CN113789314B - Application of ribosome sigma factor B and mutant thereof in improving yield of lipstatin - Google Patents

Application of ribosome sigma factor B and mutant thereof in improving yield of lipstatin Download PDF

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CN113789314B
CN113789314B CN202111260753.2A CN202111260753A CN113789314B CN 113789314 B CN113789314 B CN 113789314B CN 202111260753 A CN202111260753 A CN 202111260753A CN 113789314 B CN113789314 B CN 113789314B
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黄胜雄
颜一军
刘兴勇
马小燕
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Sichuan University of Science and Engineering
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Abstract

The invention discloses a ribosome sigma factor B and application of a mutant thereof in improving the yield of lipstatin. The nucleotide sequence of the ribosome sigma factor B is shown as SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 8. The ribosome sigma factor B and the mutant overexpression thereof obtained by screening can improve the yield of lipstatin.

Description

Application of ribosome sigma factor B and mutant thereof in improving yield of lipstatin
The application is a divisional application with the application date 2020-10-29 and the application number 202011176931.9, and the patent name is 'application of a ribosome sigma factor, mutant thereof and coded protein in improving the yield of lipstatin'.
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to application of a ribosome sigma factor and a mutant thereof in improving the yield of lipstatin.
Background
The tetrahydroderivative of lipstatin, orlistat, is currently the most popular weight-reducing product for use by more than 4 million people worldwide as an anti-obesity therapeutic agent that is currently certified by the U.S. FDA, eu EMA and chinese SFDA without suppressing appetite, acting on the central nervous system. As a long-acting specific pancreatic lipase inhibitor, it can prevent triglyceride from hydrolyzing into free fatty acid and monoacylglycerol which can be absorbed by small intestinal mucosa, thereby reducing caloric intake and controlling body weight. The lipstatin is originally isolated from streptomycete Streptomyces toxytricini and has a beta-propiolactone structural unit, wherein the 2 and 3 positions of the lipstatin are respectively replaced by a 6-carbon alkyl hydrocarbon chain and a 13-carbon alkyl hydrocarbon chain, and the hydroxyl group at the C5 position of the 13-carbon alkyl hydrocarbon chain forms an ester bond with N-formoxyl-L-leucine. Because of the complex molecular structure of lipstatin, the chemical synthesis process is complex, and a great deal of manpower and material resources are consumed. At present, the lipstatin is mainly produced by adopting a semi-biological fermentation process, so that compared with a chemical method, the use of toxic solvents is reduced, the pollution to the environment is greatly reduced, and toxic substance residues are greatly reduced.
At present, research on the synthesis of lipstatin by lipstatin producing bacteria at home and abroad mainly adopts a method of combining a single factor experiment and an orthogonal design experiment to optimize a fermentation medium and fermentation parameters, such as the influence of biotin and ATP addition on lipid and lipstatin synthesis (Dong Huijun and the like, the influence of biotin and ATP addition on the synthesis of toxic trichlungin lipid and lipstatin, journal of Chinese medical industry, 2014, 01, pages 19-24).
Ribosomal sigma transcription factors are important components of RNA polymerase and play an important role in the transcription process of prokaryotes. Overexpression of frr as in Streptomyces avermitilis increases avermectin production (Li L et al, (2010) Over expression of ribosome recycling factor causes increased production of avermectin in Streptomyces avermitilis strains.J Ind Microbiol Biotechnol 37 (7): 673-679), but no ribosomal sigma transcription factor affecting lipstatin synthesis is currently reported.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a ribosome sigma factor and a mutant thereof and application of the encoded protein in improving the yield of lipstatin, the invention compares and analyzes the ribosome factors through genome sequencing and bioinformatics analysis to find out 15 different ribosome factors, then through evaluation, the over-expression of the ribosome sigma transcription factors A and B in Streptomyces toxytricini can improve the yield of the lipstatin, and provides the mutants of the two ribosome sigma transcription factors A and B and application of the mutants in the production of the lipstatin.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
use of a ribosomal sigma factor, either ribosomal sigma factor a or ribosomal sigma factor B, for increasing the yield of lipstatin.
Further, the nucleotide sequence of the ribosome sigma factor A is shown as SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 6.
Further, the nucleotide sequence of the ribosome sigma factor B is shown as SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 8.
A mutant of the ribosome sigma factor A, the nucleotide sequence of which is shown in SEQ ID NO. 1.
Further, the amino acid sequence of the protein encoded by the nucleotide is shown as SEQ ID NO.2, and compared with the amino acid sequence of the normal ribosome sigma factor A, the amino acid at 19 th position is replaced by R, the amino acid at 327 th position is replaced by G, the amino acid at 338 th position is replaced by G, the amino acid at 344 th position is replaced by F, the amino acid at 347 th position is replaced by S, and the amino acid at 538 th position is replaced by T.
A mutant of the ribosome sigma factor B, the nucleotide sequence of which is shown in SEQ ID NO. 3.
Further, the amino acid sequence of the protein encoded by the nucleotide is shown as SEQ ID NO.4, and compared with the amino acid sequence of the normal ribosome sigma factor B, the amino acid at 95 th position is replaced by T, the amino acid at 274 th position is replaced by S, the amino acid at 275 th position is replaced by T, the amino acid at 351 th position is replaced by V, and the amino acid at 411 th position is replaced by D.
The application of the mutant of the ribosome sigma factor A or the coded protein thereof in improving the yield of lipstatin.
The application of the mutant of the ribosome sigma factor B or the coded protein thereof in improving the yield of lipstatin.
A plasmid comprising the aforementioned ribosomal sigma factor, a mutant of ribosomal sigma factor a, or a mutant of ribosomal sigma factor B.
A transgenic cell line comprising the aforementioned ribosomal sigma factor, a mutant of ribosomal sigma factor a, or a mutant of ribosomal sigma factor B.
An engineering bacterium comprising the above-mentioned ribosomal sigma factor, a mutant of ribosomal sigma factor A, or a mutant of ribosomal sigma factor B.
Further, overexpression is performed by introducing an expression vector containing the coding gene into Streptomyces Streptomyces toxytricini.
Further, the gene or mutant gene is located in a plasmid or chromosome.
The beneficial effects of the invention are as follows:
screening researches on 10 ribosomal factors show that the ribosomal sigma factors A and B and mutant genes thereof are overexpressed in streptomycete Streptomyces toxytricini, so that the method is biologically safe for genetically engineered bacteria (the overexpression in the bacteria does not influence the growth of the bacteria), and meanwhile, the capability of producing lipstatin by streptomycete Streptomyces toxytricini can be effectively improved. Experimental data indicate that overexpression of the original gene at Streptomyces toxytricini increases the ability of product epstein, especially their mutant genes, further increases the ability of product epstein.
Drawings
FIG. 1 shows a map of the plasmid vector pKC1139-liprA,
FIG. 2 is a map of the plasmid vector pKC1139-liprB,
FIG. 3 is a map of the plasmid vector pKC1139-liprA-mut,
FIG. 4 is a map of plasmid vector pKC1139-liprB-mut,
Fig. 5 is a standard plot of lipstatin concentration.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
1. Material
Streptomyces toxytricini As a typical Streptomyces, the endomycelia are branched, the aerial mycelia are slightly thick, and part of aerial mycelia are differentiated into spiral spore filaments, so that the characteristic of typical Streptomyces is achieved.
The formula of the culture medium comprises:
seed culture medium: 10g of soya bean flour, 5g of Bacto soytone, 5mL of glycidol, 10mL of soya oil, and 2mL of Triton X-100[pH 6.5]per liter.
Fermentation culture: 30g of soya bean flour, 14mL of glycidol, 1g of Bacto soytone, 1mL of Triton X-100, and 60mL of soya oil[pH 7.0]per liter.
2. Detection method
(1) Sample pretreatment
1mL of the fermentation broth was placed in a 10mL centrifuge tube, 9mL of methanol was added, the mixture was stirred in a vortex mixer, sonicated for 30min, centrifuged (12000 rpm,5 min), and then filtered through a 0.22 μm filter, and the filtrate (20. Mu.L) was taken for HPLC analysis.
(2) Preparation of standards
Lipstatin standard (0.125 g/L,0.25g/L,0.5g/L,1g/L,2 g/L) was configured
(3) HPLC detection conditions
Chromatographic column: YMC-Pack ODS-A; detection wavelength: 205nm; mobile phase: acetonitrile: water = 90:10; flow rate: 1.0mL/min; column temperature: 40 ℃.
(4) Drawing of lipstatin standard curve
And (3) carrying out HPLC detection on the standard substances with different concentrations according to the conditions, and drawing a standard curve of the concentration of the lipstatin with peak areas. And drawing a standard curve of the lipstatin by taking the measured peak area A as an ordinate and recording the mass concentration C (g/L) of the lipstatin as an abscissa. See fig. 5, yielding the regression equation y=5e+6x+101960, r 2 =0.996, absorbance was a good linear relationship with mass concentration. Calculating the sample yield according to the lipstatin standard curve after the liquid phase is ended
Example 1 obtaining of strains
Diluting and coating the frozen strain on ISP3 plate culture medium, placing in a 30 ℃ hydration incubator for inversion culture, and after bacterial colony grows out,single colonies were picked, re-cultured and transferred to 2 passages. Taking a fresh long plate, washing surface spores with 10mL of sterile water, placing in a triangular flask with glass beads, culturing at 28deg.C under shaking at 200r/min for 2-3 h, filtering, serial diluting, and making into spore with concentration of about 1×10 7 Single spore suspension per mL was used.
EXAMPLE 2 Strain genome analysis
The strain Streptomyces toxytricini is sent to the China large gene company for whole genome sequencing, the comparison analysis is carried out on the ribosomal factors through genome sequencing and bioinformatics analysis, 15 different ribosomal factors are found, and through screening, the overexpression of the ribosomal sigma transcription factors A and B can improve the yield of lipstatin.
Based on the ribosome sigma transcription factors A and B obtained by screening, respectively obtaining a mutant gene of the ribosome sigma factor A and a mutant gene of the ribosome sigma factor B through substitution of nucleotides, wherein the nucleotide sequences of the mutant genes are respectively shown as SEQ ID NO.1 and SEQ ID NO. 3;
meanwhile, corresponding amino acid mutants are obtained, specifically:
mutant of ribosomal sigma factor a: the 19 th amino acid of the amino acid sequence coded by the ribosome sigma transcription factor A is replaced by R, the 327 th amino acid is replaced by G, the 338 th amino acid is replaced by G, the 344 th amino acid is replaced by F, the 347 rd amino acid is replaced by S, and the 538 th amino acid is replaced by T.
Mutant of ribosomal sigma factor B: the 95 th amino acid of the amino acid sequence coded by the ribosome sigma transcription factor B is replaced by T, the 274 th amino acid is replaced by S, the 275 th amino acid is replaced by T, the 351 th amino acid is replaced by V, and the 411 th amino acid is replaced by D.
The effect of overexpression of the substituted mutants in the strain on lipstatin production was each verified.
EXAMPLE 3 construction of plasmid
1. Construction of pKC1139-liprA
The primers of the primer pairs liprA-XbaI-F and liprA-EcoRV-R in the table 1 are respectively utilized, streptomyces toxytricini genome is used as a template, and through PCR amplification, xbaI and EcoRV enzyme cutting sites are introduced to obtain liprA fragments, and after electrophoresis verification, dpnI enzyme treatment and electrophoresis gel recovery, the purified liprA fragments are obtained. The sequence of the liprA gene is shown as SEQ ID No.5, and the encoded amino acid sequence is shown as SEQ ID No. 6.
The purified liprA fragment and pKC1139 plasmid were double digested with XbaI and EcoRV, respectively, and the double digested product of the liprA fragment was ligated with the double digested product of pKC1139 plasmid overnight at 4 ℃ with T4 ligase. The ligation product was transformed into E.coli DH 5. Alpha. And spread on LB solid plates containing 50mg/L of apramycin, after 16h of incubation, colony PCR detection was performed, and after correct sequencing, the positive bacteria obtained were designated E.coli/pKC1139-liprA. Plasmid pKC1139-liprA was extracted with plasmid kit for use, and the plasmid map is shown in FIG. 1.
2、pKC1139-liprA-mut:
The nucleotide sequence is shown as SEQ ID NO:1 to obtain a liprA-mut fragment, and constructing with the same primers according to the construction process of pKC1139-liprA to obtain plasmid pKC1139-liprA-mut. The plasmid map is shown in FIG. 3.
3. Construction of pKC 1139-liprB:
the primers of the primer pairs liprB-XbaI-F and liprB-EcoRV-R in the table 1 are respectively utilized, streptomyces toxytricini genome is used as a template, and through PCR amplification, xbaI and EcoRV enzyme cutting sites are introduced to obtain liprB fragments, and after electrophoresis verification, dpnI enzyme treatment and electrophoresis gel recovery, purified liprB fragments are obtained. The sequence of the liprB gene is shown as SEQ ID No.7, and the encoded amino acid sequence is shown as SEQ ID No. 8.
The purified liprB fragment and pKC1139 plasmid were double digested with XbaI and EcoRV, respectively, and the double digested product of the liprB fragment was ligated with the double digested product of the pKC1139 plasmid overnight at 4℃with T4 ligase. The ligation product was transformed into E.coli DH 5. Alpha. And spread on LB solid plates containing 50mg/L of apramycin, after 16h of incubation, colony PCR detection was performed, and after correct sequencing, the obtained positive bacteria were designated E.coli/pKC1139-liprB. Plasmid pKC1139-liprB was extracted with plasmid kit and the plasmid map is shown in FIG. 2.
4、pKC1139-liprB-mut:
The nucleotide sequence is shown as SEQ ID NO:3 to obtain a liprB-mut fragment, and constructing the plasmid pKC1139-liprB-mut by using the same primer according to the construction process of pKC1139-liprB. The plasmid map is shown in FIG. 4.
TABLE 1 primers
Figure BDA0003325460000000081
EXAMPLE 4 construction of plasmid-containing vector Strain
1. Preparation of E.coli ET12567/pUZ8002 containing exogenous plasmid
And transforming the constructed plasmid into escherichia coli ET12567/pUZ8002 to obtain donor bacteria containing recombinant plasmid. The donor bacteria were inoculated in 5mL of LB medium (containing 25. Mu.g/mL Km, cm and 50. Mu.g/mL Am), incubated overnight at 37℃and transferred to 10mL of LB medium (containing 25. Mu.g/mL Km, cm and 50. Mu.g/mL Am) at 1:50-100, and incubated at 37℃to OD 600 =0.3 to 0.4, cultured for about 3 hours, collected by centrifugation, washed twice with drug-free LB, and suspended in 1mL of drug-free LB medium. ET12567/pUZ8002 containing the corresponding plasmid was obtained.
2. Treatment of spores of heterologous expression strains
Streptomyces toxytricini culturing for about 7d, washing spores with 5mL of 10% glycerol, placing into 50mL test tube, shaking, mixing, filtering absorbent cotton in clean test tube, centrifuging at 3500rpm for 10min, adding 1mL of 2 XYT, and thermally activating at 50deg.C for 10min.
3. Coli ET12567/pUZ8002 and Streptomyces toxytricini strain conjugal transfer and isolation identification of strain
Respectively mixing equal amount of Streptomyces spore liquid and Escherichia coli bacterial liquid 0.5mL, and coating with 10mM MgCl 2 Is cultured overnight at 28℃with sterile water containing Am (50. Mu.g/mL) and Tri (100. Mu.g/mL) and cultured at 28℃for 7-10 d, and the positive colonies grown are picked up on IWL4 or MS and containing Am (50. Mu.g/mL) and Tri (100. Mu.g/mL)After subculturing in R2YE medium, the corresponding colonies were picked. SK/pKC1139 (control bacterium), SK/pKC1139-liprA (abbreviated SK 1), SK/pKC1139-liprA-mut (abbreviated SK 2), SK/pKC1139-liprB (abbreviated SK 3), SK/pKC1139-liprB-mut (abbreviated SK 4).
EXAMPLE 5 evaluation of different strains
Control bacteria and SK1, SK2, SK3, SK4 were subjected to shake flask fermentation, each in 3 replicates, and then subjected to the assay and specific price of lipstatin, the results of which are shown in Table 2. From the test results in table 2, it is found that the ability of the experimental group bacteria to produce lipstatin is improved compared with the control bacteria, thereby demonstrating that the overexpression of ribosomal sigma factors a and B and mutant genes thereof can improve the yield of lipstatin.
TABLE 2 Liprastatin production by different strains
Strain Lipstatin yield (g/L)
Control bacteria 1.12±0.11
SK1 1.43±0.17
SK2 1.67±0.15
SK3 1.27±0.14
SK4 1.56±0.12
Sequence listing
<110> university of light chemical industry in Sichuan
<120> application of ribosome sigma factor B and mutant thereof in improving lipstatin yield
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<213> Streptomyces sp
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caggacgagc tggtcagcgc gtatctgccg ctggtctaca acatcgtcgg gcgcgccatg 120
aacggctccg tcgacgtgga cgacgtcgtg caggacacga tgctgcgggc cctcgacggc 180
ctcggcaccc tccgttcgga cgacagcttc cgctcgtggt tggtggccat cgcgatgaac 240
cgggtgcggg cgtactggca ggcccggcgc accgctcccg gcgagagcgg tctggaggcc 300
gcctgggagc tcgccgaccc gggggcggac ttcgtcgacc tgaccgtcgt ccggctggcc 360
ctggaggggc agcgccgcga gacggcgcgc gccacccgct ggctggagcc ggacgaccgg 420
gcgctgctgt cgctgtggtg gctggagtgc gcgggggagc tgacccgggg cgaggtggcc 480
gcggcgctgg agctgacgcc gcagcacacg gccgtgcggg tgcagcggat gaaggcgcag 540
ctggagtccg cgcgcgtcgt ggagggggcg ctggcggcgc agccgccgtg cgaggcgctg 600
gccgcggtga cggcctcctg ggacggccag ccgtccactc tgtggcgcaa gcgaatagcc 660
cggcacgccc gcgagtgcct gcggtgcgcc gggctgtgga acggtctgct cccggcggag 720
gggctgctgg cgggcctggc gctcgtcccg gtgtcggcgg cgctgctggc cggggtgcgg 780
tcggccgccg cgggcggttt cgcccccgcg ggcgcggcgt acgcggcggg cgtgcccgca 840
gacggccagg cggtgtccgc cggctgggcg cccgccgacg gccaggcggc atccggcggc 900
tggacgcccg cgtacgaagc accgcccggc ggcacgggcg gcggcggcca cggcttcgcc 960
ggcggtaccg gtacggacgg cggcaacggc ttggcttccg gtgcaagtgc gggtgacggc 1020
ggcagcggct tcggcgccgg tgccgggccg cacggcggcg gcaacggctt cgcttccggt 1080
gcgggtgacg gcggcaacgg cttggcttcc ggtgcaagtg cgggtgacgg cggcagcggc 1140
cccggcgccg gtacgcgcag tggcgacgac ggtttcggcg ccggggcgca cgggggcggc 1200
agcggcttcg gcggcggtga cggcggtccg ggtggcggcg aggccgtggc ccggcttgtg 1260
ccggccggtt ccgcggaggg aggtgccgat ggcgcggcgg gcggcggccg gagcgcgttg 1320
cgcaagcggc ggcgcagtcg gcggcgggcc gtcgggggtg ccgtgctcgc cgcctgcgtc 1380
gcgggcggcg gcctcgccta cctgggcggc ttccccggct ccgccggcga ggacggcgga 1440
gccgccccgg cagctccgct gaacgcgctg tccgcaggcg agtccgccgg cccgtcgccg 1500
agcggcccgg cccagccgtc cgcgtccgct tcggcctcgc cgtccccgtc ggccaccgcg 1560
tcgccctcgg tgtcggcgtc ggcgagccct tccggcgcgg gcaaggcgtc cactggcgcg 1620
ccgaccccgt ccccgtccgc gccgcgcccc gcgtcgccgg ccccggtgcc cgccccgcag 1680
ggaccggtgg gccaggtcgt cgccctcgtc aacgccgagc gggcgaaggc gggctgcggc 1740
ccgctgaagg acgacgcgca gctgcggaag gccgcgcagc ggcactccga ggacatggcg 1800
agccggaact tcttctcgca cacggccccg gacggctccg acccgggcga gcggacgacg 1860
gccgccggct accggtgggc gacgtacggc gagaacatag cgcgcgggca gagcaccgcc 1920
gagtcggtca tgaactcgtg gatgaacagc gacggccacc gcgcgaacat cctgaactgc 1980
tccttcaagg acataggcgt cggactccac cagggccccg gcggcccgtg gtggacgcag 2040
aacttcggcg cccgcatgtg a 2061
<210> 2
<211> 686
<212> PRT
<213> Streptomyces sp
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Met Thr Ser Glu His Thr Ala Glu Leu Val Ala Ala Ala Arg Ala Gly
1 5 10 15
Asp Leu Arg Ala Gln Asp Glu Leu Val Ser Ala Tyr Leu Pro Leu Val
20 25 30
Tyr Asn Ile Val Gly Arg Ala Met Asn Gly Ser Val Asp Val Asp Asp
35 40 45
Val Val Gln Asp Thr Met Leu Arg Ala Leu Asp Gly Leu Gly Thr Leu
50 55 60
Arg Ser Asp Asp Ser Phe Arg Ser Trp Leu Val Ala Ile Ala Met Asn
65 70 75 80
Arg Val Arg Ala Tyr Trp Gln Ala Arg Arg Thr Ala Pro Gly Glu Ser
85 90 95
Gly Leu Glu Ala Ala Trp Glu Leu Ala Asp Pro Gly Ala Asp Phe Val
100 105 110
Asp Leu Thr Val Val Arg Leu Ala Leu Glu Gly Gln Arg Arg Glu Thr
115 120 125
Ala Arg Ala Thr Arg Trp Leu Glu Pro Asp Asp Arg Ala Leu Leu Ser
130 135 140
Leu Trp Trp Leu Glu Cys Ala Gly Glu Leu Thr Arg Gly Glu Val Ala
145 150 155 160
Ala Ala Leu Glu Leu Thr Pro Gln His Thr Ala Val Arg Val Gln Arg
165 170 175
Met Lys Ala Gln Leu Glu Ser Ala Arg Val Val Glu Gly Ala Leu Ala
180 185 190
Ala Gln Pro Pro Cys Glu Ala Leu Ala Ala Val Thr Ala Ser Trp Asp
195 200 205
Gly Gln Pro Ser Thr Leu Trp Arg Lys Arg Ile Ala Arg His Ala Arg
210 215 220
Glu Cys Leu Arg Cys Ala Gly Leu Trp Asn Gly Leu Leu Pro Ala Glu
225 230 235 240
Gly Leu Leu Ala Gly Leu Ala Leu Val Pro Val Ser Ala Ala Leu Leu
245 250 255
Ala Gly Val Arg Ser Ala Ala Ala Gly Gly Phe Ala Pro Ala Gly Ala
260 265 270
Ala Tyr Ala Ala Gly Val Pro Ala Asp Gly Gln Ala Val Ser Ala Gly
275 280 285
Trp Ala Pro Ala Asp Gly Gln Ala Ala Ser Gly Gly Trp Thr Pro Ala
290 295 300
Tyr Glu Ala Pro Pro Gly Gly Thr Gly Gly Gly Gly His Gly Phe Ala
305 310 315 320
Gly Gly Thr Gly Thr Asp Gly Gly Asn Gly Leu Ala Ser Gly Ala Ser
325 330 335
Ala Gly Asp Gly Gly Ser Gly Phe Gly Ala Gly Ala Gly Pro His Gly
340 345 350
Gly Gly Asn Gly Phe Ala Ser Gly Ala Gly Asp Gly Gly Asn Gly Leu
355 360 365
Ala Ser Gly Ala Ser Ala Gly Asp Gly Gly Ser Gly Pro Gly Ala Gly
370 375 380
Thr Arg Ser Gly Asp Asp Gly Phe Gly Ala Gly Ala His Gly Gly Gly
385 390 395 400
Ser Gly Phe Gly Gly Gly Asp Gly Gly Pro Gly Gly Gly Glu Ala Val
405 410 415
Ala Arg Leu Val Pro Ala Gly Ser Ala Glu Gly Gly Ala Asp Gly Ala
420 425 430
Ala Gly Gly Gly Arg Ser Ala Leu Arg Lys Arg Arg Arg Ser Arg Arg
435 440 445
Arg Ala Val Gly Gly Ala Val Leu Ala Ala Cys Val Ala Gly Gly Gly
450 455 460
Leu Ala Tyr Leu Gly Gly Phe Pro Gly Ser Ala Gly Glu Asp Gly Gly
465 470 475 480
Ala Ala Pro Ala Ala Pro Leu Asn Ala Leu Ser Ala Gly Glu Ser Ala
485 490 495
Gly Pro Ser Pro Ser Gly Pro Ala Gln Pro Ser Ala Ser Ala Ser Ala
500 505 510
Ser Pro Ser Pro Ser Ala Thr Ala Ser Pro Ser Val Ser Ala Ser Ala
515 520 525
Ser Pro Ser Gly Ala Gly Lys Ala Ser Thr Gly Ala Pro Thr Pro Ser
530 535 540
Pro Ser Ala Pro Arg Pro Ala Ser Pro Ala Pro Val Pro Ala Pro Gln
545 550 555 560
Gly Pro Val Gly Gln Val Val Ala Leu Val Asn Ala Glu Arg Ala Lys
565 570 575
Ala Gly Cys Gly Pro Leu Lys Asp Asp Ala Gln Leu Arg Lys Ala Ala
580 585 590
Gln Arg His Ser Glu Asp Met Ala Ser Arg Asn Phe Phe Ser His Thr
595 600 605
Ala Pro Asp Gly Ser Asp Pro Gly Glu Arg Thr Thr Ala Ala Gly Tyr
610 615 620
Arg Trp Ala Thr Tyr Gly Glu Asn Ile Ala Arg Gly Gln Ser Thr Ala
625 630 635 640
Glu Ser Val Met Asn Ser Trp Met Asn Ser Asp Gly His Arg Ala Asn
645 650 655
Ile Leu Asn Cys Ser Phe Lys Asp Ile Gly Val Gly Leu His Gln Gly
660 665 670
Pro Gly Gly Pro Trp Trp Thr Gln Asn Phe Gly Ala Arg Met
675 680 685
<210> 3
<211> 1566
<212> DNA
<213> Streptomyces sp
<400> 3
atggggctca tggacgcgga ccacgcgggc ctggtcgtcg cggctcaggc cggggacgac 60
cgggcgcgcg aagagctgat cgccgcctac ctgccgctgg tctacaacat cgtccggcgg 120
gcgctgagcg cacatgccga cgtcgacgac gtcgtccagg agacgctgtt gcgcgtggtg 180
cgcgaccttc ctgccctgcg cgctcccgac agcttccgct cctggctggt gtcgatcacg 240
ctccgccaga tacagaccca ctggcagcgg cagcgcgcgg tcaccaaccg gaccgccgtc 300
atcgacgagg cgctcgacct gccggatgcc ggcttcgaac ccgaggacgc gacgatcctg 360
cgcctccgtg tgtcggacga gcgccgtcgg atcgccgaag ccggccggtg gctcgacccg 420
gaccaccgcg ccctgctgtc cctctggtgg caggagcacg ccgggctgct gacccgggag 480
gacatcgcgg ccgcgacggg cctcaccgcc gcccacgccg gagtgcgcct gcagcgcatg 540
cgcgagcagc tggacctgag ccggacgatc gtcgccgccc tggaggccca cccgcgatgc 600
ccgcagctgg gcgagaccgt cgccggctgg gacggcctgc gtacctcggt gtggcggaag 660
cggatcgcgc ggcacacccg cgactgcccg gtctgcacgg cgacgacggc gaaccgggtc 720
cctgccgagc agctgctgct cggcctcgcg ccgctggccg tccccgccgg actcctcgcc 780
acgctgaccg ccaagggcct gctgtcgggt tcggccgcga gcaccgccgc gcctgccatg 840
gcgccggtcg cggccggcgc ggcggtgaag gcaggcggtc tgcacggcgc ggcgaccggc 900
aaactccacg cggtgaccgc tcacccgctg gcaagcatcg ccgccggtgc ggtgctcatc 960
accggagccg ccacgtacgc ggcctggccg gagccgacgc cccgggcgcc cggcgtcacc 1020
gccgccccca ccgtcgcccc cacggccgcc gttcccgcgc cggtcccgtc gggcaccccc 1080
acgccggccg caccgccgcc ggcgagtccg tccgccgtcc ccgcgggcac ggttccgctg 1140
ggcgcgcaca cactggagtc cgtcgaccag cccgacctct acctgacgta cgccggcgac 1200
ttcgcgacgc tcggccgggc cgccgactcc gacagcacgc aggcacggca gcgcgtcacg 1260
ttcacggtgg tccgggggct ggccgacggg cggtgcgtca ccttccgcgc ggccgacggc 1320
cgttacctgc ggcaccacta cctgcggctg cggctgagca ccgacgacgg cagcgaactc 1380
ttccggaagg acgcgacctt ctgcccccgc cccggagcgg tcgcggggtc ggtgaccctg 1440
tactcccaca actacccggg atcggtcgtc cgccaccgcg acggcggcat ctggctcgac 1500
ggctccgacg gcacccgggc cttcgccggc caggcctcct tcgtcgtccg caaggcccgg 1560
ccctga 1566
<210> 4
<211> 521
<212> PRT
<213> Streptomyces sp
<400> 4
Met Gly Leu Met Asp Ala Asp His Ala Gly Leu Val Val Ala Ala Gln
1 5 10 15
Ala Gly Asp Asp Arg Ala Arg Glu Glu Leu Ile Ala Ala Tyr Leu Pro
20 25 30
Leu Val Tyr Asn Ile Val Arg Arg Ala Leu Ser Ala His Ala Asp Val
35 40 45
Asp Asp Val Val Gln Glu Thr Leu Leu Arg Val Val Arg Asp Leu Pro
50 55 60
Ala Leu Arg Ala Pro Asp Ser Phe Arg Ser Trp Leu Val Ser Ile Thr
65 70 75 80
Leu Arg Gln Ile Gln Thr His Trp Gln Arg Gln Arg Ala Val Thr Asn
85 90 95
Arg Thr Ala Val Ile Asp Glu Ala Leu Asp Leu Pro Asp Ala Gly Phe
100 105 110
Glu Pro Glu Asp Ala Thr Ile Leu Arg Leu Arg Val Ser Asp Glu Arg
115 120 125
Arg Arg Ile Ala Glu Ala Gly Arg Trp Leu Asp Pro Asp His Arg Ala
130 135 140
Leu Leu Ser Leu Trp Trp Gln Glu His Ala Gly Leu Leu Thr Arg Glu
145 150 155 160
Asp Ile Ala Ala Ala Thr Gly Leu Thr Ala Ala His Ala Gly Val Arg
165 170 175
Leu Gln Arg Met Arg Glu Gln Leu Asp Leu Ser Arg Thr Ile Val Ala
180 185 190
Ala Leu Glu Ala His Pro Arg Cys Pro Gln Leu Gly Glu Thr Val Ala
195 200 205
Gly Trp Asp Gly Leu Arg Thr Ser Val Trp Arg Lys Arg Ile Ala Arg
210 215 220
His Thr Arg Asp Cys Pro Val Cys Thr Ala Thr Thr Ala Asn Arg Val
225 230 235 240
Pro Ala Glu Gln Leu Leu Leu Gly Leu Ala Pro Leu Ala Val Pro Ala
245 250 255
Gly Leu Leu Ala Thr Leu Thr Ala Lys Gly Leu Leu Ser Gly Ser Ala
260 265 270
Ala Ser Thr Ala Ala Pro Ala Met Ala Pro Val Ala Ala Gly Ala Ala
275 280 285
Val Lys Ala Gly Gly Leu His Gly Ala Ala Thr Gly Lys Leu His Ala
290 295 300
Val Thr Ala His Pro Leu Ala Ser Ile Ala Ala Gly Ala Val Leu Ile
305 310 315 320
Thr Gly Ala Ala Thr Tyr Ala Ala Trp Pro Glu Pro Thr Pro Arg Ala
325 330 335
Pro Gly Val Thr Ala Ala Pro Thr Val Ala Pro Thr Ala Ala Val Pro
340 345 350
Ala Pro Val Pro Ser Gly Thr Pro Thr Pro Ala Ala Pro Pro Pro Ala
355 360 365
Ser Pro Ser Ala Val Pro Ala Gly Thr Val Pro Leu Gly Ala His Thr
370 375 380
Leu Glu Ser Val Asp Gln Pro Asp Leu Tyr Leu Thr Tyr Ala Gly Asp
385 390 395 400
Phe Ala Thr Leu Gly Arg Ala Ala Asp Ser Asp Ser Thr Gln Ala Arg
405 410 415
Gln Arg Val Thr Phe Thr Val Val Arg Gly Leu Ala Asp Gly Arg Cys
420 425 430
Val Thr Phe Arg Ala Ala Asp Gly Arg Tyr Leu Arg His His Tyr Leu
435 440 445
Arg Leu Arg Leu Ser Thr Asp Asp Gly Ser Glu Leu Phe Arg Lys Asp
450 455 460
Ala Thr Phe Cys Pro Arg Pro Gly Ala Val Ala Gly Ser Val Thr Leu
465 470 475 480
Tyr Ser His Asn Tyr Pro Gly Ser Val Val Arg His Arg Asp Gly Gly
485 490 495
Ile Trp Leu Asp Gly Ser Asp Gly Thr Arg Ala Phe Ala Gly Gln Ala
500 505 510
Ser Phe Val Val Arg Lys Ala Arg Pro
515 520
<210> 5
<211> 2061
<212> DNA
<213> Streptomyces sp
<400> 5
atgacttcag agcacacggc agagctggtc gcggcggccc gcgcgggaga cctccacgcg 60
caggacgagc tggtcagcgc gtatctgccg ctggtctaca acatcgtcgg gcgcgccatg 120
aacggctccg tcgacgtgga cgacgtcgtg caggacacga tgctgcgggc cctcgacggc 180
ctcggcaccc tccgttcgga cgacagcttc cgctcgtggt tggtggccat cgcgatgaac 240
cgggtgcggg cgtactggca ggcccggcgc accgctcccg gtgagagcgg tctggaggcc 300
gcctgggagc tcgccgaccc gggggcggac ttcgtcgacc tgaccgtcgt ccggctggcc 360
ctggaggggc agcgccgcga gacggcgcgc gcgacccgct ggctggagcc ggacgaccgg 420
gcgctgctgt cgctgtggtg gctggagtgc gcgggggagc tgacccgggg cgaggtggcc 480
gcggcgctgg agctgacgcc gcagcacacg gccgtgcggg tgcagcggat gaaggcgcag 540
ctggagtccg cgcgcgtggt ggagggggcg ctggcggcgc agccgccgtg cgaggcgctg 600
gccgcggtga cggcctcctg ggacgggcag ccgtccactc tgtggcgcaa gcgaatagcc 660
cggcacgccc gcgagtgcct gcggtgcgcc gggctgtgga acggtctgct cccggcggag 720
gggctgctgg cgggcctggc gctcgtcccg gtgtcggcgg cgctgctggc cggggtgcgg 780
tcggccgccg cgggcggttt cgcccccgcg ggcgcggcgt acgcggcggg cgtgcccgca 840
gacggccagg cggtgtccgc cggctgggcg cccgccgacg gccaggcggc atccggcggc 900
tggacgcccg cgtacgaagc accgcccggc ggcacgggcg gcggcggcca cggcttcgcc 960
ggcggtaccg gtacggacga cggcaacggc ttggcttccg gtgcaagtgc ggatgacggc 1020
ggcagcggcc tcggcgccgg tgccgggccg cacggcggcg gcaacggctt cgcttccggt 1080
gcgggtgacg gcggcaacgg cttggcttcc ggtgcaagtg cgggtgacgg cggcagcggc 1140
gcgggtgacg gcacgcgcag tggcgacgac ggtttcggcg ccggggcgca cgggggcggc 1200
agcggcttcg gcggcggtga cggcggtccg ggtggcggcg aggccgtggc ccggcttgtg 1260
ccggccggtt ccgcggaggg aggtgccgat ggcgcggcgg gcggcggccg gagcgcgttg 1320
cgcaagcggc ggcgcagtcg gcggcgggcc gtcgggggtg ccgtgctcgc cgcctgcgtc 1380
gcgggcggcg gcctcgccta cctgggcggc ttccccggct ccgccggcga ggacggcgga 1440
gccgccccgg cagctccgct gaacgcgctg tccgcaggcg agtccgccgg cccgtcgccg 1500
agcggcccgg cccagccatc cgcgtccgct tcggcctcgc cgtccccgtc ggccaccgcg 1560
tcgccctcgg tgtcggcgtc ggcgagccct tccggcgcgg gcaaggcgtc cagtggcgcg 1620
ccgaccccgt ccccgtccgc gccgcgcccc gcgtcgccgg ccccggtgcc cgccccgcag 1680
ggaccggtgg gccaggtcgt cgccctcgtc aacgccgagc gggcgaaggc gggctgcggc 1740
ccgctgaagg acgacgcgca gctgcggaag gccgcgcagc ggcactccga ggacatggcg 1800
agccggaact tcttctcgca cacggccccg gacggctccg acccgggcga gcggacgacg 1860
gccgccggct accggtgggc gacgtacggc gagaacatag cgcgcgggca gagcaccgcc 1920
gagtcggtca tgaactcgtg gatgaacagc gacggccacc gcgcgaacat cctgaactgc 1980
tccttcaagg acataggcgt cggactccac cagggccccg gcggcccgtg gtggacgcag 2040
aacttcggcg cccgcatgtg a 2061
<210> 6
<211> 686
<212> PRT
<213> Streptomyces sp
<400> 6
Met Thr Ser Glu His Thr Ala Glu Leu Val Ala Ala Ala Arg Ala Gly
1 5 10 15
Asp Leu His Ala Gln Asp Glu Leu Val Ser Ala Tyr Leu Pro Leu Val
20 25 30
Tyr Asn Ile Val Gly Arg Ala Met Asn Gly Ser Val Asp Val Asp Asp
35 40 45
Val Val Gln Asp Thr Met Leu Arg Ala Leu Asp Gly Leu Gly Thr Leu
50 55 60
Arg Ser Asp Asp Ser Phe Arg Ser Trp Leu Val Ala Ile Ala Met Asn
65 70 75 80
Arg Val Arg Ala Tyr Trp Gln Ala Arg Arg Thr Ala Pro Gly Glu Ser
85 90 95
Gly Leu Glu Ala Ala Trp Glu Leu Ala Asp Pro Gly Ala Asp Phe Val
100 105 110
Asp Leu Thr Val Val Arg Leu Ala Leu Glu Gly Gln Arg Arg Glu Thr
115 120 125
Ala Arg Ala Thr Arg Trp Leu Glu Pro Asp Asp Arg Ala Leu Leu Ser
130 135 140
Leu Trp Trp Leu Glu Cys Ala Gly Glu Leu Thr Arg Gly Glu Val Ala
145 150 155 160
Ala Ala Leu Glu Leu Thr Pro Gln His Thr Ala Val Arg Val Gln Arg
165 170 175
Met Lys Ala Gln Leu Glu Ser Ala Arg Val Val Glu Gly Ala Leu Ala
180 185 190
Ala Gln Pro Pro Cys Glu Ala Leu Ala Ala Val Thr Ala Ser Trp Asp
195 200 205
Gly Gln Pro Ser Thr Leu Trp Arg Lys Arg Ile Ala Arg His Ala Arg
210 215 220
Glu Cys Leu Arg Cys Ala Gly Leu Trp Asn Gly Leu Leu Pro Ala Glu
225 230 235 240
Gly Leu Leu Ala Gly Leu Ala Leu Val Pro Val Ser Ala Ala Leu Leu
245 250 255
Ala Gly Val Arg Ser Ala Ala Ala Gly Gly Phe Ala Pro Ala Gly Ala
260 265 270
Ala Tyr Ala Ala Gly Val Pro Ala Asp Gly Gln Ala Val Ser Ala Gly
275 280 285
Trp Ala Pro Ala Asp Gly Gln Ala Ala Ser Gly Gly Trp Thr Pro Ala
290 295 300
Tyr Glu Ala Pro Pro Gly Gly Thr Gly Gly Gly Gly His Gly Phe Ala
305 310 315 320
Gly Gly Thr Gly Thr Asp Asp Gly Asn Gly Leu Ala Ser Gly Ala Ser
325 330 335
Ala Asp Asp Gly Gly Ser Gly Leu Gly Ala Gly Ala Gly Pro His Gly
340 345 350
Gly Gly Asn Gly Phe Ala Ser Gly Ala Gly Asp Gly Gly Asn Gly Leu
355 360 365
Ala Ser Gly Ala Ser Ala Gly Asp Gly Gly Ser Gly Ala Gly Asp Gly
370 375 380
Thr Arg Ser Gly Asp Asp Gly Phe Gly Ala Gly Ala His Gly Gly Gly
385 390 395 400
Ser Gly Phe Gly Gly Gly Asp Gly Gly Pro Gly Gly Gly Glu Ala Val
405 410 415
Ala Arg Leu Val Pro Ala Gly Ser Ala Glu Gly Gly Ala Asp Gly Ala
420 425 430
Ala Gly Gly Gly Arg Ser Ala Leu Arg Lys Arg Arg Arg Ser Arg Arg
435 440 445
Arg Ala Val Gly Gly Ala Val Leu Ala Ala Cys Val Ala Gly Gly Gly
450 455 460
Leu Ala Tyr Leu Gly Gly Phe Pro Gly Ser Ala Gly Glu Asp Gly Gly
465 470 475 480
Ala Ala Pro Ala Ala Pro Leu Asn Ala Leu Ser Ala Gly Glu Ser Ala
485 490 495
Gly Pro Ser Pro Ser Gly Pro Ala Gln Pro Ser Ala Ser Ala Ser Ala
500 505 510
Ser Pro Ser Pro Ser Ala Thr Ala Ser Pro Ser Val Ser Ala Ser Ala
515 520 525
Ser Pro Ser Gly Ala Gly Lys Ala Ser Ser Gly Ala Pro Thr Pro Ser
530 535 540
Pro Ser Ala Pro Arg Pro Ala Ser Pro Ala Pro Val Pro Ala Pro Gln
545 550 555 560
Gly Pro Val Gly Gln Val Val Ala Leu Val Asn Ala Glu Arg Ala Lys
565 570 575
Ala Gly Cys Gly Pro Leu Lys Asp Asp Ala Gln Leu Arg Lys Ala Ala
580 585 590
Gln Arg His Ser Glu Asp Met Ala Ser Arg Asn Phe Phe Ser His Thr
595 600 605
Ala Pro Asp Gly Ser Asp Pro Gly Glu Arg Thr Thr Ala Ala Gly Tyr
610 615 620
Arg Trp Ala Thr Tyr Gly Glu Asn Ile Ala Arg Gly Gln Ser Thr Ala
625 630 635 640
Glu Ser Val Met Asn Ser Trp Met Asn Ser Asp Gly His Arg Ala Asn
645 650 655
Ile Leu Asn Cys Ser Phe Lys Asp Ile Gly Val Gly Leu His Gln Gly
660 665 670
Pro Gly Gly Pro Trp Trp Thr Gln Asn Phe Gly Ala Arg Met
675 680 685
<210> 7
<211> 1566
<212> DNA
<213> Streptomyces sp
<400> 7
atggggctca tggacgcgga ccacgcgggc ctggtcgtcg cggctcaggc cggggacgac 60
cgggcgcgcg aagagctgat cgccgcctac ctgccgctgg tctacaacat cgtccggcgg 120
gcgctgagcg cacatgccga cgtcgacgac gtcgtccagg agacgctgtt gcgcgtggtg 180
cgcgaccttc ctgccctgcg cgctcccgac agcttccgct cctggctggt gtcgatcacg 240
ctccgccaga tacagaccca ctggcagcgg cagcgcgcgg tcgccaaccg gaccgccgtc 300
atcgacgagg cgctcgacct gccggatgcc ggcttcgaac ccgaggacgc gacgatcctg 360
cgcctccgtg tgtcggacga gcgccgtcgg atcgccgaag ccggccggtg gctcgacccg 420
gaccaccgcg ccctgctgtc cctctggtgg caggagcacg ccgggctgct gacccgggag 480
gacatcgcgg ccgcgacggg cctcaccgcc gcccacgccg gagtgcgcct gcagcgcatg 540
cgcgagcagc tggacctgag ccggacgatc gtcgccgccc tggaggccca cccgcgatgc 600
ccgcagctgg gcgagaccgt cgccggctgg gacggcctgc gtacctcggt gtggcggaag 660
cggatcgcgc ggcacacccg cgactgcccg gtctgcacgg cgacgacggc gaaccgggtc 720
cctgccgagc agctgctgct cggcctcgcg ccgctggccg tccccgccgg actcctcgcc 780
acgctgaccg ccaagggcct gctgtcgggt tcggccgcgg gcgccgccgc gcctgccatg 840
gcgccggtcg cggccggcgc ggcggtgaag gcaggcggtc tgcacggcgc ggcgaccggc 900
aaactccacg cggtgaccgc tcacccgctg gcaagcatcg ccgccggtgc ggtgctcatc 960
accggagccg cgacgtacgc agcctggccg gagccgacgc cccgggcgcc cggcgtcacc 1020
gccgccccca ccgtcgcgcc cacggccgcc gctcccgcgc cggtcccgtc gggcaccccc 1080
acgccggccg caccgccacc ggcgagtccg tccgccgtcc ccgcgggcac ggttccgctg 1140
ggcgcgcaca cactggagtc cgtcgaccag cccgacctct acctgacgta cgccggcgac 1200
ttcgcgacgc tcggccgggc cgccgactcc ggcagcacgc aggcacggca gcgcgtcacg 1260
ttcacggtgg tccgggggct ggccgacggg cggtgcgtca ccttccgcgc ggccgacggc 1320
cgttacctgc ggcaccacta cctgcggctg cggctgagca ccgacgacgg cagcgaactc 1380
ttccggaagg acgcgacctt ctgcccccgc cccggagcgg tcgcggggtc ggtgaccctg 1440
tactcccaca actacccggg atcggtcgtc cgccaccgcg acggcggcat ctggctcgac 1500
ggctccgacg gcacccgggc cttcgccggc caggcctcct tcgtcgtccg caaggcccgg 1560
ccctga 1566
<210> 8
<211> 521
<212> PRT
<213> Streptomyces sp
<400> 8
Met Gly Leu Met Asp Ala Asp His Ala Gly Leu Val Val Ala Ala Gln
1 5 10 15
Ala Gly Asp Asp Arg Ala Arg Glu Glu Leu Ile Ala Ala Tyr Leu Pro
20 25 30
Leu Val Tyr Asn Ile Val Arg Arg Ala Leu Ser Ala His Ala Asp Val
35 40 45
Asp Asp Val Val Gln Glu Thr Leu Leu Arg Val Val Arg Asp Leu Pro
50 55 60
Ala Leu Arg Ala Pro Asp Ser Phe Arg Ser Trp Leu Val Ser Ile Thr
65 70 75 80
Leu Arg Gln Ile Gln Thr His Trp Gln Arg Gln Arg Ala Val Ala Asn
85 90 95
Arg Thr Ala Val Ile Asp Glu Ala Leu Asp Leu Pro Asp Ala Gly Phe
100 105 110
Glu Pro Glu Asp Ala Thr Ile Leu Arg Leu Arg Val Ser Asp Glu Arg
115 120 125
Arg Arg Ile Ala Glu Ala Gly Arg Trp Leu Asp Pro Asp His Arg Ala
130 135 140
Leu Leu Ser Leu Trp Trp Gln Glu His Ala Gly Leu Leu Thr Arg Glu
145 150 155 160
Asp Ile Ala Ala Ala Thr Gly Leu Thr Ala Ala His Ala Gly Val Arg
165 170 175
Leu Gln Arg Met Arg Glu Gln Leu Asp Leu Ser Arg Thr Ile Val Ala
180 185 190
Ala Leu Glu Ala His Pro Arg Cys Pro Gln Leu Gly Glu Thr Val Ala
195 200 205
Gly Trp Asp Gly Leu Arg Thr Ser Val Trp Arg Lys Arg Ile Ala Arg
210 215 220
His Thr Arg Asp Cys Pro Val Cys Thr Ala Thr Thr Ala Asn Arg Val
225 230 235 240
Pro Ala Glu Gln Leu Leu Leu Gly Leu Ala Pro Leu Ala Val Pro Ala
245 250 255
Gly Leu Leu Ala Thr Leu Thr Ala Lys Gly Leu Leu Ser Gly Ser Ala
260 265 270
Ala Gly Ala Ala Ala Pro Ala Met Ala Pro Val Ala Ala Gly Ala Ala
275 280 285
Val Lys Ala Gly Gly Leu His Gly Ala Ala Thr Gly Lys Leu His Ala
290 295 300
Val Thr Ala His Pro Leu Ala Ser Ile Ala Ala Gly Ala Val Leu Ile
305 310 315 320
Thr Gly Ala Ala Thr Tyr Ala Ala Trp Pro Glu Pro Thr Pro Arg Ala
325 330 335
Pro Gly Val Thr Ala Ala Pro Thr Val Ala Pro Thr Ala Ala Ala Pro
340 345 350
Ala Pro Val Pro Ser Gly Thr Pro Thr Pro Ala Ala Pro Pro Pro Ala
355 360 365
Ser Pro Ser Ala Val Pro Ala Gly Thr Val Pro Leu Gly Ala His Thr
370 375 380
Leu Glu Ser Val Asp Gln Pro Asp Leu Tyr Leu Thr Tyr Ala Gly Asp
385 390 395 400
Phe Ala Thr Leu Gly Arg Ala Ala Asp Ser Gly Ser Thr Gln Ala Arg
405 410 415
Gln Arg Val Thr Phe Thr Val Val Arg Gly Leu Ala Asp Gly Arg Cys
420 425 430
Val Thr Phe Arg Ala Ala Asp Gly Arg Tyr Leu Arg His His Tyr Leu
435 440 445
Arg Leu Arg Leu Ser Thr Asp Asp Gly Ser Glu Leu Phe Arg Lys Asp
450 455 460
Ala Thr Phe Cys Pro Arg Pro Gly Ala Val Ala Gly Ser Val Thr Leu
465 470 475 480
Tyr Ser His Asn Tyr Pro Gly Ser Val Val Arg His Arg Asp Gly Gly
485 490 495
Ile Trp Leu Asp Gly Ser Asp Gly Thr Arg Ala Phe Ala Gly Gln Ala
500 505 510
Ser Phe Val Val Arg Lys Ala Arg Pro
515 520
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gctctagaat gacttcagag cacacggc 28
<210> 10
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gcgatatctc acatgcgggc gccgaagt 28
<210> 11
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gctctagaat ggggctcatg gacgcgga 28
<210> 12
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gcgatatctc agggccgggc cttgcgga 28

Claims (6)

1. The application of the ribosome sigma factor B in improving the yield of lipstatin is characterized in that the nucleotide sequence of the ribosome sigma factor B is shown as SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 8.
2. A mutant of a ribosome sigma factor B, which is characterized in that the nucleotide sequence of the mutant is shown as SEQ ID NO. 3; the amino acid sequence of the protein coded by the nucleotide is shown as SEQ ID NO. 4.
3. Use of a mutant of ribosomal sigma factor B according to claim 2 for increasing the yield of lipstatin.
4. A plasmid comprising the nucleotide sequence of the mutant ribosomal sigma factor B of claim 2.
5. A transgenic cell line comprising the nucleotide sequence of the mutant ribosomal sigma factor B of claim 2.
6. An engineered bacterium comprising the nucleotide sequence of the mutant of ribosomal sigma factor B of claim 2.
CN202111260753.2A 2020-10-29 2020-10-29 Application of ribosome sigma factor B and mutant thereof in improving yield of lipstatin Active CN113789314B (en)

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Family Cites Families (8)

* Cited by examiner, † Cited by third party
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US6238887B1 (en) * 1997-10-03 2001-05-29 Brigham & Women's Hospital Ribosome recycling factor (FRR) of Staphylococcus aureus
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