• 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.

• 미생물학회지
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• 제37권2호
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• pp.164-169
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• 2001

Cephamycin C를 생산하는 몇가지 균주를 사용하여 cephalosporin C로부터 7$\alpha$-hydroxycephalosporin C로의 전환력을 조사하였다. 이들 균주중에서 cephalosporin 7$\alpha$-hydroxylase의 활성은 Streptomyces clavuligerus ATCC 27064가 가장 높게 나타났다. Streptomyces clavuligerus ATCC 27064로부터 얻은 조효소액을 정제한 후 activated DEAE-sphacel 레진에 고정화하여 기질인 cephalosporin C로부터 7$\alpha$-hydroxycephalosporin C를 생산하였다. 고정화에 의해서 생성된 물질을 ESI-Mass로 측정한 결과, 분자량 431로 나타났다. 또한 $^1H$ NMR 분석에 의해 고정화 효소에 의해 생성된 물질은 cephalosporin C로부터 얻어진 7$\alpha$-hydroxycephalosporin C임을 확인하였다.

• 생명과학회지
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• 제16권1호
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• pp.76-81
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• 2006

방선균에서 DNA 전사 억제인자로써 작용하여 이차대사산물의 생산뿐만 아니라 형태분화를 조절하는 $\gamma-butyrolactone$ autoregulator receptor를 암호화하는 유전자(scaR)를 clavulanic arid 생산 균주, Streptomyces clavuligerus로부터 클로닝하고 in vitro에서 ScaR의 특징을 연구하여 보고한 바 있다. 따라서 본 연구에서는 ScaR의 in vivo 기능을 분석하기위해 상동재조합방법을 이용하여 scaR이 제거된 변이주를 제작하고 야생균주와 표현형을 비교해 보았다. 그 결과, Streptomyces clavuligerus의 형태분화에서는 큰 차이를 나타내지 않았지만 clavulanic acid의 생산에 있어서는 scaR 파괴 변이주가 야생균주에 비해 생산이 증가된 경향을 보였다. 그러므로 ScaR이 S. clavuligerus의 형태분화에는 영향을 미치지 않지만 clavulanic acid 생합성에는 negative regulator로 작용한다는 것을 본 연구를 통해 명확하게 확인할 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.687-687
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• 2005

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.146-152
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• 2010

Streptomyces clavuligerus NRRL3585 produces a clinically important $\beta$-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap) was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into a deletion mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared with the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared with the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1638-1644
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• 2007

The effect of carbon sources on tacrolimus production by a mutant strain of Streptomyces clavuligerus CKD 1119, an isolate from soil, was examined. Among the carbohydrates and oils tested in this work, a mixed carbon source of soluble starch and com oil was the best. An analysis of the culture kinetics also showed that, in contrast to the carbohydrates, the com oil was consumed later in the antibiotic production phase, implying that the oil substrate was the principal carbon source for the biosynthesis of tacrolimus, and this was directly proven by experiments using $^{14}C$-glucose and $^{14}C$-oleate substrates. Furthermore, com oil induced the formation of lipase by the mutant strain, whereas the addition of glucose significantly repressed lipase activity. The lipase activity exhibited by the FK-506-overproducing mutants was also observed to be directly proportional to their tacrolimus yield, indicating that a high lipase activity is itself a crucial factor for tacrolimus production. A feasibility study with a 200-1 pilot-scale fermentor and the best strain (Tc-XII-15322) identified in this work revealed a high volumetric and specific productivity of about 495 mg/l and 0.34 mg/mg dry mycelium, respectively.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1170-1175
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• 2004

The involvement of the relA and rsh genes in the morphological and physiological differentiation of Streptomyces clavuligerus was evaluated with the relA and rsh genes mutants. The morphological differentiation of S. clavuligerus was greatly affected by the disruption of the relA gene, but not very much by the disruption of the rsh gene. The altered morphological characteristics were completely restored by the complementation of the corresponding disrupted genes. Thus, it was apparent that the mycelial morphology and clavulanic acid production were severely affected by the disruption of the relA gene. Production of clavulanic acid in the submerged batch culture and glycerol-limited chemostat showed that production was inversely related to the specific growth rate in the wild-type strain. However, the production of clavulanic acid in the ${\Delta}relA$ and ${\Delta}rsh$ null mutants was completely abolished. Therefore, it seems plausible that the stringent response of S. clavuligerus to starvation for amino acids is governed mainly by ReIA, rather than Rsh, and that the (p)ppGpp synthesized immediately after the depletion of amino acids triggers the initiation of pathways for both morphological and physiological differentiation in this species.

• KSBB Journal
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• 제14권4호
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• pp.440-444
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• 1999

S. clavulirerus ATCC 27064 균주는 약 200 mg/L의 clavulanic acid를 생산하였지만, 이 균주를 NTG에 의해 돌연변이 시킴으로써 얻어진 S. clavuligerus KK를 같은 배양조건에서 약 5배 많은 1150 mg/L의 clavulanic acid를 생산하였다. 무기염이 첨가되지 않은 clavulanic acid의 기본 생산 배지에서는 clavulanic acid의 생산량은 1150 mg/L였으나 다양한 무기염 중에서 MgSO$_4$가 첨가된 배지에서는 약 2000 mg/L나 생산되었다. 따라서 기본 배지조성인 1%(v/v) glycerol, 1.5%(w/v) soybean flour, 0.1%(w/v) $KH_2PO_4$, 0.2%(v/v) soybean oil에 0.4%(w/v) MgSO$_4$를 첨가함으로써 clavulanic acid의 생산을 높일 수 있었다.Airlift 와 bubble column 및 교반식 생물반응기에서 회분식 배양을 했을 경우 clavulanic acid의 생산은 airlift 생물 반응기에서는 1100 ng/L, bubble column 생물 반응기에서는 1450 mg/L, 교반식 생물 반응기에서는 1500 mg/L가 생산되어, 교반식 반응기가 적절하다고 생각되며 이는 배양액이 시간이 지날수록 점도가 높아지기 때문에 임펠러에 의한 교반 효과가 있는 교반식 반응기에서 물질전달이 가장 용이하기 때문인 것으로 보인다. 특히 교반식 생물 반응기에서 용존산소농도에 따라 교반속도를 조절하여 회분식 배양을 했을 경우가 수율이 0.18, 생산성은 250mg/Lㆍday로 가장 우수하게 나타났다.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.417-426
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• 2008

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by increasing dosoge of a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1538-1545
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• 2007

Clavulanic acid (CA) is an inhibitor of ${\beta}$-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHLl, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHLl3, and a 23.8-fold increase with pNQ1. The integrative pNQl strain has been successfully applied to enhance production.