초록
<P><B>Abstract</B></P> <P>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 <I>Methylomicrobium alcaliphilum</I> 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 (<I>budABC</I>) from <I>Klebsiella pneumoniae</I>, 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 <I>budA</I> of <I>K. pneumoniae</I> and <I>budB</I> of <I>Bacillus subtilis</I> under the control of the Tac promoter. The genome-scale metabolic network reconstruction of <I>M. alcaliphilum</I> 20Z enabled <I>in silico</I> gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The <I>ldh</I>, <I>ack</I>, and <I>mdh</I> genes in <I>M. alcaliphilum</I> 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆<I>ldh</I> ∆<I>ack</I> ∆<I>mdh</I> 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-CH<SUB>4</SUB> were obtained under O<SUB>2</SUB>-limited conditions. This study first demonstrates the strategy of <I>in silico</I> simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 2,3-Butanediol(BDO) was produced from methane using an engineered <I>Methylomicrobium alcaliphilum</I> 20Z. </LI> <LI> The expression of 2,3-BDO synthetic genes were optimized under different promoters. </LI> <LI> <I>In silico</I> gene knockout predictions were performed to improve the production of 2,3-BDO. </LI> <LI> A 86.2 mg/L 2,3-BDO with 0.0318 g-(2,3-BDO)/g-CH<SUB>4</SUB> yield were obtained under O<SUB>2</SUB>-limited condition. </LI> </UL> </P>