[논문]PET 생분해에 관여하는 방선균 유래 PETase 유전자의 이종숙주 발현

양수빈; 유연진; 김응수; 최시선

doi:10.48022/mbl.2211.11002

PET 생분해에 관여하는 방선균 유래 PETase 유전자의 이종숙주 발현
Heterologous Expression of Streptomyces PETase Gene Involved in PET Biodegradation 원문보기

Microbiology and biotechnology letters = 한국미생물·생명공학회지, v.50 no.4, 2022년, pp.501 - 507

양수빈 (인하대학교 생물공학과) , 유연진 (인하대학교 생물공학과) , 김응수 (인하대학교 생물공학과) , 최시선 (인하대학교 생물공학과)

초록
AI-Helper

대표적인 플라스틱 소재인 PET (Polyethylene terephthalate)는 높은 내구성, 경제성과 같은 유용한 물리화학적 특성으로 병, 섬유, 용기 등 다양한 산업 분야에 사용되고 있다. 최근 일회용품을 비롯한 플라스틱 사용량 증가로 인해, 이를 처리하기 위한 방법이 필요한 상황이다. 기존의 매립, 소각 등과 같이 자연상태에 노출되는 방법과 달리 최근 미생물을 이용한 친환경적인 방법이 주목받고 있다. 본 연구에서는 PETase 유전자를 가지고 있는 토양 유래 방선균 Streptomyces. javensis Inha503를 선별하고, agar plate diffusion assay를 통해 PU (Polyurethane) 가수분해 능력을 확인하였다. 해당 균주를 PET과 함께 한달 간 배양하였고, 주사전자현미경을 통해 PET 분해능력을 확인하였다. 또한, S. javensis Inha503 유전체 탐색에서 선별된 PETase 유전자를 PET 분해능이 없는 이종숙주 S. lividans와 S. coelicolor 균주에 도입하여 PET 분해능을 확인함으로써, 방선균 유래 PETase 유전자의 활성을 최초로 확인하였다.

Abstract ▼ AI-Helper

PET (Polyethylene terephthalate), a representative plastic material, has useful physicochemical properties such as high durability and economic feasibility, and is used in various industrial fields such as bottles, fibers, and containers. Due to the recent increase in plastic usage including disposable products, eco-friendly strategy using microorganisms have drawn attention differentiated from conventional landfill and incineration methods. In this study, a soil-derived Streptomyces javensis Inha503 containing a PETase gene was selected and the ability to hydrolyze PU (Polyurethane) was confirmed through agar plate diffusion assay. This strain was cultured with PET for a month, and PET decomposition ability was also confirmed through a scanning electron microscope. Moreover, cloning and heterologous expression of S. javensis Inha503 PETase gene exhibited PET activity in the PETase non-containing S. coelicolor, confirming for the first time the presence of functional PETase gene in Streptomyces species.

주제어

표/그림 (4)

그림 Fig. 1. Overall scheme of Streptomyces PETase gene cloning and heterologous expression.
그림 Fig. 2. In silico analysis of InPETase. (A) Genome information of S. javensis Inha503. Size and location on genome of InPETase; left. Mechanisms of PET degradation; right, (B) Sequence alignment of S. javensis Inha503 InPETase and I. sakaiensis IsPETase. The red box is serine hydrolase motif (GXSXG) belonging to α/β hydrolase superfamily. Catalytic triad are shown in red and oxyanion hole is shown in blue.
그림 Fig. 3. SEM image of commercial bottle-derived PET and PET film after incubation with S. javensis Inha503. (A) PET bottle (w/o culture), (B) PET bottle, R5 medium, Inha503, (C) PET bottle, GSS medium, Inha503, (D) bare PET film (with culture in TSB medium), (E) PET film, TSB medium, Inha 503, (F) PET film, GSS medium, Inha503. All the images were taken at a magnification of x50K.
그림 Fig. 4. PCL agar plate diffusion assay with heterologous expression. (A) Graphical representation of the pSE34 shuttle vector including InPETase, (B) PCL agar plate diffusion assay. a; wild type Inha503 b; S. lividans TK21 c; S. coelicolor M511 d,e; S. lividans TK21 / pSE34_- PETase f,g; S. coelicolor M511 / pSE34_PETase. The yellow circles show the clear zone created by InPETase

참고문헌 (46)

Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, et al. 2018. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat. Commun. 9: 382.

상세보기
Plastics Europe. 2008. The compelling facts about plastics: an？analysis of plastic production, demand and recovery for 2006 in？Europe.
Plastics Europe. 2021. Plastics - the Facts 2021.
Rochman CM, Browne MA, Halpern BS, Hentschel BT, Hoh E,？Karapanagioti HK, et al. 2013. Classify plastic waste as hazardous.？Nature 494: 169-171.

상세보기
Patricio Silva AL, Prata JC, Walker TR, Duarte AC, Ouyang W,？Barcelo D, et al. 2021. Increased plastic pollution due to COVID-19 pandemic: Challenges and recommendations. Chem. Eng. J.？405: 126683.

상세보기
Knoblauch D, Mederake L. 2001. Government policies combatting plastic pollution. Curr. Opin. Toxicol. 28: 87-96.
Chung MS, Lee WH, You YS, Kim HY, Park KM. 2003. Manufacturing multi-degradable food packaging films and their degradability. Korean J. Food. Sci. Technol. 35: 877-883.
Organization for Economic Co-operation and Development.？2022. Global Plastic Outlook.
Teuten EL, Saquing JM, Knappe DR, Barlaz MA, Jonsson S, Bjorn？A, et al. 2009. Transport and release of chemicals from plastics to？the environment and to wildlife. Philos. Trans. R. Soc. Lond. B. Biol.？Sci. 364: 2027-45.
Ragaert K, Delva L, Van Geem K. 2017. Mechanical and chemical？recycling of solid plastic waste. Waste. Manag. 69: 24-58.

상세보기
Drzyzga O, Prieto A. 2019. Plastic waste management, a matter？for the 'community'. Microb. Biotechnol. 12: 66-68.

상세보기
Almeida EL, Carrillo Rincon AF, Jackson SA, Dobson ADW. 2019.？In silico screening and heterologous expression of a polyethylene terephthalate hydrolase (PETase)-like enzyme (SM14est)？with polycaprolactone (PCL)-degrading activity, from the？marine sponge-derived strain Streptomyces sp. SM14. Front.？Microbiol. 10: 2187.
Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y,？et al. 2016. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351: 1196-1199.

상세보기
Wei R, Zimmermann W. 2017. Biocatalysis as a green route for？recycling the recalcitrant plastic polyethylene terephthalate.？Microb. Biotechnol. 10: 1302-1307.

상세보기
Kawai F, Kawabata T, Oda M. 2019. Current knowledge on enzymatic PET degradation and its possible application to waste？stream management and other fields. Appl. Microbiol. Biotechnol.？103: 4253-4268.

상세보기
Bornscheuer UT. 2016. Feeding on plastic. Science 351: 1154-1155.

상세보기
Chen CC, Han X, Ko TP, Liu W, Guo RT. 2018. Structural studies？reveal the molecular mechanism of PETase. FEBS J. 285: 3717-3723.

상세보기
Fecker T, Galaz-Davison P, Engelberger F, Narui Y, Sotomayor M,？Parra LP, et al. 2018. Active site flexibility as a hallmark for efficient PET degradation by I. sakaiensis PETase. Biophys J. 114:？1302-1312.

상세보기
Son HF, Cho IJ, Joo S, Seo H, Sagong HY, Choi SY, et al. 2019.？Rational protein engineering of thermo-stable PETase from？Ideonella sakaiensis for highly efficient PET degradation. ACS？Catal. 9: 3519-3526.

상세보기
Liu B, He L, Wang L, Li T, Li C, Liu H, et al. 2018. Protein crystallography and site-direct mutagenesis analysis of the poly(ethylene？terephthalate) hydrolase PETase from Ideonella sakaiensis.？Chembiochem. 19: 1471-1475.

상세보기
Ma Y, Yao M, Li B, Ding M, He B, Chen S, et al. 2018. Enhanced？poly(ethylene terephthalate) hydrolase activity by protein engineering. Engineering 4: 888-893.

상세보기
Kim HR, Lee HM, Yu HC, Jeon E, Lee S, Li J, Kim DH. 2020. Biodegradation of polystyrene by Pseudomonas sp. isolated from the？gut of superworms (Larvae of Zophobas atratus). Environ. Sci.？Technol. 54: 6987-6996.

상세보기
Woo S, Song I, Cha HJ. 2020. Fast and facile biodegradation of？polystyrene by the gut microbial flora of Plesiophthalmus davidis？larvae. Appl. Environ. Microbiol. 86: e01361-20.
Kim JW, Park SB, Tran QG, Cho DH, Choi DY, Lee YJ, et al. 2020.？Functional expression of polyethylene terephthalate-degrading？enzyme (PETase) in green microalgae. Microb. Cell Fact. 19: 97.

상세보기
Choi SS, Katsuyama Y, Bai L, Deng Z, Ohnishi Y, Kim ES. 2018.？Genome engineering for microbial natural product discovery.？Curr. Opin. Microbiol. 45: 53-60.

상세보기
Kim HY, Han CY, Park JS, Oh SH, Kang SH, Kim ES, et al. 2018.？Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic. Sci. Rep. 8: 13584.

상세보기
Kang HS, Kim ES. 2021. Recent advances in heterologous？expression of natural product biosynthetic gene clusters in？Streptomyces hosts. Curr. Opin. Biotechnol. 69: 118-127.

상세보기
Kim SH, Lee HN, Kim HJ, Kim ES. 2011. Transcriptome analysis of？an antibiotic downregulator mutant and synergistic actinorhodin？stimulation via disruption of a precursor flux regulator in？Streptomyces coelicolor. Appl. Environ. Microbiol. 77: 1872-1877.

상세보기
Duong CTP, Lee HN, Choi SS, Lee SY, Kim ES. 2009. Functional？expression of SAV3818, a putative TetR-family transcriptional？regulatory gene from Streptomyces avermitilis, stimulates antibiotic production in Streptomyces species. J. Microbiol. Biotechnol.？19: 136-139.

원문보기 상세보기
Lee HN, Kim JS, Kim P, Lee HS, Kim ES. 2013. Repression of antibiotic downregulator WblA by AdpA in Streptomyces coelicolor.？Appl. Environ. Microbiol. 79: 4159-4163.

상세보기
Hwang YB, Lee MY, Park HJ, Han K, Kim ES. 2007. Isolation of？putative polyene-producing actinomycetes strains via PCR-based genome screening for polyene-specific hydroxylase？genes. Process. Biochem. 42: 102-107.

상세보기
Nah JH, Park SH, Yoon HM, Choi SS, Lee CH, Kim ES. 2012. Identification and characterization of wblA-dependent tmcT regulation during tautomycetin biosynthesis in Streptomyces sp.？CK4412. Biotechnol. Adv. 30: 202-209.

상세보기
Park HJ, Kim ES. 2003. An inducible Streptomyces gene cluster？involved in aromatic compound metabolism. FEMS Microbiol.？lett. 226: 151-157.
Pyeon H, Nah HJ, Kang SH, Choi SS, Kim ES. 2017. Heterologous？expression of pikromycin biosynthetic gene cluster using？Streptomyces artificial chromosome system. Microb. Cell. Fact.？16: 96.

상세보기
Kim ES, Hopwood DA, Sherman DH. 1994. Analysis of type II？polyketide beta-ketoacyl synthase specificity in Streptomyces？coelicolor A3 (2) by trans complementation of actinorhodin？synthase mutants. J. Bacteriol. 176: 1801-1804.

상세보기
Park HS, Kang SH, Park HJ, Kim ES. 2005. Doxorubicin productivity？improvement by the recombinant Streptomyces peucetius with？high-copy regulatory genes cultured in the optimized media？composition. J. Microbiol. Biotechnol. 15: 66-71.
Park NS, Myeong JS, Park HJ, Han KB, Kim SN, Kim ES. 2005. Characterization and culture optimization of regiospecific cyclosporin？hydroxylation in rare actinomycetes species. J. Microbiol. Biotechnol. 15: 188-191.

원문보기 상세보기
Im JH, Kim MG, Kim ES. 2007. Comparative transcriptome analysis？for avermectin overproduction via Streptomyces avermitilis？microarray system. J. Microbiol. Biotechnol. 17: 534-538.

원문보기 상세보기
Kim CY, Park HJ, Kim ES. 2003. Heterologous expression of？hybrid type II polyketide synthase system in Streptomyces？species. J. Microbiol. Biotechnol. 13: 819-822.
Park NS, Park HJ, Han KB, Kim ES. 2006. Heterologous expression？of novel cytochrome P450 hydroxylase genes from Sebekia？benihana. J. Microbiol. Biotechnol. 16: 295-298.
Ma L, Du L, Chen H, Sun Y, Huang S, Kim ES, et al. 2015. Reconstitution of the in vitro activity of the cyclosporine-specific P450？hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system. Appl. Environ.？Microbiol. 81: 6268-6275.

상세보기
Choi SS, Lee HN, Park E, Lee SJ, Kim ES. 2020. Recent advances in？microbial production of cis,cis-muconic acid. Biomolecules 10: 1238.

상세보기
Shin WS, Lee D, Lee SJ, Chun GT, Choi SS, Kim ES, et al. 2018.？Characterization of a non-phosphotransferase system for cis,？cis-muconic acid production in Corynebacterium glutamicum.？Biochem. Biophys. Res. Commun. 499: 279-284.

상세보기
Brott S, Pfaff L, Schuricht J, Schwarz JN, Bottcher D, et al. 2021.？Engineering and evaluation of thermostable IsPETase variants？for PET degradation. Eng. Life. Sci. 22: 192-203.
Molitor R, Bollinger A, Kubicki S, Loeschcke A, Jaeger KE, Thies S.？2019. Agar plate-based screening methods for the identification？of polyester hydrolysis by Pseudomonas species. Microb. Biotechnol.？13: 271-284.
Nishida H, Tokiwq Y. 1993. Distribution of poly(β-hydroxybutyrate) and poly(ε-caprolactone) aerobic degrading microorganisms in different environments. World J. Microbiol. Biotechnol. 28:？2929-2935.？

상세보기

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증