• Journal of Microbiology
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• 제38권3호
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• pp.160-168
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• 2000

A-factor I a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. to identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by Streptomyces griseus IFO 13350 and its A-factor deficient mutant strain, Streptomyces griseus HH1, as well as Streptomyces griseus HH1 transformed with the afsA gene were sturdied. In general Streptomyces griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of Streptomyces griseus IFO 13350 was greatly enhanced more than twice compared with that of Streptomyces griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of Streptomyces griseus HH1 was greatly enhanced more than twice compared with that of Streptomyces griseus IFO 13350, and this observation was reversed in the presence of thiostreptione, However, Streptomyces griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between Streptomyces griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-amionpeptidase activity was 2 times higher in Streptomyces griseus HH1 than in strain IFO 13350 . Streptomyces griseus HH1 harboring afsA showed a similar level of enzyme activity , however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morpholofical differentiation, the formation of aerial meycelium and spores was delayed by two or three days.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1350-1355
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• 2004

The sprA and sprB genes, encoding the chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB), and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from S. griseus and were overexpressed in various strains of S. griseus. When the sprT gene was introduced into S. griseus, trypsin activity increased 2-fold in the A-factor deficient mutant strain, S. griseus HH1, and increased 4-fold in the wild strain, S. grise us IFO 13350. However, there was no detectable increase of chymotrypsin activity in the transformants of S. griseus with either sprA or sprB, in contrast to the results obtained from S. lividans as a heterologous host. To solve the negative gene dosage effects in S. griseus, either the sprA or the sprB genes with their own ribosome binding sites were linked to the downstream of the entire sprT gene, and the coexpression efficiency was examined in S. lividans and S. griseus. The transformants of S. lividans with either pWHM3-TA (sprT+sprA) or pWHM3­TB (sprT+sprB) showed 3-fold increase of trypsin activity over that of the control, however, only the transformant of pWHM3-TB demonstrated 7-fold increase in chymotrypsin activity, indicating that the pWHM3-TB has a successful construction for the overexpression of chymotrypsin in Streptomyces. When the coexpression vectors were introduced into S. griseus IFO 13350, the trypsin level sharply increased by more than 4-fold, however, the chymotrypsin level did not increase. These results strongly suggest that the overexpression of the sprA and sprB genes is tightly regulated in S. griseus.

• 한국식품영양학회지
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• 제15권4호
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• pp.364-369
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• 2002

방선균은 토양 속에 다양하게 존재하는 미생물의 일종으로 그람 양성 진정세균으로 이차대사산물을 생산하는 시기와 포자 착생이 시작되는 세포분화의 시기가 밀접한 관련이 있다. S. griseus는 streptomycin을 비롯한 다양한 종류의 endopeptidase 및 exopeptidase들을 생산한다. 방선균에서의 protease 생산은 많은 경우에 이차대사산물이 형성되거나 형태분화가 유도되는 시기에 동시에 시작된다는 점에서 Pretense가 이차대사물질 생산 및 세포분화에 일정한 기능을 수행할 것이 라는 점을 시사하고 있다. 본 연구에서는 S. griseus IFO 13350에서 클로닝한 SGPD protease가 각 strain에서 형태학적으로나 생리적으로 어떠한 gene dosage 효과를 미치는지 조사하는 것이었다. sprD 유전자가 S.lividans를 숙주로 사용한 시스템에서 대량발현이 성공적으로 되는 것을 확인한 후, 본 유전자를 클로닝한 S. griseus IFO13350 균주와 이의 A-factor 결손주인 S. griseus HH1에 형질전환하였다. S. griseus HH1과 S. griseus IFO13350에서는 protease activity가 벡터만 도입된 대조군과 sprD 유전자가 들어간 형질전환체에서 큰 차이를 보이지 않았다. 또한 S. griseus IFO 13350 및 HH1 모두에서 생리학적·형태학적 분화의 차이를 발견하지 못하였다. Chymotrypsin계열의 pretense를 암호화하는 유전자만이 S. griseus에서 발현이 repression된다는 사실을 본 연구 결과를 통하여 알게 되었다. 이를 바탕으로 sprD유전자와 동일계열의 chymotrypsin 계열의 유전자들이 공통적으로 S. griseus에서 repression 되는 일반적인 기전이 있을 것으로 판단, chymotrypsin계열 유전자들의 promoter부분의 염기 상동성을 조사하였다 번역개시부위 바로 상부 유전자부터 상동성을 조사한 결과 적어도 상당부분의 염기배열이 잘 보존된 지역이 존재함을 알게 되었다. 향후 이들 발현기구의 조절기구를 연구함으로서 protease의 기능을 밝히는데 좋은 단서를 제공할 것으로 판단된다.

• 미생물학회지
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• 제41권1호
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• pp.87-92
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• 2005

방선균 Streptomyces griseus에서 상업적 목적으로 생산되는 protease인 protease는 serine protease, alkaline protease, aminopeptidase및 carboxypeptidase로 구성되어 있는 복합체로서 의 약용 소염제로 널리 사용되어지고 있다. 본 연구에서는 기존에 개발되어 있는 방선균용 integration vector인 pSET152로부터 목적산물의 대량발현을 위해 방선균용 promoter ermE가 cloning된 새로운 integration vector인 pHJ101을 개발하였고, pretense의 생산량 증대에 사용하였다. 새로 개발된 integration vector에 S. griseus protease A를 코드하고 있는 유전자, sprA와 S. griseus pretense B유전자, sprB를 각각 cloning하여 plasmid pHJ201과 pHJ202를 구축하였다. 이들 plasmid들을 S. griseus IFO 13350에 형질전환하여 발현용plasmid가 chromosome에 integration된 재조합 균주 S. gliseus HA와 S. griseus HB를 얻었다. 이들 재조합균주로부터 전체 protease의 생산량을 확인한 결과, 모균주보다 각각 S. griseus HA는 약 5.3 배, S. griseus HB는 약 5 배 정도 생산량이 증대되었다. 이들 결과로부터 특정유전자의 고발현용 integration vector의 제작이 확인되었으며, 전체 protease의 생산량 증대의 가능성이 시사되었다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.81-88
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• 2007

The sprC gene encodes Streptomyces griseus protease C (SGPC), a bacterial chymotrypsin-like serine protease. Because the published data on sprC was not complete, we cloned and analyzed a new DNA fragment spanning downstream to upstream of the sprC gene from S. griseus IFO13350. The cloned 2.3-kb DNA fragment was placed on a high-copy number plasmid and introduced into Streptomyces lividans TK24. Chymotrypsin activity of the transformant was 8.5 times higher than that of the control after 3 days of cultivation and stably maintained until 9 days of cultivation, which dearly indicated that the cloned 2.3-kb fragment contained the entire sprC gene with its own promoter. When the same construct was introduced in the S. griseus IFO13350 (wild strain) and its two mutant strains in the A-factor regulatory cascade, ${\Delta}adpA$ and HO1, the chymotrypsin activity increased fivefold only in the ${\Delta}adpA$ strain. Transcriptional analysis based on RT-PCR revealed that the sprC gene is normally transcribed in both strains; however, earlier transcription was observed in the wild strain compared with the ${\Delta}adpA$ strain. A gel mobility shift assay showed that the AdpA protein did not bind to the promoter region of sprC. All these data clearly indicate that the expression of sprC is not dependent on the AdpA protein, but is distinctly regulated from other chymotrypsin genes composing an AdpA regulon. Earlier morphological differentiation was observed in S. lividans TK24, and S. griseus IFO13350 and HO1, transformed with the expression vector. The transformant of S. griseus ${\Delta}adpA$ formed markedly larger colonies. Antisense repression of sprC resulted in severe decrease of chymotrypsin activity, down to one-third of the control, and delayed morphological differentiation. All these data suggest that SGPC is related to normal morphogenesis in S. griseus.

• Journal of Microbiology and Biotechnology
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• 제21권7호
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• pp.675-678
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• 2011

DNA methylation in Streptomyces griseus IFO 13350 was analyzed by high-performance liquid chromatographic analysis and bisulfite-based analysis to reveal two methylation sites, 5'-$GC^{5m}$ CGGC-3' and 5'-$GAG^{5m}$ CTC-3'. The methylation was reconstituted in Escherichia coli by simultaneous expression of S. griseus SGR4675 and S. achromogenes M.SacI. The E. coli cells produced plasmids that mimicked the methylation profile of S. griseus DNA, which was readily introduced into S. griseus. The results of this study raise the possibility of a promising approach to establish efficient transformation in several streptomycetes.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• 한국미생물·생명공학회지
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• 제36권1호
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• pp.28-33
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• 2008

Streptomyces griseus trypsin (SGT)을 코드하는 sprT 유전자와 그 하류에 존재하는 두 개의 조절 유전자 rsgtR1 및 sgtR2를 동시에 갖고 있는 재조합 벡터 pWHM3-TR1R2를 S. lividans TK24 및 S. griseus IFO 13350에 도입하여, 트립신의 생산성을 더욱 증대시킬 수 있는 배지를 조사하였다. S. lividans TK24/pWHM3-TR1R2의 경우 배양 5일을 기준으로 R2YE에서 가장 높은 생산성(0.74 unit/mL)을 나타냈고, C5/L. (0.66 unit/mL), Livid (0.08 unit/mL), NDSK(0.06 unit/mL) 순으로 나타났다 S. griseus IFO 13350/pWHM3-TR1R2의 경우에는 전반적으로 배양 7일에 트립신 활성이 가장 높았으며, C5/L (1.518 unit/mL), R2YE(1.284 unit/mL), NDSK (0.932 unit/mL), Livid (0.295 unit/mL) 순으로 나타났다. S. griseus IFO 13350/pWHM3-TR1R2를 C5/L 배지에서 7일간 배양한 배양액으로부터 $25%{\sim}60%$ ammonium sulfate 침전, CM-sepharose 및 Sp-sepharose column chromatography를 통하여 트립신을 고순도로 정제할 수 있었다. 최종 purification fold는 6.5배, 순수 정제된 트립신의 specific activity는 69,252 unit/mg, 회수율은 1.4%이었다.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.312-317
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• 2006

The chromosomal sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was disrupted in Streptomyces griseus by insertion of the neomysin-resistance gene. The production of chymotrypsin activity of sprD disruptant was not completely abolished, but delayed by 24 h, compared with that of wild-type strain. The aerial mycelial formation of sprD disruptant was retarded, and specifically the formation of spores was not observed in the central region of colonies. However, normal morphological development into spores was observed in the marginal region of colonies. In addition, the production of yellow pigment that might be dependent on A-factor was also decreased in the sprD disruptant, compared with that of the wild-type strain. Introduction of the sprD gene, which was placed on a high copy-numbered plasmid into S. griseus ${\Delta}sprD$, partially restored the ability of morphological development, and a significant level of sporulation was observed. When the overexpression vector for sprD, pWHM3-D, was introduced in S. griseus, there was no significant change in the chymotrypsin activity or colonial morphology, in contrast to Streptomyces lividans, indicating the presence of a tight regulation system for the overexpression of the sprD gene in S. griseus.