Application of ribosome sigma factor B and mutant thereof in increasing yield of lipstatin

文档序号：1961368 发布日期：2021-12-14 浏览：14次中文

阅读说明：本技术 一种核糖体σ因子B及其突变体在提升利普司他汀产量中的应用 (Application of ribosome sigma factor B and mutant thereof in increasing yield of lipstatin ) 是由黄胜雄颜一军刘兴勇马小燕于 2020-10-29 设计创作，主要内容包括：本发明公开了一种核糖体σ因子B及其突变体在提升利普司他汀产量中的应用。该核糖体σ因子B的核苷酸序列如SEQ ID NO.7所示,其编码的蛋白质的氨基酸序列如SEQ ID NO.8所示。本申请筛选得到的核糖体σ因子B及其突变体过表达均能提高利普司他汀的产量。(The invention discloses application of ribosome sigma factor B and 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 obtained by screening and the overexpression of the mutant thereof can improve the yield of lipstatin.)

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 ribosomal sigma factor B as defined in claim 1, characterized in that the nucleotide sequence of said mutant is a nucleotide sequence represented by SEQ ID No.6, substituted, deleted and/or added with one or more nucleotides and encoding the same functional protein, the specific sequence being represented by 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 ribosomal sigma factor B of claim 1 or the nucleotide sequence of a mutant of ribosomal sigma factor B of claim 2.

5. A transgenic cell line comprising the nucleotide sequence of ribosomal sigma factor B of claim 1 or the nucleotide sequence of a mutant of ribosomal sigma factor B of claim 2.

6. An engineered bacterium comprising the nucleotide sequence of ribosomal sigma factor B of claim 1 or the nucleotide sequence of a mutant of ribosomal sigma factor B of claim 2.

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to application of ribosome sigma factors and mutants thereof in improving the yield of lipstatin.

Background

The lipstatin tetrahydro derivative orlistat is used as an obesity treatment drug which does not suppress appetite and does not act on the central nervous system and is currently certified by the US FDA, the European Union EMA and the Chinese SFDA, is taken by more than 4 million people all over the world and successfully reduces weight, and is the most popular weight-reducing product at present. As a long-acting specific pancreatic lipase inhibitor, it can prevent the hydrolysis of triglyceride into free fatty acid and monoacylglycerol which can be absorbed by small intestine mucosa, thereby reducing calorie intake and controlling body weight. Lipstatin was originally isolated from Streptomyces toxytricini and has a beta-propiolactone structural unit, which is substituted at the 2-and 3-positions with a 6-and 13-carbon alkyl hydrocarbon chain, respectively, and the hydroxyl group at the C5 position of the 13-carbon alkyl hydrocarbon chain forms an ester bond with N-formyl-L-leucine. Because the lipstatin molecule structure is complex, the chemical synthesis process is complex, and a large amount of manpower and material resources are consumed. At present, the lipstatin is mainly produced by adopting a semi-biological fermentation process, and compared with a chemical method, the use of toxic solvents is reduced, so that the pollution to the environment and the toxic substance residue are greatly reduced.

At present, the research on the synthesis of lipstatin from lipstatin producing bacteria at home and abroad mainly adopts a method combining a single-factor test and an orthogonal design test to optimize a fermentation medium and fermentation parameters, such as the influence of biotin and ATP addition on the synthesis of lipid and lipstatin (Donghuijun and the like, the influence of biotin and ATP addition on the synthesis of lipid and lipstatin of Streptomyces toxytriacus, J.Med.Med.C., 2014, No. 01, pages 19-24).

The ribosomal sigma transcription factor is an important component of RNA polymerase, and plays an important role in the transcription process of prokaryotes. Overexpression of frr, for example in Streptomyces avermitilis, increases the production of avermectins (Li L et al, (2010) Over expression of ribosomal transduction genes involved in production of avermectin in Streptomyces avermitilis strain J Ind Microbiol Biotechnol 37(7):673 and 679), but currently ribosomal sigma transcription factors affecting the synthesis of lipstatin are not reported.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a ribosome sigma factor, a mutant thereof and application of a protein obtained by coding in the improvement of the yield of lipstatin.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

an application of ribosome sigma factor in improving the yield of lipstatin is ribosome sigma factor A or ribosome sigma factor B.

Furthermore, the nucleotide sequence of ribosome sigma factor A is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 6.

Furthermore, the nucleotide sequence of ribosome sigma factor B is shown in SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 8.

A mutant of ribosome sigma factor A as described above, the nucleotide sequence of the mutant is shown in SEQ ID NO. 1.

Further, the amino acid sequence of the nucleotide-encoded protein is shown in SEQ ID No.2, in which amino acid at position 19 is replaced with R, amino acid at position 327 is replaced with G, amino acid at position 338 is replaced with G, amino acid at position 344 is replaced with F, amino acid at position 347 is replaced with S, and amino acid at position 538 is replaced with T, as compared to the normal ribosomal sigma factor a amino acid sequence.

A mutant of ribosomal sigma factor B as described above, the nucleotide sequence of which is shown in SEQ ID No. 3.

Further, the amino acid sequence of the nucleotide-encoded protein is shown in SEQ ID No.4, in which amino acid at position 95 is replaced with T, amino acid at position 274 is replaced with S, amino acid at position 275 is replaced with T, amino acid at position 351 is replaced with V, and amino acid at position 411 is replaced with D, as compared with the normal ribosomal sigma factor B amino acid sequence.

The mutant of the ribosome sigma factor A or the protein coded by the mutant is applied to the improvement of the yield of lipstatin.

The mutant of the ribosome sigma factor B or the protein coded by the mutant can be applied to the improvement of the yield of lipstatin.

A plasmid comprising the ribosomal sigma factor, a mutant of the ribosomal sigma factor A, or a mutant of the ribosomal sigma factor B.

A transgenic cell line comprising the ribosomal sigma factor, a mutant of ribosomal sigma factor a, or a mutant of ribosomal sigma factor B.

An engineering bacterium comprising the ribosome sigma factor, a mutant of the ribosome sigma factor A, or a mutant of the ribosome sigma factor B.

Further, the expression vector containing the coding gene is introduced into Streptomyces toxytricini for overexpression.

Further, the gene or mutant gene is located in a plasmid or chromosome.

The invention has the beneficial effects that:

screening research on 10 ribosome factors shows that ribosome sigma factors A and B and mutant genes thereof are over-expressed in Streptomyces toxytricini, so that the genetic engineering bacteria are biologically safe (the over-expression in the bacteria does not influence the growth of the bacteria), and the capacity of producing lipstatin by Streptomyces toxytricini can be effectively improved. The experimental data indicate the ability to increase lipstatin production, among others, by overexpression of the original gene in Streptomyces toxytricini, and especially their mutant genes.

Drawings

FIG. 1 shows a map of a plasmid vector pKC1139-liprA,

FIG. 2 shows a map of a plasmid vector pKC1139-liprB,

FIG. 3 is a map of plasmid vector pKC1139-liprA-mut,

FIG. 4 is a map of plasmid vector pKC1139-liprB-mut,

Fig. 5 is a standard curve of lipstatin concentration.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the 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 it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

1. Material

Streptomyces toxytricini, a typical Streptomyces species, has a branched intrabasal hyphae, a slightly thicker aerial hyphae, and a part of the aerial hyphae differentiated into spiral hyphae, and has characteristics typical of Streptomyces.

The formula of the culture medium is as follows:

seed culture medium: 10g of soya bean flow, 5g of Bacto soytone, 5mL of glycerol, 10mL of soya oil, and 2mL of Triton X-100[ pH 6.5] perlite.

Fermentation culture: 30g of soya bean flow, 14mL of glycerol, 1g of Bacto soytone, 1mL of Triton X-100, and 60mL of soya oil [ pH 7.0] perliter.

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 mixed in a vortex mixer, sonicated for 30min, centrifuged (12000rpm, 5min), filtered through a 0.22 μm filter, and the filtrate (20 μ L) was used for HPLC analysis.

(2) Preparation of standards

Preparing lipstatin standard substance (0.125g/L,0.25g/L,0.5g/L,1g/L,2g/L)

(3) HPLC detection conditions

A chromatographic column: YMC-Pack ODS-A; detection wavelength: 205 nm; mobile phase: acetonitrile: water 90: 10; flow rate: 1.0 mL/min; column temperature: at 40 ℃.

(4) Drawing of lipstatin standard curve

And (4) carrying out HPLC detection on the standard substances with different concentrations according to the conditions, and drawing a peak area lipstatin concentration standard curve. And (4) taking the measured peak area A as a vertical coordinate, taking the mass concentration C (g/L) of the lipstatin as a horizontal coordinate, and drawing a lipstatin standard curve. See fig. 5, resulting in regression equation y-5E +6x +101960, R²The absorbance has a good linear relationship with mass concentration at 0.996. After the liquid phase is combined, the sample yield is calculated according to the lipstatin standard curve

Example 1 obtaining of the Strain

The frozen strains are diluted and spread on an ISP3 plate culture medium, placed in a hydration incubator at 30 ℃ for inverted culture, and after bacterial colonies grow out, single bacterial colonies are picked out for re-culture and transferred to 2 generations. Taking a fresh plate, washing surface spores with 10mL sterile water, placing the plate in a triangular flask filled with glass beads, performing shaking culture at 28 ℃ and 200r/min for 2-3 h, filtering, and performing serial dilution to obtain spores with the concentration of about 1 × 10⁷Single spore suspension per mL is ready for use.

Example 2 genomic analysis of the strains

The strain Streptomyces toxytricini is sent to Huahua Dageney company for whole genome sequencing, ribosome factors of the strain are compared and analyzed through genome sequencing and bioinformatics analysis, 15 different ribosome factors are found, and the yield of lipstatin can be improved by over-expressing ribosome sigma transcription factors A and B through screening.

Based on the ribosome sigma transcription factor A and B obtained by screening, respectively obtaining mutant genes of the ribosome sigma factor A and mutant genes of the ribosome sigma factor B by nucleotide substitution, wherein the nucleotide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 3;

simultaneously obtaining corresponding amino acid mutants, which specifically comprise:

mutant of ribosomal sigma factor a: the ribosome sigma transcription factor A encodes an amino acid sequence in which the 19 th amino acid 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 th amino acid is replaced by S, and the 538 th amino acid is replaced by T.

Mutant of ribosomal sigma factor B: the ribosome sigma transcription factor B encodes an amino acid sequence in which the 95 th amino acid 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.

And respectively verifying the influence of the over-expression of the substituted mutant in the strain on the yield of lipstatin.

EXAMPLE 3 construction of plasmid

1. Construction of pKC1139-liprA

Respectively utilizing the primer pairs of liprA-XbaI-F and liprA-EcoRV-R in the table 1, taking Streptomyces toxytricini genome as a template, carrying out PCR amplification, introducing XbaI and EcoRV enzyme cutting sites to obtain a liprA fragment, carrying out electrophoresis verification, carrying out enzymatic treatment by DpnI, and recycling electrophoresis gel to obtain a purified liprA fragment. The gene sequence of the liprA is shown in SEQ ID No.5, and the coded amino acid sequence is shown in SEQ ID No. 6.

The above purified liprA fragment and pKC1139 plasmid were double digested with XbaI and EcoRV, respectively, and the double digested products of the liprA fragment and pKC1139 plasmid were ligated by T4 ligase overnight at 4 ℃. The ligation products are transformed into Escherichia coli DH5 alpha, spread on LB solid plate containing 50mg/L apramycin, cultured for 16h, colony PCR detection is carried out, Jinzhi sequencing is carried out, and after the sequencing is correct, the obtained positive bacteria are named as E. Plasmid pKC1139-liprA was extracted with the 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 as a template to obtain a fragment of liprA-mut, and constructing the plasmid pKC1139-liprA-mut by using the same primers according to the construction process of pKC 1139-liprA. The plasmid map is shown in FIG. 3.

3. Construction of pKC 1139-liprB:

respectively utilizing the primer pairs liprB-XbaI-F and liprB-EcoRV-R in the table 1, taking Streptomyces toxytricini genome as a template, carrying out PCR amplification, introducing XbaI and EcoRV enzyme cutting sites to obtain a liprB fragment, carrying out electrophoresis verification, carrying out enzymatic treatment by DpnI, and recycling electrophoresis gel to obtain a purified liprB fragment. The liprB gene sequence is shown in SEQ ID No.7, and the coded amino acid sequence is shown in SEQ ID No. 8.

The above purified liprB fragment and pKC1139 plasmid were double digested with XbaI and EcoRV, respectively, and the double digested products of the liprB fragment and pKC1139 plasmid were ligated with T4 ligase overnight at 4 ℃. The ligation products are transformed into Escherichia coli DH5 alpha, spread on LB solid plate containing 50mg/L apramycin, cultured for 16h, colony PCR detection is carried out, Jinzhi sequencing is carried out, and after the sequencing is correct, the obtained positive bacteria are named as E. Plasmid pKC1139-liprB was extracted with the plasmid kit for use, and the plasmid map is shown in FIG. 2.

4、pKC1139-liprB-mut:

The nucleotide sequence is shown as SEQ ID NO: 3 as a template, and constructing the plasmid pKC1139-liprB-mut by using the same primers according to the construction process of pKC 1139-liprB. The plasmid map is shown in FIG. 4.

TABLE 1 primers

EXAMPLE 4 construction of plasmid vector-containing Strain

1. Preparation of E.coli ET12567/pUZ8002 containing the exogenous plasmid

And (3) transforming the constructed plasmid into escherichia coli ET12567/pUZ8002 to obtain a donor bacterium containing the recombinant plasmid. The donor bacteria are inoculated into 5mL LB culture medium (containing 25 mug/mL Km, Cm and 50 mug/mL Am), cultured overnight at 37 ℃, and transferred to 10mL LB culture medium according to the ratio of 1: 50-100In medium (containing 25. mu.g/mL Km, Cm and 50. mu.g/mL Am), incubated at 37 ℃ to OD₆₀₀The cells were cultured for about 3 hours under the condition of 0.3 to 0.4, centrifuged, 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 is cultured for about 7 days, 2 plates are taken, spores are washed by 5mL of 10% glycerol, the washed spores are placed into a 50mL test tube, absorbent cotton is filtered in a clean test tube after shaking and mixing, centrifugation is carried out for 10min at 3500rpm, 1mL of 2 XYT is added, and thermal activation is carried out for 10min at 50 ℃.

3. Joint transfer of Escherichia coli ET12567/pUZ8002 and Streptomyces toxytricini strain and isolation and identification of the strain

Respectively taking 0.5mL of equivalent streptomycete spore liquid and escherichia coli liquid, uniformly mixing and coating the mixture containing 10mM MgCl₂The conjugative transfer medium (IWL4 or MS and R2YE) of (1), culturing at 28 ℃ overnight, covering with sterile water containing Am (50. mu.g/mL) and Tri (100. mu.g/mL), culturing at 28 ℃ for 7-10 days, subculturing grown positive colonies in IWL4 or MS and R2YE medium containing Am (50. mu.g/mL) and Tri (100. mu.g/mL), and picking corresponding colonies. SK/pKC1139 (control), SK/pKC1139-liprA (abbreviated as SK1), SK/pKC1139-liprA-mut (abbreviated as SK2), SK/pKC1139-liprB (abbreviated as SK3), and SK/pKC1139-liprB-mut (abbreviated as SK 4).

Example 5 evaluation of different strains

Control bacteria were subjected to shake flask fermentations with SK1, SK2, SK3, SK4, each in 3 replicates, followed by lipstatin determinations and titers, with the results shown in table 2. According to the detection results in table 2, the experimental group bacteria are found to have improved ability to produce lipstatin compared with the control bacteria, so that the over-expression of the ribosome sigma factors A and B and the mutant genes thereof can improve the yield of lipstatin.

TABLE 2 lipstatin production by different strains

Bacterial strains	Lipstatin yield (g/L)
		Control bacterium	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> Sichuan university of light chemical industry

<120> application of ribosome sigma factor B and mutant thereof in improving yield of lipstatin

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2061

<212> DNA

<213> Streptomyces sp

<400> 1

atgacttcag agcacacggc agagctggtc gcggcggccc gcgcgggaga cctccgcgcg 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 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

<400> 2

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