Report membrane transport of lactic acid in the filamentous fungus rhizopus

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.