• Journal of Microbiology and Biotechnology
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• 제33권12호
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• pp.1681-1691
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• 2023

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

• BMB Reports
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• 제29권1호
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• pp.88-91
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• 1996

The anaerobically expressed gene aeg-46.5, which had been identified by the operon fusion technique with a hybrid bacteriophage of ${\lambda}$ and Mu, ${\lambda}$placMu53, was studied for its expression pattern and growth. The expression of aeg-46.5 was studied in the wild-type cell and mutant cells that have mutation (s) in the control gene of anaerobic respiration (fnr) and nitrate response (narL and narP). The ${\beta}$-galactosidase reporter gene showed maximum expression in narL host after two hours of aerobic to anaerobic switch in M9-Glc-nitrate medium. Both 40 mM and 100 mM concentrations of nitrate ion in the medium had little effect on expression level. We propose that aeg-46.5 is subject to multiple regulations of anaerobic activation by Fnr, nitrate activation by NarP and repression mediated by NarL.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.762-766
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• 2001

The nar promoter of Escherichia coli was known to induce maximally under anaerobic or microaerobic conditions in the presence of nitrate. In this study, the nar promoter was tested to see whether the expression level of a reporter gene which fused lacZ gene at nar promoter's downstream, in the some gram negative host strains(Agrobacterium, Pseudomonas and Rhizobium). A nar promoter system(Combination of nar promoter and gram negative strain) was grown under aerobic conditions to absorbance at 600 nm of nearly 2.0 and then, the nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic condition in the fermentor cultures, using different gram negative hosts. For a wild type nar promoter (pNW61), it was possible to maintain production of ${\beta}-galactosidase$ activity per cell(specific ${\beta}-galactosidase$ activity) at 14,000, 9600, 45 Miller units in the presence of 1% nitrate. and for a nitrate - independent nar promoter (pNW618) at 12,000, 10,400 and 58 Miller units in the absence of nitrate ion, respectively.

• 한국지하수토양환경학회:학술대회논문집
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• 한국지하수토양환경학회 2000년도 추계학술대회
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• pp.207-208
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• 2000

Since trichloroethylene (TCE), dichloroethylene (DCE), and vinyl chloride (VC) arise from anaerobic degradation of tetrachloroethylene (PCE) and TCE, there is interest in creating aerobic remediation systems that avoid the highly toxic VC and cis-DCE which predonominate in anaerobic degradation. However, it seemed TCE could not be degraded aerobically without an inducing compound (which also competitively inhibits TCE degradation). It has been shown that TCE induces expression of both the toluene dioxygenase of p. putida F1 as well as toluene-p-monooxygenase of P.mendocina KRI. We investigated here the ability of PCE, TCE, and chlorinated phenols to induce toluene-o-xylene monooxygenase (ToMO) from P.stutzeri OX1. ToMO has a relaxed regio-specificity since it hydroxylates toluene in the ortho, meta, and para positions; it also has a broad substrate range as it oxidizes o-xylene, m-xylene, p-xylene, toluene, benzene, ethylbenzene, styrene, and naphthalene; chlorinated compounds including TCE, 1, 1-DCE, cis-DCE, trans-DCE, VC, and chloroform : as well as mixtures of chlorinated aliphatics (Pseudomonas 1999 Maui Meeting). ToMO is a multicomponent enzyme with greatest similarity to the aromatic monooxygenases of Burkholderia pickettii PKO1 and P.mendocina KR1. Using P.sturzeri OX1, it was found that PCE induces P.mendocina KR1 Using P.situtzeri OX1, it was found that PCE induces ToMO activity measured as naphthalene oxygenase activity 2.5-fold, TCE induces 2.3-fold, and toluene induces 3.0 fold. With the mutant P.stutzeri M1 which does not express ToMO, it was also found there was no naphthalene oxygenate activity induced by PCE and TCE; hence, PCE and TCE induce the tow path. Using P.putida PaW340(pPP4062, pFP3028) which has the tow promoter fused to the reporter catechol-2, 3-dioxygenase and the regulator gene touR, it was determined that the tow promoter was induced 5.7-, 7.1-, and 5.2-fold for 2-, 3-, 4-chlorophenol, respectively (cf. 8.9-fold induction with o-cresol) : however, TCE and PCE did not directly induce the tou path. Gas chromatography and chloride ion analysis also showed that TCE induced ToMO expression in P.stutzeri OX1 and was degraded and mineralized. This is the first report of significant PCE induction of any enzyme as well as the first report of chlorinated compound induction of the tou operon. The results indicate TCE and chlorinated phenols can be degraded by P.stutzeri OX1 without a separate inducer of the tou pathway and without competitive inhibition.

• KSBB Journal
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• 제11권4호
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• pp.431-437
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• 1996

본 연구에서는 질산염(nitrate) 존재하에서 혐기 조건이 되면 최대로 발현되는 변형된 naT 프로모터 를 대장균내에서 단백질을 대량 생산하기 위한 발현 운반체로 쓰일 수 있는지를 조사하였다. 이를 위해 용존산소농도에 따라 naT 프로모터의 유도에 영향을 미치는 염색체 fnT 유전자가 발현되지 못하는 대장 균(ES200l)에 pBR322 플라스미드에 프로모터상 의 10 지역에서 특정부위돌연변이화에 의해 con­s sensus sequence로 바핀 변형된(modified) naT 프 로모터(pMW616)가 도입되어 있는 계(pMW616/ E ES2001 )가 이용되었다. 이 변형된 naT 프로모터의 하류에는 구조유전자(structural gene) 인 질산염 환 원효소대 신에 ${\beta}$-galactosidase를 발현하는 lacZ 유 전자가 클로닝되어 있다. 이 변형된 naT 프로모터의 유도에 대한 최적조건을 찾기 위해 변형된 naT 프로 모터를 유도시키는 방법, 변형된 naT 프로모터가 최 대로 유도되는 질산엽의 농도, 말현된 $\beta$galactosid a ase의 양 빛 변형된 naT 프로모터가 유도되는 특성 들이 조사되었다. 이에 대한 실험으로부터 다음과 같은 결론들이 얻어졌다; 이 변형된 naT 프로모터로 부터 ${\beta}$-galactosidase의 말현은 질산염의 농도에 의 해서는 크게 영향을 받지 않았다. 한편, 변형된 naT 프로모터로부터 ${\beta}$-galactosidase의 말현은 성장단계 에서 대장균을 호기상태에서 OD600이 약 2.27~ 될 때까지 키우다가 유도시 혐기상태로 만드는 것이 가 장 유리하였으며, 이 때 발현된 ${\beta}$-galactosidase의 비활성도는 약 13,000 Miller unit였다. 하지만, 이 변형된 naT 프로모터는 이켓을 유도시키기 전에도 발현된 ${\beta}$-galactosidase의 비활성도가 약 6,000 M Miller unit로 매우 높음으로 인하여 이 변형된 naT 프로모터는 원하는 단백질을 유도성보다는 구성적으 로 발현시키는데 더 적합한 프로모터였다.

• 생명과학회지
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• 제16권4호
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• pp.632-639
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• 2006

본 연구에서는 lacZ transcriptional fusion plasmid를 이용하여 광합성 세균인 Rhodobacter sphaeroides에서의 7가지 광합성유전자 (puf, puc, puhA, bchC, bchE, bchF, bchI) 발현의 경향과 조절을 조사하였다. R. sphaeroides에서 puhA와 bchI를 제외한 모든 광합성유전자들이 호기적 조건과 비교했을 때 혐기적 조건에서 더욱 강하게 발현되었다. puhA 유전자는 bchFNBHLM-RSP0290과 operon을 형성하며, bchI 유전자는 crtA와 operon을 이루는 것으로 나타났다. 광합성 조건에서 자란 R. sphaeroides의 puf, puc, bchCXYZ operon의 발현은 빛의 세기에 비례하는 반면, bchFNBHLM(RSP0290 puhA) operon의 발현은 빛의 세기에 반비례 하였다. bchEJG의 발현은 $10\;W/m^2$의 빛이 조사된 광합성 조건에서 제일 낮았으며, $100\;W/m^2$의 빛의 광합성 조건에서 가장 높았다. R. sphaeroides의 산소인지와 빛 인지에 관련된 세 가지 주요 조절기작에 의한 광합성유전자 조절은 다음과 같다. puf와 bchC는 PpsR repressor와 PrrBA two-component system에 의해 조절된다. 그리고 puc operon은 PpsR, FnrL, PrrBA system에 의해 조절된다. bchE의 발현은 FnrL과 PrrBA system에 의해 조절되는 반면, bchF는 오로지 PpsR에 의해서만 조절된다. PpsR repressor는 강한 세기의 빛 조건에서 bchf 발현억제의 원인이 되며, FnrL은 그 자체가 산소를 인지하는 기능 이외에도 세포질의 산화/환원 상태의 인지에 관련될 것으로 보인다.