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item Skory, Christopher - Chris

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/2003
Publication Date: 11/18/2003
Citation: Skory, C.D. 2004. Lactic acid production by Rhizopus oryzae transformants with lactate dehydrogenase activity. Applied Microbiology and Biotechnology. 64:237-242.

Interpretive Summary: Lactic acid has long been used by the food industry as an additive for preservation, flavor, and acidity. Recently, it has gained popularity for the manufacture of environmentally friendly products which include the biodegradable plastic, poly-lactic acid (PLA), and the chlorine-free solvent, ethyl lactate. Lactic acid is typically made by microorganisms that are able to convert, or ferment sugars obtained from agricultural crops, such as corn. The lactic acid market for the U.S. is currently about 50,000 tons/yr. and could increase substantially if the market for PLA develops as expected. Fermentations with the fungus Rhizopus are often preferred because the quality of the final product is superior to that obtained by bacterial fermentations. Furthermore, this organism produces lactic acid in the chemical form that is more desirable for making PLA. We have genetically modified Rhizopus strains to overproduce an enzyme critical in the synthesis of lactic acid. This improvement resulted in considerably higher lactic acid productivity with less unwanted byproducts. This work is expected to be a significant contribution towards the development of improved technologies for producing lactic acid.

Technical Abstract: Rhizopus oryzae is capable of producing high levels of lactic acid with fermentation of glucose. Yields typically vary from 60-80% with the remaining glucose diverted primarily into ethanol fermentation. The goal of this work was to increase lactate dehydrogenase (LDH) activity, so lactic acid fermentation could more effectively compete for available pyruvate. Three different constructs, pLdhA71X, pLdhA48XI, and pLdhA89VII, containing various lengths of the ldhA gene fragment were transformed into R. oryzae. This fungus rarely integrates DNA used for transformation, but instead relies on extra-chromosomal replication in high copy number. Plasmid pLdhA48XI was linearized prior to transformation in order to facilitate integration into the pyrG gene used for selection. Isolates transformed with ldhA containing plasmid were compared to both the wild-type parent strain and the auxotrophic recipient strain containing vector only. All isolates transformed with pLdhA71X or pLdhA48XI had multiple copies of the ldhA gene that resulted in ldhA transcript accumulation, LDH specific activity, and lactic acid production higher than the controls. Integration of plasmid pLdhA48XI increased stability of the strain, but did not seem to offer any benefit for increasing lactic acid production. Since lactic acid fermentation competes with ethanol and fumaric acid production, it was not unexpected that increased lactic acid production was always concomitant with decreased ethanol and fumaric acid. Plasmid pLdhA71X containing a large 6.1 kb ldhA fragment routinely yielded higher levels of lactic acid than the smaller 3.3 kb region used to construct plasmid pLdhA48XI. The greatest levels ldhA transcript and enzyme production occurred with isolates transformed with plasmid pLdhA89VII. However, these transformants always produced less lactic acid and higher amounts of ethanol, fumaric, and glycerol compared to the control.