초록
<P>Crude glycerol, the major by-product of biodiesel production, is an attractive bioprocessing feedstock owing to its abundance, low cost, and high degree of reduction. In line with the advent of the biodiesel industry, <I>Clostridium pasteurianum</I> has gained prominence as a result of its unique capacity to convert waste glycerol into <I>n</I>-butanol, a high-energy biofuel. However, no efforts have been directed at abolishing the production of 1,3-propanediol (1,3-PDO), the chief competing product of <I>C. pasteurianum</I> glycerol fermentation. Here, we report rational metabolic engineering of <I>C. pasteurianum</I> for enhanced <I>n</I>-butanol production through inactivation of the gene encoding 1,3-PDO dehydrogenase (<I>dhaT</I>). In spite of current models of anaerobic glycerol dissimilation, culture growth and glycerol utilization were unaffected in the <I>dhaT</I> disruption mutant (<I>dhaT</I>::Ll.LtrB). Metabolite characterization of the <I>dhaT</I>::Ll.LtrB mutant revealed an 83% decrease in 1,3-PDO production, encompassing the lowest <I>C. pasteurianum</I> 1,3-PDO titer reported to date (0.58 g liter<SUP>−1</SUP>). With 1,3-PDO formation nearly abolished, glycerol was converted almost exclusively to <I>n</I>-butanol (8.6 g liter<SUP>−1</SUP>), yielding a high <I>n</I>-butanol selectivity of 0.83 g <I>n</I>-butanol g<SUP>−1</SUP> of solvents compared to 0.51 g <I>n</I>-butanol g<SUP>−1</SUP> of solvents for the wild-type strain. Unexpectedly, high-performance liquid chromatography (HPLC) analysis of <I>dhaT</I>::Ll.LtrB mutant culture supernatants identified a metabolite peak consistent with 1,2-propanediol (1,2-PDO), which was confirmed by nuclear magnetic resonance (NMR). Based on these findings, we propose a new model for glycerol dissimilation by <I>C. pasteurianum</I>, whereby the production of 1,3-PDO by the wild-type strain and low levels of both 1,3-PDO and 1,2-PDO by the engineered mutant balance the reducing equivalents generated during cell mass synthesis from glycerol. </P><P><B>IMPORTANCE</B> Organisms from the genus <I>Clostridium</I> are perhaps the most notable native cellular factories, owing to their vast substrate utilization range and equally diverse variety of metabolites produced. The ability of <I>C. pasteurianum</I> to sustain redox balance and glycerol fermentation despite inactivation of the 1,3-PDO pathway is a testament to the exceptional metabolic flexibility exhibited by clostridia. Moreover, identification of a previously unknown 1,2-PDO-formation pathway, as detailed herein, provides a deeper understanding of fermentative glycerol utilization in clostridia and will inform future metabolic engineering endeavors involving <I>C. pasteurianum</I>. To our knowledge, the <I>C. pasteurianum dhaT</I> disruption mutant derived in this study is the only organism that produces both 1,2- and 1,3-PDOs. Most importantly, the engineered strain provides an excellent platform for highly selective production of <I>n</I>-butanol from waste glycerol.</P>