• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.116-120
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• 2006

We constructed four recombinant plasm ids to enhance the production of clavulanic acid (CA) in Streptomyces clavuligerus NRRL3585: (1) pIBRHL1, which includes ccaR, a pathway-specific regulatory gene involved in cephamycin C and CA biosynthesis; (2) pIBRHL2, containing claR, again a regulatory gene, which controls the late steps of CA biosynthesis; (3) pGIBR containing afsR-p, a global regulatory gene from Streptomyces peucetius; and (4) pKS, which harbors all of the genes (ccaR/ claR/ afsR-p). The plasmids were expressed in S. clavuligerus NRRL3585 along with the $ermE^*$ promoter. All of them enhanced the production of CA; 2.5-fold overproduction for pIBRHL1, 1.5-fold for pIBRHL2, 1.6-fold for pGIBR, and 1.5-fold for pKS compared to the wild type.

• 한국미생물·생명공학회지
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• 제34권3호
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• pp.211-215
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• 2006

S. lividans에서 클로닝된 조절유전자인 afsR2를 과발현시 나타나는 up-regulated protein과 down-regulated protein 관련 유전자들을 proteomics 방법으로 선별하였고[3], 이를 방선균 발현벡터인 pSE34를 이용하여 제작된 pPNP, pGPD를 S. avermitilis에 형질전환시켜 avermectin 및 이차대사산물의 생산량 차이를 비교 분석하였다. 그 결과 up-regulated protein으로 예상되던 PNP는 wild-type S. avermitilis에서만 제한적으로 avermetin-type 대사산물의 생산성 향상을 촉진시켰으며, 이와 반대로 down-regulated protein으로 예상되었던 GPD는 avermectin-type 대사산물의 생산량을 wild type S. avermitilis에서는 4배, over-producer S. avermitilis에서는 2.5배 증가시켰다. 본 연구결과는, 방선균 조절 단백질들이 이차대사산물의 종류 및 생합성 기작에 따라 전혀 다르게 작용될 수 있음을 암시하며, 향후 이들 조절 단백질들에 대한 보다 구체적인 분자수준에서의 연구 필요성을 제시하고 있다.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.325-332
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• 1998

In vitro phosphorylation experiments with a cell extract of Streptomyces coelicolor A3(2) M130 in the presence of ${\gamma}-[^32P]$]ATP revealed the presence of multiple phosphorylated proteins, including the AfsR/AfsK kinases which control the biosynthesis of A-factor, actinorhodin, and undecylprodigiosin. Phosphorylation of AfsR by a cell extract as an AfsK source was significantly inhibited by Ser/Thr protein kinase inhibitors, staurosporine and K-252a, at concentrations giving 50% inhibition ($IC_50$) of $1{\mu}M\;and\;0.1{\mu}M$, respectively. Further in vitro experiments with the cell extracts showed that phosphorylation of multiple proteins was inhibited by various protein kinase inhibitors with different inhibitory profiles. Manganese and calcium ions in the reaction mixture also modulate phosphorylation of multiple proteins. Manganese at 10 mM greatly enhanced the phosphorylation and partially circumvented the inhibition caused by staurosporine and K-252a. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, which are known as tyrosine kinase inhibitors, did not show any significant inhibition of AfsR phosphorylation. Consistent with the in vitro effect of the kinase inhibitors, they inhibited aerial mycelium formation and pigmented antibiotic production on solid media. On the contrary, when assayed in liquid culture, the amount of actinorhodin produced was increased by staurosporine and K-252a and greatly decreased by manganese. All of these data clearly show that the genus Streptomyces possesses several protein kinases of eukaryotic types which are involved in the regulatory network for morphogenesis and secondary metabolism.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.52-58
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• 2014

Structurally, herboxidiene contains the tetrahydropyran acetic acid moiety and a side chain including a conjugated diene, and has been isolated from Streptomyces chromofuscus ATCC 49982. Its production was significantly elevated nearly 13.5-fold (0.74 g/l) in a medium supplemented with glycerol (medium No. 6A6), and was more efficacious (1.08 g/l; 19.8-fold) in fed-batch fermentation at 36 h in medium No. 6A6, from Streptomyces chromofuscus. For further enhancement, regulatory genes metK1-sp and afsR-sp from Streptomyces peucetius were overexpressed using an expression vector, pIBR25, and similarly ACCase from Streptomyces coelicolor and two genes, metK1-sp and afsR-sp, were also overexpressed using an integration vector, pSET152, under the control of the strong $ermE^*$ promoter in Streptomyces chromofuscus. Only the recombinant strains S. chromofuscus SIBR, S. chromofuscus GIBR, and S. chromofuscus AFS produced more herboxidiene than the parental strain in optimized medium No. 6A6 with an increment of 1.32-fold (0.976 g/l), 3.85-fold (2.849 g/l), and 1.7-fold(1.258 g/l) respectively.

• 한국식품위생안전성학회지
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• 제35권5호
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• pp.438-446
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• 2020

메주와 누룩은 한국 전통 발효 식품에 사용되는 스타터로, Aspergillus속 곰팡이나 aflatoxin에 노출되기 쉽다. 본 연구에서는 우리나라에서 시판되는 57개의 메주 시료와 18개의 누룩 시료로부터 Aspergillus 속 곰팡이를 분리하고 동정하였다. 분리주의 aflatoxin 생성 가능성을 평가하기 위하여 multiplex PCR을 통해 aflatoxin 생합성 유전자(aflO, aflP, aflR)를 확인하고, 이들 분리주에 의해 생성되는 aflatoxin 함량을 HPLC로 조사하였다. 뿐만 아니라 시판 메주와 누룩 시료 중 aflatoxin 함량을 분석하였다. 그 결과, 메주 시료로부터 130개, 누룩 시료로부터 47개 균주가 분리되어 총 177개의 분리주를 확인 및 동정하였다. 각각 메주와 누룩으로부터 분리된 19.2% (25/130), 10.6% (5/47)의 분리주가 3 종류의 aflatoxin 생합성 유전자를 모두 보유하였으며, 그 중 메주로부터 분리된 5개의 분리주가 실제로 aflatoxin을 생성하였다. 시판 메주와 누룩 시료 중 aflatoxin 함량을 분석한 결과, 88% (51/58)의 메주 시료의 aflatoxin 오염 수준은 모두 검출한계 미만으로 나타났고, 누룩 또한 시료의 39% (7/18)가 검출한계 미만으로 확인되었다. 메주와 누룩에서 분리된 분리주 중 aflatoxin 생합성 유전자를 모두 보유하거나 배지 상에서 aflatoxin 생성을 보여준 aflatoxigenic 균주는 존재하였으나 유통되고 있는 시료에서 aflatoxin 오염 빈도는 낮은 수준임을 확인할 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.