Streptomyces griseus HH1, An A-factor Deficient Mutant Produces Diminished Level of Trypsin and Increased Level of Metalloproteases

Kim, Jung-Mee,Hong, Soon-Kwang The Microbiological Society of Korea 2000 The journal of microbiology Vol.38 No.3

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.

Streptomyces griseus IFO13350 유래 sprA 및 sprB 유전자를 이용한 Pretense 생산균주 개발

황지환,이창권,이강무,조병기,박해룡,황용일,Hwang Ji-Hwan,Lee Chang-Kwon,Lee Kang-Mu,Jo Byoung-Kee,Park Hae-Ryong,Hwang Yong-Il 한국미생물학회 2005 미생물학회지 Vol.41 No.1

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp. 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.

Enhancement of Protein Secretion by TatAC Overexpression in Streptomyces griseus

지원재,오은아,Jong-Hee Kim,홍순광 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.1

Production of proteins in secretary form is one of the important factors affecting fermentation. The Tat (twin arginine translocation) protein secretion system, which includes the proteins TatA, TatB, and TatC, was identified in the genomic sequence of Streptomyces griseus IFO13350. The tatA and tatC genes were organized into a polycistronic operon, whereas tatB was located separately on the chromosome. Comparison of amino acid sequences suggested that TatC was a membrane-spanning protein, whereas TatA and TatB were found to be cytoplasmic proteins. Analysis of extracellular proteins and N-terminal amino acid sequencing revealed that secretion of SGR5556 was significantly enhanced by overexpression of TatAC in S. griseus HH1. Further, enzymatic study showed that SGR5556 encoded a glycerophosphoryl diester phosphodiesterase. In addition,other hydrolase activities, such as those of amylase, total protease, metalloprotease, trypsin, chymotrypsin, and Leuaminopeptidase,were also enhanced by 3, 3, 2.6, 2.3, 5.4,and 2.5 fold, respectively, in S. griseus upon TatAC overexpression. Overexpression of TatAC induced the production of a greenish-yellow pigment in S. griseus HH1 as well as more abundant sporulation at an earlier stage in Streptomyces coelicolor A3(2). In silico analysis by TatFIND, SignalP, and TMHMM identified 19 binding proteins, 28 enzymatic proteins, and 27 other proteins with unknown functions as putative TatAC-dependent secretary proteins. These results clearly indicate that TatA and TatC constitute a functional Tat system in S. griseus. Additionally,the S. griseus Tat system can be useful for the production of valuable proteins, including many hydrolytic enzymes and candidates of Tat-dependent secretary proteins, under industrial conditions.

Genome-wide screening antifungal genes in <i>Streptomyces griseus</i> S4-7, a Fusarium wilt disease suppressive microbial agent

Hong, Sung Won,Kim, Da-Ran,Kwon, Youn Sang,Kwak, Youn-Sig Published by Elsevier/North Holland on behalf of t 2019 FEMS microbiology letters Vol.366 No.12

<P> <I>Streptomyces</I> is a widely studied bacterial genus, particularly with regard to secondary metabolites and antibiotics production. <I>Streptomyces griseus</I> S4-7 was isolated from a strawberry Fusarium wilt disease suppressive soil, and its biological control ability has been well established. However, the antifungal mechanism of strain S4-7 is not yet fully understood at the molecular and biochemical level. Therefore, in this study we created a random mutant library for strain S4-7 with the Tn5 transposon element to investigate antifungal traits on a genome-wide scale. In total 4646 individual mutant strains were created and 13 mutants were selected based on loss of antifungal activity. The knockout genes were identified as electron transfer oxidoreductase (eto),sigma factor-70(sig70) and nrps by Inverse PCR (I-PCR). eto regulates the <I>geranylgeranyl</I> reductase gene, which is involved in terpenoid-quinone biosynthesis, an important factor in cell fitness. In the <I>△eto</I> strain, expression of <I>wbl,</I> a master regulator of the production of secondary metabolites, was significantly reduced. sig70 is responsible for the cell differentiation sensing mechanism in genus <I>Streptomyces. △nrps</I> showed decreased production of hybrid peptide-polyketide siderophores. These results suggest that <I>S. griseus</I> S4-7 may have various antifungal mechanisms, and each mechanism is essential to maximal antifungal activity. <P>

Streptomyces griseus의 특이적 포자형성에 관여하는 유전자의 전사량 분석

지원재 ( Won-jae Chi ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.4

S. griseus wild type에서 dasA 유전자의 과발현에 의해 유도된 기저균사의 ectopic sporulation 관련 유전자를 알아 보기 위해서, empty vector가 삽입된 균주와 dasA가 과발현된 균주의 전사체를 DNA microarray법으로 비교하였다. DNA microarray 결과를 토대로 dasA 유전자 과발현 균주에서 2배이상 발현량이 증가되었으며 p-value가 0.05 미만 (p-value < 0.05)인 유전자들 중에서 false positive 를 제외 시키는 작업을 통하여 최종적으로 4개의 유전자(SGR794, SGR2469, SGR3656, SGR3657)와 3개의 cluster (SGR795-797, SGR2377-2378, SGR6997-6998)를 선발하였다. 이들의 전사량은 low resolution Sl nuclease mapping 법을 통하여dasA 유전자 과발현 균주에서 증가된 것을 확인하였다. Two Streptomyces griseus strains, a wild-type strain and an A-factor-dependent transcriptional activator mutant strain harboring multiple copies of a gene, dasA, that encodes a substrate-binding protein of the ATP-binding cassette transporter, showed severe ectopic sporulation of young substrate hyphae in response to glucose. The effect of dasA overexpression on the ectopic sporulation of Streptomyces strains was evaluated by comparing the transcriptomes of the strain harboring multiple copies of dasA and a strain harboring empty vector. By DNA microarray, 4 genes (SGR794, SGR2469, SGR3656, and SGR3657) and 3 clusters (SGR795-797, SGR2377-2378, and SGR6997-6998) were differentially expressed by more than 2-fold in S. griseus strains harboring dasA. The DNA microarray result was validated by low-resolution S1 nuclease mapping.

Streptomyces속 균주가 생성하는 Alkaline Protease의 생산 및 정제

최청,정영건,성삼경,최광수,이재성,조영제,권오진 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.2

토양으로부터 alkaline protease 생성능이 강한 Streptomyces griseus HC-1141을 분리하였으며, 효소생산의 최적 배양조건은 0.5% casein, 0.05% ammonium chloride, 0.1% ferrous sulfate, 2.0%의 lactose, pH 8.0에서 84시간 배양했을 때이다. 효소의 정제는 ammonium sulfate 침전, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, crystallization으로 하여 53.23배 정제할 수 있었으며 polyacrylamide gel 전기영동상 단일밴드를 나타내었다. 정제효소의 분자량은 sodium dodecyl sulfate-polyacrylamide gel electrophoresis에 의해 31,000 정도로 추정되었고, 결정구조는 판상형태이었으며 아미노산 조성은 glycine과 glutamic acid 함량이 많고 arginine함량이 적었으며, 또한 말단아미노산 분석에서 N-말단은 leucine이고 C-말단은 histidine이었다. An alkaline protease producing microorganism was isolated from soil and identified as Streptomyces griseus HC-1141. The optimum culture condition of Streptomyces griseus HC-l141 for the production of alkaline protease was as follows; 0.5% casein, 0.05% ammonium chloride, 0.1% ferrous sulfate, 2.O% lactose, pH 8.0 and 84 hrs. The enzyme was purified about 53 folds by ammonium sulfate treatment, DEAE-cellulose ion exchange chromatography and gel filtration on Sephadex G-150. The homogeneity of the purified enzyme was verified by polyacrylamide gel electrophoresis. The molecular weight was estimated to be 31,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme consists of glycine and glutamic acid as major amino acids. The N-terminal and C-terminal residues of the alkaline protease were leucine and histidine respectively.

Cloning and Characterization of a Heterologous Gene Stimulating Antibiotic Production in Streptomyces lividans TK-24

Kwon, Hyung-Jin,Lee, Seung-Soo,Hong, Soon-Kwang,Park, Uhn-Mee,Suh, Joo-Won The Microbiological Society of Korea 1999 The journal of microbiology Vol.37 No.2

Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

Isolation and Physico-Chemical Properties of Cycloheximide-isomer from Streptomyces sp.NA-52

Yoon,Won-Ho,Lim,Dae-Seog,Kim,Chang-Han,Yoon,Sang-Kun 건국대학교 동물자원연구센터 1999 動物資源硏究誌 Vol.20 No.-

새로운 분리균주 Streptomyces sp. NA-52는 Pyricularia oryzae와 Chlorella regularis에 대해 항균활성물질을 생산하는 것으로 밝혀졌다. 그 균주에 의해 생산되는 항생물질 복합물 중 (compound A 및 B), 활성이 좋은 화합물 A가 분리되었으며, 그것의 이화학적 성질이 조사되었다. 그 화합물의 분자량은 281(EI-MS)로 결정되었고, UV, IR 및 ¹H, 13C NMR 스펙트럼 분석결과, cycloheximide(4-[2-(3, 5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione)의 이성체와 같은 glutarimide 그룹의 항생물질로 분류되었다. 한편, 백자무우 씨앗의 발아시험결과, compound A는 cycloheximide와 마찬가지로 백자무우씨앗에 대해 강한 독성을 가지고 있었으나, compound B는 그 씨앗을 100% 발아시켰다. 지금까지의 cycloheximide 이성체와 비교해 볼 때, compound B는 백자무우씨앗에 대해 전혀 독성을 나타내지 않는, glutarimide 그룹내의 새로운 cycloheximide 이성체로 추정되었다. 결과적으로 cycloheximide와 같은 glutarimide 그룹의 항생물질이 Streptomyces griseus가 아닌 다른 방선균종에서도 생산된다는 것으로 밝혀졌다. A new isolate of Streptomyces sp. NA-52 was found to produce antibiotics with antimicrobial spectra against Pyricularia oryzae and Chlorella regularis. The main active component, compound A, of the antibiotic complex (compounds A and B) produced by the strain was isolated, and its physico-chemical properties were investigated. The molecular weight of the compound was determined to be 281 (EI-MS). UV, 1R, and ¹H and 13C NMR spectra suggested that the compound is a cycloheximide (4-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione) isomerlike glutarimide group antibiotic. Ina germination test using BAIKJA radish seed, neither compound A nor cycloheximide germinated the seeds, but compound B completely germinated them. Compared with known cycloheximide-isomers, compound B may be thought of as a new cycloheximide-isomer, without toxicity for BAIKJA radish seed, in the glutarimide group.

Improvement of Transformation Efficiency by Strategic Circumvention of Restriction Barriers in Streptomyces griseus

( Suzuki Hirokazu ),( Shunji Takahashi ),( Hiroyuki Osada ),( Ken Ichi Yoshida ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.7

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`-GC5mCGGC-3` and 5`-GAG5mCTC-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.

Cloning, DNA Sequence Determination, and Analysis of Growth-Associated Expression of sodF Gene Coding for Fe-and Zn-Containing Superoxide Dismutase of Streptomyces griseus

Kim, Ju Sim,Lee, Jeong Kug 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.5

Iron- and zinc-containing superoxide dismutase (FeZnSOD) and nickel-containing superoxide dismutase (NiSOD) are cytoplasmic enzymes in Streptomyces griseus. The sodF gene coding for FeZnSOD was cloned from genomic Southern hybridization analysis with a 0.5-kb DNA probe, which was PCR-amplified with facing primers corresponding to the N-terminal amino acids of the purified FeZnSOD of S. griseus and a C-terminal region which is conserved among bacterial FeSODs and MnSODs. The sodF open reading frame (ORF) was comprised of 213 amino acids (22,430Da), and the deduced sequence of the protein was highly homologous (86% identity) to that of FeZnSOD of Streptomyces coelicolor. The FeZnSOD expression of exponentially growing S. griseus cell was approximately doubled as the cell growth reached the early stationary phase. The growth-associated expression of FeZnSOD was mainly controlled at the transcriptional level, and the regulation was exerted through the 110bp regulatory DNA upstream from the ATG initiation codon of the sodF gene.