(487d) Characterization of a Butanol Producing Mutant of Clostridium Pasteurianum Evolved on Crude Glycerol

Crude glycerol derived from the transesterification of fatty acids in biodiesel production is a waste product (~10% w/w). Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. However, crude glycerol contains byproducts which inhibit growth, feedstock consumption, and butanol production. To overcome this limitation, we employed a traditional random mutagenesis and directed evolution selection technique. A wild type C. pasteurianum (ATCC 6013) culture was mutagenized with NTG (N-methyl-N′-nitro-N-nitrosoguanidine) (two rounds of mutation and recovery) and was subsequently selected upon over 10 days in increasing concentrations of crude glycerol (from 80 to 150 g/L). We isolated the best performing mutant (dubbed M150B). It showed a 72% increase in butanol production in 100 g/L crude glycerol compared to the wild type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol).

Both the wild type and M150B strains were sequenced and assembled via Single Molecule Real-Time (SMRT) sequencing. The wild type was assembled into 2 contigs (4.4 Mb) and compared to the previously published version of the same strain, which exists in 37 contigs with a total length of 4.28 Mb (our data contained an additional 120 kb region). Mutations introduced to the M150B genome were identified by sequence comparison to the wild type. Furthermore, strand-specific RNAseq was employed to compare the transcriptomes of the two strains during growth on crude glycerol.

In addition, methylation patterns on genomic DNA identified with SMRT sequencing were used to aid in optimization of transformation efficiency of plasmid DNA, as C. pasteurianum contains endogenous restriction endonuclease genes.