Functional Analysis of SGR4635-Induced Enhancement of Pigmented Antibiotic Production in Streptomyces lividans

지원재,이순열,이재학 한국미생물학회 2011 The journal of microbiology Vol.49 No.5

The Gram-positive mycelium-producing bacterium Streptomyces undergoes complex morphological differentiation after autolytic degradation of the vegetative mycelium. Cell-wall breakdown during growth stimulates cell development and secondary metabolite production by Streptomyces. N-acetylglucosamine (GlcNAc) produced by cell-wall lysis acts as a signal molecule, triggering the production of secondary metabolites in S. coelicolor A3(2). Here, we report that introduction of multiple copies of the GlcNAc-internalizing gene (sgr4635, encoding nagE2) of S. griseus activates actinorhodin and undecylprodigiosin production during the late growth of S. lividans in the absence of GlcNAc. Furthermore, the repressor-type transcriptional regulator DasR binds to two operator sites upstream of sgr4635. Our findings indicate that sgr4635 induces DasR-mediated antibiotic production by internalizing the GlcNAc accumulated from cell-wall lysis.

Streptomyces griseus ATCC10137에서 Trypsin 유전자 sprT의 주변 유전자군 분석

지원재,김미순,김종희,강대경,홍순광,Chi Won-Jae,Kim Mi-Soon,Kim Jong-Hee,Kang Dae-Kyung,Hong Soon-Kwang 한국미생물학회 2005 미생물학회지 Vol.41 No.4

Streptomyces griseus trypsin(SGT)을 코드하는 sprT 유전자를 포함하여 약 6.7 kb의 DNA 단편을 Streptomyces griseus ATCC 10137의 염색체 DNA로부터 클로닝 하여 염기서열을 결정하였다. 염기서열 분석 결과, 염색체 DNA를 EcoRI-HindIII 제한효소로 완전 분해하여 클로닝한 약6.7 kb의 단편에는 sprT유전자를 포함하여 총6개의 완전한 ORF (open reading frame)와 1개의 불완전한 ORF가 존재하는 것으로 밝혀졌으며, 순서대로 ORF1, SGT, ORF2, ORF3, ORF4, ORF5, ORF6로 명명하였다. 예상 단백질의 아미노산 서열 분석 결과, ORF1은 oxidoreductase, ORF3는 ArsR family의 transcription regulator, ORF4는 Listeria monocytogenes의 LPXTG motif를 갖는 peptidoglycan bound protein, ORF5는 transmembrane helix를 갖는 막단백질, ORF6는 Streptomyces avermitilis에서 보고된 lipoprotein과 높은 상동성을 보였으며, ORF2는 기능을 예측 할 수 없었다. 이와 같은 분석결과, sprT 유전자 주위에는 세포막이나 세포벽 구성성분을 코드하는 유전자가 존재하고 있으며, 따라서 SGT Pretense는 이러한 세포막 또는 세포벽 합성이나 분해과정에서 어떤 기능을 담당할 가능성 이 있는 것으로 추측되었다. A 6.7kb DNA fragment containing the sprT gene encoding Streptomyces griseus trypsin (SGT) was cloned from Streptomyces griseus ATCC 10137, and the complete nucleotide sequence was determined. Nucleotide sequence and deduced amino acid or the EcoRI-HindIII fragment revealed the presence or the six complete ORFs containing the sprT gene and one incomplete ORF, which were named ORF1, SGT, ORF2, ORF3, ORF4, ORF5, and ORF6, respectively. ORF1 has homology with the oxidoreductases from several organisms. ORF2 and ORF3 show similarity with unknown proteins and transcription regulator that belongs to the ArsR family, respectively. ORF4 and ORF5 show homology with the peptidoglycan bound protein with LPXTG motif from Listeria monocytogenes and the membrane protein with transmembrane helix from several organisms, respectively. The last ORF, ORF6, shows homology with the lipoprotein from Streptomyces avermitilis.

Characterization of Sgr3394 Produced only by the A-Factor-Producing Streptomyces griseus IFO 13350, not by the A-Factor Deficient Mutant HH1

지원재,Xue-Mei Jin,Sung-Cheol Jung,오은아,홍순광 한국미생물학회 2011 The journal of microbiology Vol.49 No.1

Protein D (9.7 kDa) is an extracellular protein detected in the culture broth of A-factor-producing Streptomyces griseus IFO 13350, but not of the A-factor-deficient mutant strain S. griseus HH1. Comparison of the N-terminal amino acid sequence with the genomic sequencing data of S. griseus IFO 13350 identified protein D as Sgr3394, which encodes a putative secretory protein with unknown function. The premature Sgr3394 consisted of 128 amino acids (13.5 kDa), showed 87.5% identity with SACT1DRAFT-0503, from Streptomyces sp. ACT-1, and 68.8% identity with SrosN15-18634, from S. roseosporus NRRL15998, and was confirmed to be matured for secretion by a peptide cleavage between the Ala-38 and Ala-39 bond. RT-PCR anaylsis of Sgr3394 clearly showed that it can be transcribed in the wild-type strain, but not in the A-factor-deficient strain. However, a gel-mobility shift assay of the promoter region of sgr3394 with A-factor-dependent transcriptional regulator (AdpA) showed that AdpA could not specifically recognize the putative AdpA-binding site (5′-TCCCCCGAAT-3′). All of these data strongly suggest that the expression of sgr3394 is not directly induced by AdpA but is regulated indirectly by an A-factor dependent protein. Introduction of sgr3394 on a high-copy-numbered plasmid (pWHM3-sgr3394) into S. lividans TK21 induced massive production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment). Compared to the control, production of each pigment increased by 6.1 and 2.6 times, respectively, on R2YE agar, and 3.1and 1.4 times, respectively, in R2YE broth; there was little influence on morphogenesis. In S. coelicolor A3(2)/pWHM3-sgr3394, actinorhodin and undecylprodigiosin productions were enhanced to 1.8 and 1.1 times those observed in the control, respectively, suggesting that overexpression of sgr3394 can stimulate secondary metabolism, especially actinorhodin biosynthesis, in S. lividans and S. coelicolor.

Streptomyces griseus의 형태분화 및 이차대사 관련 protease

지원재 명지대학교 생명공학연구소 1999 생명공학 논문집 Vol.1 No.-

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.

비파괴 계장화 압입시험기법을 통한 API X65 배관 용접부 잔류응력 평가

지원재,이윤희,김우식,김철만,권동일 대한용접접합학회 2003 대한용접·접합학회지 Vol.21 No.5

Apparent mechanical properties in structural components can be different from the initially designed values due to the formation of the residual stress in metal forming and welding. Therefore, the evaluation of residual stress has great importance in the reliability diagnosis of structural components. A nondestructive instrumented indentation technique has been proposed to evaluate various strength concerning mechanical properties from the analysis of load-depth curve. In this study, quantitative residual stress estimation on API X65 welded joints for natural gas pipeline was performed by analyzing the variation of indentation loading curve by residual stress through a new proposed theoretical model. The residual stress from the indentation method was compared with that from the saw-cutting method.

Characterization of Two Thermostable β-agarases from a Newly Isolated Marine Agarolytic Bacterium, Vibrio sp. S1

지원재,서주원,홍순광 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.5

An agar-degrading bacterium, strain S1, was isolated from the coastal seawater of Jeju Island, Korea, and identified as a novel species of the genus Vibrio. The isolate, Vibrio sp. S1, produced at least five kinds of extracellular agarases in artificial sea water broth containing yeast extract and bacto peptone, and two of them were purified to homogeneity. Both agarases, AgaA33 and AgaA29, with apparent molecular weights of 33 kDa and 29 kDa, respectively, exhibited an optimum temperature and pH of 45°C and 7.0, respectively. AgaA33 and AgaA29 showed acidophilic properties and maintained 93% and 87% of the maximum agarase activity at 50°C, respectively, displaying their thermostability. Moreover, more than 80% activity was retained after heat treatment at 45°C for 1 h. Their agarase activities were inhibited by the presence of EDTA and remarkably stimulated by the presence of Mn2+ in a concentration-dependent manner, indicating that both agarases required the Mn2+ ion as a cofactor. The AgaA33 enzyme exhibited Km and Vmax values of 4.02 mg/mL and 27 U/mg, respectively. AgaA29 exhibited Km and Vmax values of 3.26 mg/mL and 200 U/mg, respectively. The instrumental analysis demonstrated that both are new β-agarases that can hydrolyze agarose and agaro-oligomers into neoagarotetraose and neoagarohexaose. In addition, AgaA33 coproduced neoagarooctaose as the major final product. Both thermostable enzymes are expected to be useful for the industrial application of agar.

Periplasmic Expression, Purification, and Characterization of an Anti-epidermal Growth Factor Receptor Antibody Fragment in Escherichia coli

지원재,김혜림,유희정,김영필,홍순광 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.2

New structural designs of antibody fragments have considerable biotechnological and therapeutic potential. In this study, we describe the construction and functional expression of a cetuximab-based antibody fragment (scFv- CH3, minibody) that exhibits activity against human colon cancer. Heterologous expression in Escherichia coli (E. coli) was improved by optimizing the host cells, signal peptides, induction conditions, and culture media. The recombinant minibody was expressed successfully in the periplasm of E. coli BL21(DE3) and purified by immobilized metal affinity chromatography using a Ni2+-NTA resin. The purified minibody showed high binding affinity to cell-surface epidermal growth factor receptor (EGFR) and exhibited inhibition of EGFR-mediated signal transduction in the human colon cancer cell line HT29 in a similar way by the cetuximab. The minibody also showed significant level of anti-cancer ability in the HT29 colorectal cancer xenograft model, which was lower than that by the cetuximab.

대한민국 울진 연안 해양에서 분리한 해양 미생물 Ruegeria sp. 50C-3의 동정 및 내열성 효소 생산

지원재,김종희,박재선,홍순광,Chi, Won-Jae,Kim, Jong-Hee,Park, Jae-Seon,Hong, Soon-Kwang 한국미생물학회 2016 미생물학회지 Vol.52 No.3

대한민국 동해안 울진 앞 바닷물로부터 50-C로 명명한 해양 미생물을 분리하였다. 50-C 균주는 그람-음성, 호기성 세균이며, 노란색 집락을 형성하고, 극성편모를 갖는 박테리아이다. 이 균주는 $20-50^{\circ}C$, pH 5.5-8.5 범위에서 자라며, 비교적 고온인 $40-50^{\circ}C$, pH 6.5-7.5, 2% (w/v) NaCl에서 최적 성장을 보인다. 16S rRNA 유전자 서열 분석결과 50C-3 균주는 Ruegeria 속에 속하는 R. intermedia CC-GIMAT-$2^T$, R. lacuscaerulensis ITI-$1157^T$의 16S rRNA 유전자 서열과 각각 99.4%, 96.98% 상동성을 보였다. 그러나 50C-3 균주는 운동성, 탄소이용능력, 효소생산능력 등의 생리학적 특성에서 두 균주와는 명확히 다른 특성을 보였다. 50C-3 균주의 DNA G+C content는 66.7 mol%이고, 주요한 respiratory quinone은 ubiquinone-10 (Q-10)이었다. 이와 같은 형태학적, 생리학적, 유전학적 특성을 비교하여, 50C-3 균주는 R. intermedia CC-GIMAT-$2^T$와 같은 종에 속하는 새로운 변종으로 판단되며 Ruegeria sp. 50C-3으로 명명하였다(KCTC23890 =DSM25519). 50C-3 균주는 cellulase, agarase 활성은 없었지만, alkaline phosphatase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase를 생산하였고 이들 모두 $50^{\circ}C$ 에서도 활성이 좋은 내열성 효소일 것으로 판단되었다. 특히, ${\beta}$-galactosidase의 경우 $37^{\circ}C$에서 보다 $50^{\circ}C$에서의 활성이 1.9배 증가하여 산업적으로 활용성이 클 것으로 예상된다. A marine bacterium, designated as strain 50C-3, was isolated from a seawater sample collected from the East Sea of South Korea. The strain is a Gram-negative, aerobic, yellow colored polar-flagellated bacterium that grows at $20-50^{\circ}C$ and pH 5.5-8.5. Optimal growth occurred at $40-50^{\circ}C$, at pH 6.5-7.5, and in the presence of 2% (w/v) NaCl. Based on 16S rRNA gene sequence similarity, the isolate was considered to represent a member of the genus Ruegeria. The result of this analysis showed that strain 50C-3 shared 99.4% and 96.98% sequence similarity with Ruegeria intermedia CC-GIMAT-$2^T$ and Ruegeria lacuscaerulensis ITI-$1157^T$, respectively. Furthermore, strain 50C-3 showed clear differences from related strains in terms of several characteristics such as motility, carbon utilization, enzyme production, etc. The DNA G+C content was 66.7 mol%. Chemotaxonomic analysis indicated ubiquinone-10 (Q-10) as the predominant respiratory quinone. Based on phenotypic, chemotaxonomic, and phylogenetic characteristics, the isolate represents a novel variant of the Ruegeria intermedia CC-GIMAT-$2^T$, for which we named Ruegeria sp. 50C-3 (KCTC23890=DSM25519). Strain 50C-3 did not produce cellulase and agarase, but produced alkaline phosphatase, ${\alpha}$-galactosidase, and ${\beta}$-galactosidase. The three enzymes showed stable activities even at $50^{\circ}C$ and thus regarded as thermostable enzymes. Especially, the ${\beta}$-galactosidase activity enhanced by 1.9 times at $50^{\circ}C$ than that at $37^{\circ}C$, which may be very useful for industrial application.

서해안 해수로부터 분리한 한천분해 해양미생물 Pseudoalteromonas sp. H9의 동정 및 특성 연구

지원재,윤영상,김종희,홍순광,Chi, Won-Jae,Youn, Young Sang,Kim, Jong-Hee,Hong, Soon-Kwang 한국미생물·생명공학회 2015 한국미생물·생명공학회지 Vol.43 No.2

대한민국 대천 해수로부터 agarase를 생산하는 균주 H9을 분리하였다. 본 균주는 16S rRNA 염기 염기서열 분석결과로부터 Pseudoalteromonas espejiana NCIMB2127^T (98.98%), Pseudoalteromonas carrageenovora ATCC12662^T (98.78%), Pseudoalteromonas atlantica IAM12927^T (98.64%), Pseudoalteromonas issachenkonii KMM3549^T (98.63%) 등과 높은 상동성을 보였다. 균주 H9은 genomic DNA 내 G+C 농도가 41.56%이고 주요 퀴논으로 quinone-8을 포함하고 있다. 균주 H9의 주요 지방산으로 C_16:1ω7c (34.3%), C_16:0 (23.72%), C_18:1ω7c (13.64%) 등이 포함되었다. 이러한 유전적, 생리적 특성에 따라 균주 H9은 Pseudoalteromonas 속의 균으로 분류하여 Pseudoalteromonas sp. H9으로 명명하였다. 균주 H9이 세포외부로 분비하는 총 agarase는 40-45℃와 pH 7.0-8.0의 조건에서 높은 효소 활성을 갖으며, agarose를 분해하여 (neo)agarotetraose와 (neo)agarohexaose를 생산하였다. 균주 H9은 한천분해를 위해 유용하게 사용될 수 있으며, 다양한 생리활성을 갖는 (neo)agarooligosaccharide는 기능성 식품, 화장품 등의 산업에 유용하게 사용될 수 있을 것으로 기대된다. An agarolytic marine bacterium (H9) was isolated from the coastal seawater of the West Sea, South Korea. The isolate, H9, was gram-negative and rod-shaped with a smooth surface and polar flagellum. Cells grew at 20-30℃, between pH 5.0 and 9.0, and in ASW-YP (Artificial Sea Water-Yeast extract, Peptone) media containing 1-5% (w/v) NaCl. The G+C content was 41.56 mol%. The predominant isoprenoid quinone in strain H9 was ubiquinone-8. The major fatty acids (>10%) were C_16:1ω7c (34.3%), C_16:0 (23.72%), and C_18:1ω7c (13.64%). Based on 16S rRNA gene sequencing, and biochemical and chemotaxonomic characterization, the strain was designated as Pseudoalteromonas sp. H9 (=KCTC23887). In liquid culture supplemented with 0.2% agar, the cell density and agarase activity reached a maximum level of OD = 4.32 (48 h) and OD = 3.87 (24 h), respectively. The optimum pH and temperature for the extracellular crude agarases of H9 were 7.0 and 40℃, respectively. Thin-layer chromatography analysis of the agarase hydrolysis products revealed that the crude agarases hydrolyze agarose into neoagarotetraose and neoagarohexaose. Therefore, the new agar-degrading strain, H9, can be applicable for the production of valuable neoagarooligosaccharides and for the complete degradation of agar in bio-industries.