Title: Analysis of a Functional Lactate Permease in the Fungus Rhizopus Authors
Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 21, 2009
Publication Date: February 10, 2010
Citation: Skory, C.D., Hector, R.E., Gorsich, S., Rich, J.O. 2010. Analysis of a functional lactate permease in the fungus Rhizopus. Enzyme and Microbial Technology. 46(1):43-50. Interpretive Summary: This research describes the discovery of a protein from the fungus Rhizopus involved in transporting lactic acid into and out of the cell, thereby providing a better understanding of the mechanisms that this fungus utilizes to produce significant quantities of this industrial chemical. The fungus Rhizopus is frequently used to convert, or ferment sugars obtained from agricultural crops to lactic acid. This natural product is used extensively by the food industry and is now being applied to the manufacture of environmentally friendly products, which include the biodegradable plastic, poly-lactic acid (PLA), and the chlorine-free solvent, ethyl lactate. In order to allow the market potential of lactic acid to continue expanding at the current rapid pace, it is important that the production costs are minimized by the development of new and improved technologies. Lactic acid normally requires special proteins to help facilitate the transport into and out of the cell. Such proteins have never been previously described from filamentous fungi. In this work, we characterize several of those proteins from two different Rhizopus species and show that one of them is capable to transporting lactic acid. The results of this study will allow new strategies to be developed for improving synthesis of lactic acid, thereby benefiting the agricultural grower and ultimately the consumer.
Technical Abstract: The fungus Rhizopus is frequently used for fermentative production of lactic acid, but little is known about the mechanisms or proteins for transporting this carboxylic acid. Since transport of the lactate anion across the plasma membrane is critical to prevent acidification of the cytoplasm, we evaluated the functionality of two lactate-proton symport paralogs, LacA and LacB, from R. delemar. Both of these proteins showed significant ancestral homology to bacterial lactate permease with 46-50% identity to similar homologs from the genus Burkholderia. Based on qPCR, the highest level of expression in Rhizopus for the lacA gene was on complex medium containing pyruvate, while lacB transcript was barely detected with all of the tested culture conditions. A Saccharomyces cerevisiae jen1 deletion strain lacking the ability to transport monocarboxylates was restored for growth on lactate and pyruvate with the expression of LacA. Expression of the LacB in this same strain did not confer the ability to grow on either carbon source. LacA expression also allowed active transport of L-[**14C(U)]-lactate into yeast cells and this accumulation was inhibited by the proton uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. Translation fusions with GFP showed that LacA accumulates primarily in the plasma and vacuolar membrane, while LacB is dispersed throughout the cytoplasm. These results indicate that the Rhizopus LacA is a functional lactate symport that is probably involved in uptake of pyruvate or lactate, while the physiological role of LacB is unknown.