Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6494091 | Metabolic Engineering | 2018 | 40 Pages |
Abstract
Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66â¯mg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7â¯mg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain âldh âack âmdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8â¯mg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2â¯mg/L with a yield of 0.0318â¯g-(2,3-BDO) /g-CH4 were obtained under O2-limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs.
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Authors
Anh Duc Nguyen, In Yeub Hwang, Ok Kyung Lee, Donghyuk Kim, Marina G. Kalyuzhnaya, Rina Mariyana, Susila Hadiyati, Min Sik Kim, Eun Yeol Lee,