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 35.66 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 57.7 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 ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8 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 86.2 mg/L with a yield of 0.0318 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.
journal_name
Metab Engjournal_title
Metabolic engineeringauthors
Nguyen AD,Hwang IY,Lee OK,Kim D,Kalyuzhnaya MG,Mariyana R,Hadiyati S,Kim MS,Lee EYdoi
10.1016/j.ymben.2018.04.010subject
Has Abstractpub_date
2018-05-01 00:00:00pages
323-333eissn
1096-7176issn
1096-7184pii
S1096-7176(18)30110-1journal_volume
47pub_type
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