The fumR gene encoding fumarase in the filamentous fungus Rhizopus oryzae: cloning, structure and expression

The filamentous fungus Rhizopus oryzae (Ro) is known for its ability to overproduce and accumulate high levels of fumaric acid (FA) under stress conditions. In order to study the molecular mechanisms involved in the increased biosynthesis of FA, the gene (designated fumR) encoding Ro fumarase was cloned and analysed for its structure and expression. Nucleotide (nt) sequence and comparison of the fumR product with fumarases from various sources established that fumR contains nine introns and encodes a deduced product of 494 amino acids (aa), related to class-II fumarases. A fumarase protein of 50 kDa was immuno-detected in crude Ro extracts. Primer extension experiments mapped the 5′ end of the fumR RNA 159 nt upstream from the putative translation start codon. Both primer extension and Northern analysis showed the existence of one transcript of fumR. The level of fumR RNA increased in cells producing FA under stress conditions (high carbon and low nitrogen levels in the medium), suggesting that transcriptional regulation of fumR might be involved in the overproduction and accumulation of FA by Ro cells under stress conditions. The possibility that additional mechanisms are responsible for this phenomenon is discussed.