Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Substrate chain-length specificities of polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa P-5

Pseudomonas aeruginosa P-5에 존재하는 polyhydroxyalkanoate synthase PhaC1과 PhaC2의 기질특이성

  • Woo, Sang Hee (Department of Microbiology and Molecular Biology, Chungnam National University) ;
  • Lee, Sun Hee (Department of Microbiology and Molecular Biology, Chungnam National University) ;
  • Rhee, Young Ha (Department of Microbiology and Molecular Biology, Chungnam National University)
  • 우상희 (충남대학교 미생물.분자생명과학과) ;
  • 이선희 (충남대학교 미생물.분자생명과학과) ;
  • 이영하 (충남대학교 미생물.분자생명과학과)
  • Received : 2016.09.13
  • Accepted : 2016.09.23
  • Published : 2016.12.31
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Abstract

Pseudomonas aeruginosa P-5 is an unusual organism capable of synthesizing polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyvalerate (3HV) and medium-chain-length (MCL) 3-hydroxyalkanoate (3HA) monomer units when C-odd alkanoic acids are fed as the sole carbon source. Evaluation of the substrate chain-length specificity of two P. aeruginosa P-5 PHA synthases ($PhaC1_{P-5}$ and $PhaC2_{P-5}$) by heterologous expression of $PhaC1_{P-5}$ and $PhaC2_{P-5}$ genes in Pseudomonas putida GPp104 revealed that $PhaC2_{P-5}$ incorporates both 3HV and MCL 3HAs into PHA, whereas $PhaC1_{P-5}$ favors only MCL 3HAs for polymerization. In order to obtain $PhaC2_{P-5}$ mutants with altered substrate specificity, site-specific mutagenesis for $PhaC2_{P-5}$ was conducted. Amino acid substitutions of $PhaC2_{P-5}$ at two positions (Ser326Thr and Gln482Lys) were very effective for synthesizing copolymers with a higher 3HV fraction. When recombinant P. putida GPp104 harboring double mutated $phaC2_{P-5}$ gene ($phaC2_{P-5}QKST$) was grown on nonanoic acid, 2.5-fold increase of copolymer content with 3.8-fold increase of 3HV fraction was observed. The $phaC2_{P-5}QKST$-containing Ralstonia eutropha PHB-4 supplemented with valeric acid also produced copolymers consisting of 3HV and 3-hydroxyheptanoate with a high 3HV fraction. These results suggest that recombinants containing $phaC2_{P-5}QKST$ could be useful for production of new PHA copolymers with improved material properties.

Pseudomonas aeruginosa P-5 균주는 홀수개의 탄소수를 갖는 지방산으로부터 3-hydroxyvalerate (3HV)와 medium-chain-length (MCL) 3-hydroxyalkanoates (3HAs) 단위체로 구성된 popolyhydroxyalkanoate (PHA) 공중합체를 생산하는 특이한 성질을 갖고 있다. 이 균주가 갖고 있는 2개의 MCL-PHA synthases ($PhaC1_{P-5}$와 $PhaC2_{P-5}$)의 탄소길이에 따른 기질특이성을 비교하기 위하여 각각의 유전자를 PHA 생합성능이 결여된 돌연변이주 Pseudomonas putida GPp104에 도입하고 발현시킨 결과, $PhaC2_{P-5}$는 3HV와 MCL 3HAs로 이루어진 공중합체를 생산하지만 $PhaC1_{P-5}$는 단지 MCL 3HAs로 구성된 공중합체를 생산하였다. 이는 $PhaC2_{P-5}$가 $PhaC1_{P-5}$과는 달리 보다 짧은 탄소길이의 3-hydroxyvaleryl Co-A를 기질로 인지하여 합성반응에 이용할 수 있음을 보여주는 것이다. 또한 $PhaC2_{P-5}$의 효소활성 및 기질특이성의 변화를 유도하기 위하여 위치지정 돌연변이생성을 수행하고 P. putida GPp104과 다른 PHA 생합성능 결여 돌연변이주인 Ralstonia eutropha $PHB^-4$에서 발현시킨 결과, $PhaC2_{P-5}$ 내 두 개 아미노산의 치환(Ser326Thr과 Gln482Lys)이 공중합체의 3HV 함량을 크게 증진시키는 효과를 보였다. 두 개 아미노산이 모두 치환된 $PhaC2_{P-5}$ 유전자($phaC2_{P-5}QKST$)를 갖는 P. putida GPp104를 nonanoic acid가 탄소원으로 함유된 배지에서 배양하였을 때, 모균주에 비해 공중합체 함량과 공중합체 내 3HV 함량이 각각 2.5배 및 3.5배 증가하였다. 따라서 $phaC2_{P-5}QKST$를 포함하는 재조합 균주는 개량된 물성의 신규PHAs 생산에 유용할 것으로 기대된다.

Keywords

References

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