Author
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Skory, Christopher - Chris |
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Bothast, Rodney |
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Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only Publication Acceptance Date: 8/14/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Recent events have demonstrated that there is a growing interest in using lactic acid for the production of biodegradable polymers. It has been estimated that the lactic acid market for the U.S., which produces about 50,000 tons/year, could exceed one million tons within a decade. Rhizopus oryzae is often preferred for manufacturing high quality lactic acid because it synthesizes only stereo-chemically pure L-(+)-lactic acid and has minimal growth requirements, allowing for improved product clean-up. In order to increase the efficiency of lactic acid conversion through genetic modification, we have developed a transformation system based on auxotrophic complementation of the OMP-decarboxylase, pyrG, gene. Uridine auxotrophs of R. oryzae were isolated by selection on 5-fluoroorotic acid, following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene were detected by assaying cell extracts for the ability to convert [14C] labeled OMP to UMP. The pyrG gene was identified by first designing degenerate PCR primers based on conserved regions from the Mucoraceae fungi, R. niveus, Mucor circinelloides, and Phycomyces blakesleeanus. A 500 bp fragment was amplified from R. oryzae genomic DNA and sequenced to reveal that it was part of the pyrG gene. This fragment was then used to screen a genomic library for the gene in its entirety. We were unable to transform the fungus by protoplast methods because of the recalcitrant nature of the cell wall. However, untreated sporangiospores could easily be transformed by micro-projectile bombardment. Specific data regarding stability and integration will be presented. |
