(798d) Combinatorial Metabolic Engineering of Clostridium Cellulolyticum Using Clonable Genomic Library Fragments Generated From Degenerate Oligonucleotide Primed PCR

The anaerobic mesophile Clostridium cellulolyticum ATCC 35319 is an efficient cellulose utilizer that is characterized by pyruvate secretion and a slow growth rate.  Resolving a critical metabolic bottleneck in C. cellulolyticum has been performed using an over-expression genomic library to identify gene fragments that impart a growth advantage on cellulosic substrates. A novel, template-independent approach involving degenerated oligonucleotide primed PCR was used to generate genomic libraries reliably and rapidly from the genomic DNA of multiple bacteria with different methylation characteristics.  This approach allowed for isolation and amplification of genomic fragments from environmental sources rich in biological and cellulolytic activity.  Also unique to our new method is that error-prone PCR is supported in library generation, dramatically expanding the search space for improved biological activity.  We show that the use of single-stranded degenerate oligonucleotide primers consisting of a 10-15bp 3’ tail, a 6-15bp degenerate interior, and a 2-6bp 5’ anchor allow for random binding and amplification of pure or mixed DNA samples using two separate PCR cycling parameters. These DNA fragments clone at high efficiency to commercial TOPO™ cloning vectors and are easily transposed into other vectors using the Gateway™ cassette system. This new methodology has made generating and cloning a genomic library a routine and easily repeatable procedure in our laboratory.  A genomic library was generated from C. cellulolyticum DNA as well as DNA amplified from several environmental sampling sites.  Expressing the library in continuously sub-cultured C. cellulolyticum grown on different cellulosic carbon sources (including recycled paper) allowed for identification of unique gene fragments that resolved metabolic bottlenecking and improved cellulose degradation rates.  Ultimately, this new strain will enable the conversion of cellulose into valuable chemicals and fuels.