Genetic Enhancement of Microbial Production of 2,3-Butanediol (Bacillus Polymyxa)

187 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.The subject of this study was the genetic enhancement of 2,3-butanediol production by Bacillus polymyxa. Mutagenesis and cloning techniques were studied.A series of spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations were used to create a mutant strain of B. polymyxa ATCC 12321 which showed constitutive production of catabolic (alpha)-acetolactate synthase, a key enzyme in the 2,3-butanediol pathway. This mutant strain was shown to produce 2,3-butanediol and its immediate precursor acetoin somewhat earlier and in considerably greater quantities than the wild-type strain under a wide variety of fermentation conditions. The mutant strain also grew more slowly and displayed a different colony type than the wild-type strain.Since very little molecular biology work had been done with B. polymyxa, it was necessary to develop these techniques. Procedures were developed for growth of B. polymyxa on minimal media and for isolation of plasmid DNA from the microorganism.A penicillin-injured-cell transformation system was developed to transfer plasmid DNA to B. polymyxa. The system included transformation in a buffer containing Mg('++) ions and growth on a peptone-glucose-yeast extract selection medium. Chloramphenicol-resistance plasmids pC194 and pBD64 were transferred by this system, and plasmid bands from pC194 could be visualized on agarose gels of plasmid DNA from transformed cells of B. polymyxa.Plasmid DNA could also be transformed into protoplasted cells of B. polymyxa. However, cell walls could not be regenerated on the protoplasted cells.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD