Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/27/2004
Publication Date: 5/27/2004
Citation: Skory, C.D. 2004. Silencing of lactate dehydrogenase gene of Rhizopus oryzae [abstract]. American Society for Microbiology. p. 79.
Technical Abstract: Rhizopus is a filamentous fungus that is used for production of fermented foods, industrial enzymes (e.g., glucoamylase, protease, and lipase), organic acids (e.g., lactate and fumarate), and corticosteroids; all the while being a food spoilage organism, a plant pathogen, and an opportunistic human pathogen. Despite its importance, the techniques for genetic manipulation of this organism are still in an early stage of development compared to those used for many other fungi. One of the more difficult techniques has been eliminating gene expression by targeted deletion. This is because DNA transformed into Rhizopus rarely integrates and is autonomously replicated in a high molecular weight concatenated arrangement. We felt that RNA interference (RNAi) might be a way to circumvent this difficulty. RNAi technology is based on a recently discovered cascade mechanism of silencing the expression of target genes by using double stranded RNA (dsRNA). In this pathway, dsRNA is processed into 20-25 nt small interfering RNAs that subsequently form silencing complexes that destroy complementary target DNA. Several types of gene expression systems for Rhizopus were designed to produce altered transcript that are capable of forming dsRNA of the lactate dehydrogenase gene, ldhA. This gene was chosen as a test model because we have shown that it is primarily responsible for lactic acid production. We were not able to achieve any significant reduction of lactic acid for isolates containing short (20-25 nt) synthetic ldhA RNAi in the expression plasmids. However, expression of a 430 nt inverted repeat of ldhA resulted in a 25% decrease in lactic acid production. Interestingly, the greatest reduction of acid production was with a translational fusion construct using the phosphoglycerate kinase promoter. Isolates had significantly lower LDH enzymatic activity and up to 95% less lactic acid accumulation. Fermentation products shifted primarily to ethanol and fumaric acid, while growth was relatively unaffected. We are optimistic that the success of this approach can be applied to the manipulation of other genes of interest.