Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Production of a hypothetical polyene substance by activating a cryptic fungal PKS-NRPS hybrid gene in Monascus purpureus

홍국Monascus purpureus에서 진균 PKS-NRPS 하이브리드 유전자의 발현 유도를 통한 미지 polyene 화합물의 생성

  • Suh, Jae-Won (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Balakrishnan, Bijinu (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Lim, Yoon Ji (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Lee, Doh Won (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Choi, Jeong Ju (Department of Biosciences and Bioinformatics, Myongji University) ;
  • Park, Si-Hyung (Department of Oriental Medicine Resources and Institute for Traditional Korean Medicine Industry, Mokpo National University) ;
  • Kwon, Hyung-Jin (Department of Biosciences and Bioinformatics, Myongji University)
  • Received : 2018.02.06
  • Accepted : 2018.03.06
  • Published : 2018.03.31
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Abstract

Advances in bacterial and fungal genome mining uncover a plethora of cryptic secondary metabolite biosynthetic gene clusters. Guided by the genome information, targeted transcriptional derepression could be employed to determine the product of a cryptic gene cluster and to explore its biological role. Monascus spp. are food grade filamentous fungi popular in eastern Asia and several genome data belong to them are now available. We achieved transcription activation of a cryptic fungal polyketide synthase-nonribosomal peptide synthase gene Mpfus1 in Monascus purpureus ${\Delta}MpPKS5$ by inserting Aspergillus gpdA promoter at the upstream of Mpfus1 through double crossover gene replacement. The gene cluster with Mpfus1 show a high similarity to those for the biosynthesis of conjugated polyene derivatives with 2-pyrrolidone ring and the mycotoxin fusarin is the representative member of this group. The ${\Delta}MpPKS5$ is incapable of producing azaphilone pigment, providing an excellent background to identify chromogenic and UV-absorbing compounds. Activation of Mpfus1 resulted in a yellow hue on mycelia and its methanol extract exhibit a maximum absorption at 365 nm. HPLC analysis of the organic extracts indicated the presence of a variety of yellow compounds in the extract. This implies that the product of MpFus1 is metabolically or chemically unstable. LC-MS analysis guided us to predict the MpFus1 product and to propose that the Mpfus1-containing gene cluster encode the biosynthesis of a desmethyl analogue of fusarin. This study showcases the genome mining in Monascus and the possibility to unveil new biological activities embedded in it.

박테리아와 진균의 유전체 정보 탐색을 통하여 이차대사 생합성을 지정하는 다수의 잠재 유전자군을 찾을 수 있으며, 유전체 정보를 기반으로 특정 유전자의 발현을 활성화하여 잠재 유전자군의 생성물을 추론하고, 해당 물질의 생물학적 기능을 연구하는 것이 가능하다. 동아시아 지역에서 잘 알려진 식용 사상진균 홍국에 대하여 몇 몇 유전체 정보가 공개되어있으며, 본 연구에서는 Monascus purpureus ${\Delta}MpPKS5$ 균주에서 polyketide synthase-nonribosomal peptide synthase 유전자 Mpfus1 상단에 Aspergillus gpdA 프로모터를 삽입하는 방식으로 이 유전자의 발현을 활성화하였다. Mpfus1 유전자군은 2-pyrrolidone/conjugated polyene 구조를 갖는 물질의 생합성 유전자군들과 높은 유사성을 보이며, 이들 화합물 그룹에서 진균 독소인 fusarin이 잘 알려져 있다. ${\Delta}MpPKS5$ 균주는 홍국 azaphilone 색소 생산 능력이 소실된 균주이며 색소 및 자외선 흡수 특성을 보이는 화합물들의 동정에 적절한 균주이다. Mpfus1 활성화는 균사체가 노란색을 띠도록 유도하며, 균사체의 methanol 추출액은 365 nm에서 최대 흡광도를 보임을 확인할 수 있었다. 해당 추출액의 HPLC 분석을 통하여 다수의 화합물들이 포함되어 있음을 확인할 수 있었으며 이를 통하여 MpFus1 효소의 생성물이 대사적, 화학적으로 불안정함을 추론할 수 있다. Mpfus1 활성화 균주 추출물을 LC-MS로 분석하여 MpFus1 생성물의 구조를 유추하여 Mpfus1 유전자군이 fusarin의 탈메틸 유사체 생합성을 지정하는 것으로 제안할 수 있었다. 본 연구는 홍국 균주에서 유전체 기반-미지 화합물 발굴 연구의 예를 제시하고 홍국 균주에서 새로운 생리활성의 동정 가능성을 시사하여 준다.

Keywords

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