin derivative Antibody coupling drug development, but AP-3 component only accounts for 60% or so in general fermentation liquid.In addition, the fermentation of ansamitocin AP-3 Yield is also relatively low, seriously affects the industrialized production of ansamitocin, also for the new drug development of ansamitocin antibody coupling drug and Clinical application brings obstruction.Therefore research and development ansamitocin AP-3 superior strain has very important industrial value.
The ansamitocin AP-3 yield of wild type Actinosynnema bacterium is very low, so existing Actinosynnema bacterium work Industry bacterial strain is all the mutant strain obtained by more taking turns mutagenesis screening, but the efficiency of routine mutagenesis breeding technique is lower, becomes system An about bottleneck of ansamitocin AP-3 production technology raising.
Acyltransferase is the key enzyme of ansamitocin precursor addition isobutyryl side chain synthesis ansamitocin AP-3, the enzyme Activity improve when can be improved to ansamitocin addition isobutyryl side chain catalytic efficiency, therefore by acyltransferase into Row genetic modification is expected to obtain ansamitocin AP-3 Producing Strain.But acyltransferase not only has catalyzing iso-butane acyl group modification peace The activity of silk rhzomorph, moreover it is possible to be catalyzed the activity of other acyl groups modification ansamitocin, have no knowledge about the special of these modifications at present One property is related with those sites.Since the site of genetic modification is difficult to select, there has been no carry out about to acyltransferase at present Genetic modification and the report for obtaining ansamitocin AP-3 Producing Strain.
Summary of the invention
For the above-mentioned prior art, the first purpose of the invention is to provide a plant height Substratspezifitaet acyltransferases Actinosynnema bacterium and its application.Using the strategy of directed evolution, the acetylated enzyme of carat RⅡ is oriented and is changed It makes, and cooperates High Throughput Screening Assay to obtain high Substratspezifitaet acyltransferase, and then obtain ansamitocin AP-3 Producing Strain Strain.
To achieve the above object, the present invention adopts the following technical scheme:
One aspect of the present invention is related to a kind of high Substratspezifitaet acyltransferase, has such as SEQ ID NO.1 institute The amino acid sequence shown or the sequence are one or several amino acids formed with same function through replacement, missing or addition Amino acid sequence.
Another aspect of the present invention is related to a kind of gene for encoding amino acid sequence shown in SEQ ID NO.1.
Another aspect of the present invention be related to it is a kind of coding SEQ ID NO.1 shown in amino acid sequence gene have such as Nucleotide sequence shown in SEQ ID NO.2.
Another aspect of the invention is related to a kind of recombinant vector, is by amino acid shown in the coding SEQ ID NO.1 The gene of sequence is inserted into recombination table of the expression containing acyl transferase gene mutant obtained in coli expression carrier Up to carrier.The coli expression carrier is pHLY12 plasmid.
Another aspect of the invention is related to a kind of recombinant host cell, is that recombinant vector described above importing host is thin In born of the same parents, the recombinant host cell that screens.
The host cell can be yeast, bacterium, algae or fungi.
Preferably, the host cell is bacterium.
Further, it is preferred that the host cell is specially escherichia coli DH5a.
Another aspect of the invention is related to a kind of host strain, is containing the high Substratspezifitaet acyltransferase of above-mentioned coding Gene Actinosynnema bacterium.
Another aspect of the invention is related to the Actinosynnema bacterium of a plant height Substratspezifitaet acyltransferase, the bacterial strain Specially Actinosynnema bacterium X4732.Guangdong Province's Culture Collection has been preserved on 2 5th, 2018 (referred to as GDMCC, address are the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, Guangdong Microbes Inst), deposit number is GDMCC NO.60327。
Actinosynnema bacterium (Actinosynnema pretiosum) X4732 and starting strain preciousness beam of the invention Silk actinomyces X47 is compared, and the gene of acyltransferase has 6 bases to change, and being respectively the 108th becomes T from C, the 548 become C from T, and the 563rd becomes G from C, and the 574th becomes A from G, and the 728th becomes C from T, and the 1044th is become from G A.Wherein the 108th bit base mutation causes codon to become TTT from TTC, is same sense mutation, amino acid does not change, and is all benzene Alanine；The mutation of 548th bit base causes codon to become GCG from GTG, and amino acid becomes alanine from valine；563rd alkali Base mutation causes codon to become CGG from CCG, and amino acid becomes arginine from proline；The mutation of 574th bit base causes password Son becomes AGG from GGG, and amino acid becomes arginine from glycine；The mutation of 728th bit base causes codon to be become from GTG GCG, amino acid become alanine from valine；The mutation of 1044th bit base causes codon to become GGA from GGG, is synonymous prominent Become, it is all glycine that amino acid, which does not change,.
Actinosynnema bacterium (Actinosynnema pretiosum) X4732 is in preparing ansamitocin AP-3 Application also belong to protection scope of the present invention.
Third object of the present invention is to provide the Actinosynnema bacterium (Actinosynnema pretiosum) The preparation method of X4732 is mutated the gene of acyltransferase using continuous error-prone PCR method in vitro, with the transfer of existing acyl group Enzyme deletion mycopremna is bacterium germination, establishes the mutant library containing acyl transferase gene by engagement transfer techniques, passes through fermentation Verifying, secondary culture obtain Actinosynnema bacterium (Actinosynnema pretiosum) X4732 of stabilization characteristics of genetics.
Fourth object of the present invention is to provide a pair of for being mutated the acetylated enzyme gene of carat RⅡ in vitro The primer of fallibility PCR, primer sequence is respectively as shown in SEQ ID NO.3 in sequence table and SEQ ID NO.4.
Upstream primer ASM19Forward sequence: GCCATATGACCCCCGGTCCCGTCCTCCCT (SEQ ID NO.3)；
Downstream primer ASM19Reverse sequence: CGTCTAGATCACCCCGCCGGGTCCGGGGCG (SEQ ID NO.4).
The continuous error-prone PCR method is mutated the gene of acyltransferase in vitro method particularly includes:
(1) PCR reaction system: template DNA 2ul, 10 × PCR buffer 5ul, Taq archaeal dna polymerase 1ul, dATP 1~ 1~4ul of 4ul, dTTP, 1~4ul of dGTP, 1~4ul of dCTP, upstream primer 2ul, downstream primer 2ul, 25mM MgSO4 1 ~8ul, 5mM MnCl20~1ul, 1~3ul of DMSO, add ultrapure water to 50ul.
(2) PCR condition: 95 DEG C of initial denaturation 8 minutes, 95 DEG C of denaturation 20 seconds, 50~65 DEG C of annealing 20 seconds, extend 72 DEG C 2 points Clock recycles 30 times, extends 8 minutes eventually.
The template DNA of 1st PCR derives from starting strain Actinosynnema bacterium X47, the 2nd PCR's and subsequent PCR Template DNA uses last time PCR product.
The construction method of the mutant library containing acyl transferase gene, the specific steps are as follows:
(1) continuous error-prone PCR product is subjected to double digestion with NdeI and XbaI, recycles enzyme after Agar Gel sugar electrophoretic separation Product is cut, and is attached with the shuttle vector pHLY12 of same double digestion, escherichia coli DH5a is converted, picking transformant carries out Identification, must recombinate pHLY12；
(2) recombination pHLY12 is transferred to the acyl transferase gene of Actinosynnema bacterium X47 using engagement transfer techniques Deletion mycopremna obtains a series of joint elements, i.e. building obtains the mutant library of acyl transferase gene.
Screen method used by positive mutating strain are as follows: shake flask fermentation and HPLC detection method are used, to connecing in mutant library Zygote carries out high flux screening.
Beneficial effects of the present invention:
(1) present invention screens to obtain the mutant of acyl transferase gene for the first time by the way of orthomutation, passes through company Continuous fallibility PCR is in vitro mutated acyl transferase gene, while obtaining ansamitocin AP- by high-throughput screening method 3 superior strain X4732, ansamitocin AP-3 synthesis are horizontal 2.7 times higher than starting strain X47, and it is raw to can be applied to industrial fermentation It produces, there is important economic value.
(2) preparation method of Actinosynnema bacterium engineering bacteria of the invention, building acyl transferase gene mutant Speed is fast, while the screening efficiency of direct mutation bacterial strain is high, meets the breeding needs of industrial producing strain.
Detailed description of the invention
The chemical structure of Fig. 1 ansamitocin.
Fig. 2 is X4732 compared with the ansamitocin AP-3 fermentation level of X47.
Specific embodiment
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, following the description is merely to explain this Invention, is not defined its content.
Embodiment 1: continuous error-prone PCR obtains the acyl transferase gene containing mutating alkali yl
Acyl transferase gene is mutated in vitro using continuous error-prone PCR.Specifically:
1. the primer of fallibility PCR are as follows:
Upstream primer ASM19Forward sequence: GCCATATGACCCCCGGTCCCGTCCTCCCT；
Downstream primer ASM19Reverse sequence: CGTCTAGATCACCCCGCCGGGTCCGGGGCG.
2.PCR system: template DNA 2ul, 1~4ul of 10 × PCR buffer 5ul, Taq archaeal dna polymerase 1ul, dATP, 1~4ul of dTTP, 1~4ul of dGTP, 1~4ul of dCTP, upstream primer 2ul, downstream primer 2ul, 25mM MgSO41~ 8ul、5mM MnCl20~1ul, 1~3ul of DMSO, add ultrapure water to 50ul.
3.PCR condition: 95 DEG C of initial denaturation 8 minutes, 95 DEG C of denaturation 20 seconds, 50~65 DEG C of annealing 20 seconds, extend 72 DEG C 2 points Clock recycles 30 times, extends 8 minutes eventually.
1st time pcr template DNA derives from starting strain Actinosynnema bacterium X47, the mould of the 2nd PCR and subsequent PCR Plate DNA uses last time PCR product.
PCR product directly send sequencing, as the result is shown: as dATP 3ul, dTTP 3ul, dGTP 1ul, dCTP in PCR system 1ul, upstream primer 2ul, downstream primer 2ul, 25mM MgSO4 5ul、5mM MnCl21ul, DMSO 2ul, PCR annealing temperature It is 52 DEG C, base mismatch number is 3~12 when 2 consecutive PCRs, and the frequency of mutation is relatively appropriate for directional transformation.
Embodiment 2: the building in Actinosynnema bacterium acylase mutant library
PCR product in embodiment 1 is subjected to double digestion with NdeI and XbaI, recycles enzyme after Agar Gel sugar electrophoretic separation Product is cut, and is connected to shuttle vector pHLY12 (pHLY12 is conventional plasmid in the prior art), converts escherichia coli DH5a, Picking transformant extracts plasmid and is identified.
The recombination pHLY12 acyl transferase gene for being transferred to Actinosynnema bacterium X47 is lacked using engagement transfer techniques Bacterial strain (it is that starting strain is obtained by conventional gene knockout technology that the gene deletion strains, which are with strain X 47), 160 plants of picking connect The mutant library of zygote building acyl transferase gene.Then 160 plants of joint elements are transferred to respectively 25 on ISP2 solid plate DEG C culture 14 days, spore and number X4701~X47160 respectively are saved, the shake flask fermentation for carrying out mutant strain screens.
ISP2 culture medium is conventional commercial product.
Embodiment 3: the shake flask fermentation screening of mutant strain
The spore for the 160 plant mutant strains that embodiment 2 is selected is diluted to 10 respectively7A/ml is inoculated in liquid by 5% (V/V) In body culture medium, shake flask fermentation (every bottled 50ml fluid nutrient medium of 250ml triangle), 25 DEG C, 200rpm shaken cultivation 8 days, 3000rpm is centrifuged 10min and collects thallus, is extracted with the ethyl acetate of 3 times of volumes, then detects ansamitocin with HPLC method The fermentation level of AP-3.Ansamitocin AP-3 content is significantly higher than starting strain in 14 plant mutant strain fermentation liquids as the result is shown, just Mutation rate reaches 8.75%, as a result such as table 1.Final one plant of acquisition ansamitocin AP-3 yield ratio under conditions of flask fermentation sets out The mutant strain that 47 high 2.4 times of strain X, is named as X4732 for this mutant strain.
The composition of fluid nutrient medium are as follows: the composition of fluid nutrient medium are as follows: dextrin 50g/L, corn pulp 10g/L, peptone 2g/ L, anhydrous calcium chloride 10g/L, calcium carbonate 5g/L, pH 7.5,0.15MPa sterilizing 30min.
Wherein, corn pulp, peptone and dextrin are conventional commercial product.
The ansamitocin AP-3 yield result (mg/L fermentation liquid) of 1 positive mutating strain shake flask fermentation of table
Embodiment 4: the genetic stability of superior strain X4732 is investigated
The spore suspension dilution spread of superior strain X4732 is cultivated 14 days for 25 DEG C on ISP4 solid medium, separation The monospore bacterium colony of strain X4732 simultaneously carries out expansion culture, continuous separation monospore five times, and verifies per generation X4732 spore with shake flask fermentation The ansamitocin AP-3 fermentation level of son, the results are shown in Table 2, the results showed that and superior strain X4732 has preferable genetic stability, Meet industrial demand.
The ansamitocin AP-3 fermentation level (mg/L fermentation liquid) of 2 X4732 difference passage number of table
Passage number Repeat 1 Repeat 2 Repeat 3 Average value
Starting 890.4 902.3 872.4 888.4
The first generation 873.9 913.1 884.2 890.4
The second generation 912.3 884.6 881.7 892.9
The third generation 876.1 906.8 902.4 895.1
Forth generation 887.3 894.6 897.1 893.0
5th generation 865.4 904.6 891.2 887.1
Embodiment 5: the determined dna sequence of acylase mutant
The genomic DNA of engineering bacteria X4732 is extracted as template, ASM19Forward and ASM19Reverse primer carries out High-fidelity PCR amplification, PCR product are separated and recovered from through agarose gel electrophoresis, and conversion large intestine bar is connect with pCR-Blunt carrier Bacterium DH5a, picking positive colony carry out DNA sequencing.SEQ ID NO.2 in acyl transferase gene sequence such as sequence table in X4732 Shown, acyl transferase gene sequence is as shown in SEQ ID NO.5 in sequence table in X47.The results show that acyl group turns in X4732 Move enzyme gene has 6 bases to change compared with wild type gene, and respectively the 108th becomes T from C, and the 548th is become by T For C, the 563rd becomes G from C, and the 574th becomes A from G, and the 728th becomes C from T, and the 1044th becomes A from G.548th, 563, the mutation of 574 and 728 bit bases causes 4 amino acid variations, is V183A, P188R, G192R and V243A respectively.X4732 The amino acid sequence of middle acyltransferase is as shown in SEQ ID NO.1 in sequence table, the amino acid sequence of acyltransferase in X47 As shown in SEQ ID NO.6 in sequence table.
Actinosynnema bacterium X4732 was preserved in Guangdong Province's Culture Collection on 2 5th, 2018 (referred to as GDMCC, address are the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, Guangdong Microbes Inst), deposit number is GDMCC NO.60327。
Embodiment 6: fermented and cultured superior strain X4732 produces ansamitocin AP-3
1. seed liquor culture:
By the spore inoculating of strain X 4732 to liquid seeds shaking flask, 25 DEG C 200rpm shaken cultivation 2 days, be then seeded into 50L seeding tank, 25 DEG C of cultures, 2 days acquisition seed liquors；Simultaneously using starting strain X47 as control.
2. fermented and cultured:
Seed liquor is inoculated into 1m by 5% (volume fraction) inoculum concentration3Fermentor, ventilatory capacity 1.3VVM, speed of agitator 250rpm, 25 DEG C fermented and cultured 8 days, put the fermentation level that ansamitocin AP-3 is detected after tank.
The composition of the fluid nutrient medium of seed culture are as follows: glucose 20g/L, soluble starch 30g/L, soybean cake powder 10g/ L, corn pulp 10g/L, peptone 5g/L, sodium chloride 3g/L, calcium carbonate 5g/L, pH 7.5,0.15MPa sterilizing 30min.Fermentation training The composition of feeding fluid nutrient medium are as follows: dextrin 50g/L, corn pulp 10g/L, peptone 2g/L, anhydrous calcium chloride 10g/L, carbonic acid Calcium 5g/L, pH 7.5,0.15MPa sterilizing 30min.
Wherein, soybean cake powder, corn pulp, peptone and dextrin are conventional commercial product.
3. ansamitocin AP-3 assay:
Using the content of ansamitocin AP-3 in HPLC method measurement fermentation liquid, as a result are as follows: pacify silk in 4732 fermentation liquid of strain X Rhzomorph AP-3 content is 977mg/L, and ansamitocin AP-3 content is 362mg/L in 47 fermentation liquid of strain X, and strain X 4732 is fermented The ansamitocin AP-3 fermentation level of liquid improves 2.7 times (see Fig. 2) than starting strain X47.
Finally it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not limited to this hair It is bright, although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still It can modify to technical solution documented by previous embodiment, or part is equivalently replaced.It is all in this hair Within bright spirit and principle, any modification, equivalent replacement, improvement and so on should be included in protection scope of the present invention Within.Although above-mentioned in conjunction with specific embodiments of the present invention have been described, not to the limit of the scope of the present invention System, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art do not need to pay The various modifications or changes that creative work can be made out are still within protection scope of the present invention.
SEQUENCE LISTING
<110>Shandong Provincial Pharmaceutical Biological Tech. Research Center (institute of viruses, Shandong Province)
The Actinosynnema bacterium of<120>one plant height Substratspezifitaet acyltransferases and its application
<130> 2010
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 378
<212> PRT
<213>artificial synthesized
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Met Thr Pro Gly Pro Val Leu Pro Pro Arg Leu Pro Ser Leu Thr Gly
1 5 10 15
Ile Arg Ala Pro Leu Ala Leu Leu Val Phe Val Ala His Ala Leu Gly
20 25 30
Ser Ala Arg Phe Phe Ala Asp Glu Ser Val Asn Ser Leu Gly Phe Leu
35 40 45
Leu Pro Tyr Gly Pro Ala Ala Leu Ser Leu Phe Phe Val Leu Ser Gly
50 55 60
Phe Val Leu Val Trp Ser Glu Pro Trp Arg Glu Gly Val Gly Pro Tyr
65 70 75 80
Phe Arg Arg Arg Val Val Arg Ile Leu Pro Thr His Val Leu Thr Trp
85 90 95
Ala Ala Val Leu Leu Leu Leu Ala Ala Leu Gly Pro Leu Pro Leu Leu
100 105 110
Gly Pro Leu Pro Glu Val Gly Pro Ala Leu Val Asn Leu Ser Leu Leu
115 120 125
Gln Ser Leu Val Pro Leu Pro Asp Tyr Leu Leu Ser Val Asn Gly Ile
130 135 140
Asn Trp Ser Val Ser Cys Glu Val Val Phe Tyr Leu Leu Leu Pro Leu
145 150 155 160
Leu Ser Arg Pro Leu Leu Arg Val Pro Asp His Arg Leu Trp Ala Cys
165 170 175
Phe Gly Val Leu Ala Ala Ala Val Leu Ala Leu Arg Gly Val Ile Arg
180 185 190
Ala Leu Val Asp Gly Pro Pro Trp Ala Leu Trp Pro Pro Leu Ser Phe
195 200 205
Glu Gln Ala Trp Leu Val Asn Phe Phe Pro Leu Thr Arg Leu Pro Glu
210 215 220
Phe Leu Met Gly Val Val Leu Ala Arg Ile Val Ala Thr Gly Arg Trp
225 230 235 240
Arg Pro Ala Arg Ala Trp Trp Pro Leu Leu Gly Val Ala Ala Val Trp
245 250 255
Ala Leu Leu Pro Val Leu Pro Gln Val Tyr Ala Arg Ser Ala Ile Ala
260 265 270
Ala Val Pro Leu Ala Leu Leu Val Pro Val Ile Ala Val Arg Asp Leu
275 280 285
Glu Gly Arg Arg Ser Val Leu Ser Arg Arg Trp Val Arg Val Ala Gly
290 295 300
Asp Met Ser Tyr Ala Thr Tyr Leu Leu His Trp Pro Leu Leu Ala Val
305 310 315 320
Ala Lys His Val Ala Gly Asp Arg Leu Leu Gly Leu Gly Glu Gly Leu
325 330 335
Leu Leu Val Ala Ala Leu Tyr Ala Leu Thr Gln Gly Leu Ser Leu Leu
340 345 350
Leu His Arg Trp Val Glu Arg Pro Leu Leu Arg Arg Ala His Arg Pro
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Arg Arg Ser Pro Ala Pro Asp Pro Ala Gly
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tcggtcaact cgctcgggtt cctcctgccc tacgggcccg cggcgctgtc gttgttcttc 180
gtgctcagcg ggttcgtgct ggtgtggtcg gagccgtggc gcgagggcgt gggcccctac 240
ttccgacgcc gggtcgtgcg catcctgccc acccacgtgc tgacgtgggc cgcggtgctg 300
ctgctcctgg cggcgctcgg cccgctgccg ctgctggggc cgctgcccga ggtggggccc 360
gcgctggtca acctgtcgct gctccagtcg ctcgtgccgc tgcccgacta cctgctgtcg 420
gtcaacggca tcaactggtc ggtgtcctgc gaggtggtgt tctacctgct gctgccgctg 480
ctgtcccggc cgctgctgcg ggtgcccgac caccggttgt gggcgtgctt cggcgtgctc 540
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gcgctgtggc cgccgctgtc gttcgagcag gcgtggctgg tgaacttctt cccgctgacc 660
cggttgccgg agttcctgat gggcgtggtg ctcgccagga tcgtggccac cgggcggtgg 720
cggccggcgc gggcgtggtg gccgctgctg ggggtggcgg cggtgtgggc gctgctgccg 780
gtgctgccgc aggtgtacgc gcgcagcgcg atcgcggcgg tcccgctggc gctgctcgtc 840
cccgtcatcg ccgtgcggga cctggagggg cggcggtccg tgctgtcccg gcggtgggtg 900
cgggtcgcgg gggacatgag ctacgcgacc tacctgctgc actggccgct gctcgcggtc 960
gccaagcacg tggcggggga ccggctgctc gggctggggg aggggctgct gctggtggcg 1020
gcgctgtacg cgctcaccca gggactcagc ctgctgctgc accggtgggt ggagcgtccg 1080
ctgctgcggc gcgcccaccg gccgaggcgg tcgcccgccc cggacccggc ggggtga 1137
<210> 3
<211> 29
<212> DNA
<213>artificial synthesized
<400> 3
gccatatgac ccccggtccc gtcctccct 29
<210> 4
<211> 30
<212> DNA
<213>artificial synthesized
<400> 4
cgtctagatc accccgccgg gtccggggcg 30
<210> 5
<211> 1137
<212> DNA
<213>artificial synthesized
<400> 5
atgacccccg gtcccgtcct ccctcctcgg cttccctccc tcaccggcat ccgcgcgccg 60
ctggccctgc tcgtgttcgt ggcccacgcg ctcggctccg cccggttctt cgccgacgaa 120
tcggtcaact cgctcgggtt cctcctgccc tacgggcccg cggcgctgtc gttgttcttc 180
gtgctcagcg ggttcgtgct ggtgtggtcg gagccgtggc gcgagggcgt gggcccctac 240
ttccgacgcc gggtcgtgcg catcctgccc acccacgtgc tgacgtgggc cgcggtgctg 300
ctgctcctgg cggcgctcgg cccgctgccg ctgctggggc cgctgcccga ggtggggccc 360
gcgctggtca acctgtcgct gctccagtcg ctcgtgccgc tgcccgacta cctgctgtcg 420
gtcaacggca tcaactggtc ggtgtcctgc gaggtggtgt tctacctgct gctgccgctg 480
ctgtcccggc cgctgctgcg ggtgcccgac caccggttgt gggcgtgctt cggcgtgctc 540
gccgcggtgg tgctcgcgct gccgggcgtg atcggggcgc tggtggacgg gccgccgtgg 600
gcgctgtggc cgccgctgtc gttcgagcag gcgtggctgg tgaacttctt cccgctgacc 660
cggttgccgg agttcctgat gggcgtggtg ctcgccagga tcgtggccac cgggcggtgg 720
cggccggtgc gggcgtggtg gccgctgctg ggggtggcgg cggtgtgggc gctgctgccg 780
gtgctgccgc aggtgtacgc gcgcagcgcg atcgcggcgg tcccgctggc gctgctcgtc 840
cccgtcatcg ccgtgcggga cctggagggg cggcggtccg tgctgtcccg gcggtgggtg 900
cgggtcgcgg gggacatgag ctacgcgacc tacctgctgc actggccgct gctcgcggtc 960
gccaagcacg tggcggggga ccggctgctc gggctggggg aggggctgct gctggtggcg 1020
gcgctgtacg cgctcaccca ggggctcagc ctgctgctgc accggtgggt ggagcgtccg 1080
ctgctgcggc gcgcccaccg gccgaggcgg tcgcccgccc cggacccggc ggggtga 1137
<210> 6
<211> 378
<212> PRT
<213>artificial synthesized
<400> 6
Met Thr Pro Gly Pro Val Leu Pro Pro Arg Leu Pro Ser Leu Thr Gly
1 5 10 15
Ile Arg Ala Pro Leu Ala Leu Leu Val Phe Val Ala His Ala Leu Gly
20 25 30
Ser Ala Arg Phe Phe Ala Asp Glu Ser Val Asn Ser Leu Gly Phe Leu
35 40 45
Leu Pro Tyr Gly Pro Ala Ala Leu Ser Leu Phe Phe Val Leu Ser Gly
50 55 60
Phe Val Leu Val Trp Ser Glu Pro Trp Arg Glu Gly Val Gly Pro Tyr
65 70 75 80
Phe Arg Arg Arg Val Val Arg Ile Leu Pro Thr His Val Leu Thr Trp
85 90 95
Ala Ala Val Leu Leu Leu Leu Ala Ala Leu Gly Pro Leu Pro Leu Leu
100 105 110
Gly Pro Leu Pro Glu Val Gly Pro Ala Leu Val Asn Leu Ser Leu Leu
115 120 125
Gln Ser Leu Val Pro Leu Pro Asp Tyr Leu Leu Ser Val Asn Gly Ile
130 135 140
Asn Trp Ser Val Ser Cys Glu Val Val Phe Tyr Leu Leu Leu Pro Leu
145 150 155 160
Leu Ser Arg Pro Leu Leu Arg Val Pro Asp His Arg Leu Trp Ala Cys
165 170 175
Phe Gly Val Leu Ala Ala Val Val Leu Ala Leu Pro Gly Val Ile Gly
180 185 190
Ala Leu Val Asp Gly Pro Pro Trp Ala Leu Trp Pro Pro Leu Ser Phe
195 200 205
Glu Gln Ala Trp Leu Val Asn Phe Phe Pro Leu Thr Arg Leu Pro Glu
210 215 220
Phe Leu Met Gly Val Val Leu Ala Arg Ile Val Ala Thr Gly Arg Trp
225 230 235 240
Arg Pro Val Arg Ala Trp Trp Pro Leu Leu Gly Val Ala Ala Val Trp
245 250 255
Ala Leu Leu Pro Val Leu Pro Gln Val Tyr Ala Arg Ser Ala Ile Ala
260 265 270
Ala Val Pro Leu Ala Leu Leu Val Pro Val Ile Ala Val Arg Asp Leu
275 280 285
Glu Gly Arg Arg Ser Val Leu Ser Arg Arg Trp Val Arg Val Ala Gly
290 295 300
Asp Met Ser Tyr Ala Thr Tyr Leu Leu His Trp Pro Leu Leu Ala Val
305 310 315 320
Ala Lys His Val Ala Gly Asp Arg Leu Leu Gly Leu Gly Glu Gly Leu
325 330 335
Leu Leu Val Ala Ala Leu Tyr Ala Leu Thr Gln Gly Leu Ser Leu Leu
340 345 350
Leu His Arg Trp Val Glu Arg Pro Leu Leu Arg Arg Ala His Arg Pro
355 360 365
Arg Arg Ser Pro Ala Pro Asp Pro Ala Gly
370 375

Claims (9)

1. a kind of high Substratspezifitaet acyltransferase, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.
2. encoding the gene of the amino acid sequence of high Substratspezifitaet acyltransferase described in claim 1.
3. gene as claimed in claim 2, which is characterized in that its nucleosides of amino acid sequence shown in coding SEQ ID NO.1 Acid sequence gene is as shown in SEQ ID NO.2.
4. a kind of recombinant vector, which is characterized in that be inserted into coli expression carrier for gene described in claim 2 or 3 Obtained in expression the recombinant expression carrier containing acyl transferase gene mutant；The coli expression carrier is PHLY12 plasmid.
5. a kind of recombinant host cell, which is characterized in that import recombinant vector as claimed in claim 4 in host cell, sieve Choosing obtains recombinant host cell.
6. recombinant host cell as claimed in claim 5, which is characterized in that host cell is escherichia coli DH5a.
7. a kind of host strain, which is characterized in that the host strain is that the precious synnema containing gene described in Claims 2 or 3 is put Line bacterium.
8. the Actinosynnema bacterium of a plant height Substratspezifitaet acyltransferase, which is Actinosynnema bacterium (Actinosynnema pretiosum) X4732 was preserved in Guangdong Province's Microbiological Culture Collection on 2 5th, 2018 The heart, deposit number are GDMCC NO.60327.
9. Actinosynnema bacterium as claimed in claim 8 is preparing the application in ansamitocin AP-3.
CN201810191854.0A 2018-03-08 2018-03-08 The Actinosynnema bacterium of one plant height Substratspezifitaet acyltransferase and its application Expired - Fee Related CN108330113B (en)

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US6790954B2 (en) * 2002-01-29 2004-09-14 Immunogen, Inc. Mutant Actinosynnema pretiosum strain with increased maytansinoid production
US7432088B2 (en) * 2003-05-08 2008-10-07 Immunogen Inc. Methods for the production of ansamitocins
CN103255184B (en) * 2012-02-15 2016-01-27 百奥泰生物科技(广州)有限公司 Produce the fermention medium of ansamitocin
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CN104894183B (en) * 2015-06-25 2018-04-27 齐鲁制药有限公司 A kind of method that ansamitocin P-3 is prepared with precious orange synnema actinomycetes
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