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Chem. Eng. J., Available online 21 October 2022 | https://doi.org/10.1016/j.cej.2022.139911 A highly efficient and versatile genetic engineering toolkit for a methanotroph-based biorefinery

Jiyeong Jeong^a,1, Tae Hyun Kim^a,b,1, Nulee Jang^a, Minji Ko^a,b, Seong Keun Kim^a, Ji In Baek^a,c, Georgii Emelianov^a,b, Eugene Rha^a, Kil Koang Kwon^a, Haseong Kim^a,b, Eun Yeol Lee^d, Dae-Hee Lee^a,b,e, Hyewon Lee^a, Seung-Goo Lee^a,b

^aSynthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
^bDepartment of Biosystems and Bioengineering, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
^cDepartment of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, Republic of Korea
^dDepartment of Chemical Engineering (Integrated Engineering), Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea
^eDepartment of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon-si, Gyeonggi-do 16419, Republic of Korea

¹These authors contributed equally to this work.
Corresponding authors: D. Lee, H. Lee and S.-G. Lee

Abstract

Methanotrophs are promising and sustainable cell factory platforms owing to their ability to convert the most potent greenhouse gas, methane to valuable bioproducts. Genetic engineering toolkits for methanotrophs are extremely limited. Here, we present a phenol-inducible promoter for the high-level expression of exogenous genes in methanotrophs. The phenol-inducible gene expression system showed high dose-dependency and homogeneity in methanotrophs. Using the phenol-inducible CRISPR-base editor (BE), we developed a highly efficient methanotroph genome editing system. The CRISPR-BE system efficiently introduced an early stop codon into the target gene, enabling one-step markerless genome editing in Methylococcus capsulatus Bath. We adopted this simple and efficient genome editing tool to produce mevalonate in the engineered M. capsulatus Bath. The native phosphoketolase pathway was reinforced in M. capsulatus Bath to increase the carbon flux via acetyl-CoA towards mevalonate. This engineered M. capsulatus Bath produced the maximum concentration of 2,090 mg/L mevalonate from methane, which is the highest amount of synthetic biochemicals produced from methane in methanotrophs. Here we present not only an efficient addition to the genetic engineering toolkit for methanotrophs but also a useful platform for the development of a methanotroph cell factory.

논문정보

형식Research article
게재일2022년 12월 (BRIC 등록일 2022-12-30)
연구진국내 연구진
분야 생명과학 > 생물공학

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