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Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/13/2002 Publication Date: 1/1/2003 Citation: SKORY, C.D. LACTIC ACID PRODUCTION BY SACCHAROMYCES CEREVISIAE EXPRESSING A RHIZOPUS ORYZAE LACTATE DEHYDROGENASE GENE. JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. 2003. V. 30. P. 22-27. 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 safe 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 markets for these new products develop as expected. However, improved methods of fermentation must be developed in order to meet these expected demands. This work involved genetically modifying the common baking yeast, Saccharomyces cerevisiae, to have the ability to convert corn derived sugars to lactic acid. This yeast is generally considered a robust organism that is well accepted for use as an industrial strain in numerous applications. Several different versions of modified yeast strains were developed in this study in an effort to optimize the efficiency of lactic acid production. This work is expected to be a significant contribution towards the development of improved technologies for producing lactic acid. Technical Abstract: This work demonstrates the first example of a fungal LDH expressed in yeast. A L(+)-lactate dehydrogenase gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adhl promoter and terminator, then placed in a 2 micron containing yeast replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/L in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/L and a yield of 0.44 g lactic acid/per g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 unit/mg protein throughout the fermentation. The majority of carbon was still converted to ethanol as indicated by ethanol yields between 0.25-0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. |
