Abstract:CrgA has been identified as a negative regulator of the carotenoid biosynthesis in Blakeslea trispora and some other zygomycetes. The presence of the ring finger domains suggest that CrgA may function as an ubiquitin ligase, a key enzyme in the ubiquitination regulation system. In order to test this hypothesis, one ubiquitin activating enzyme(BtE1) and 18 putative ubiquitin-conjugation enzymes(UBC) from B.trispora were isolated and identified. Bioinformatics analysis indicated that each of the 18 UBC proteins contained a conserved domain of UBC, indicating that all of them belong to ubiquitin binding enzymes. Phylogenetic relationship analysis showed that 18 UBC proteins belong to 7 protein subfamilies, homologous with those of Saccharomyces cerevisiae. 6 candidate UBC proteins were screened based on phylogenetic analysis, and BtE1, 6 BtUBC proteins, BtCrgA and BtWC-1b were obtained by heterologous expression and affinity purification, which were ubiquitinated in vitro. BtCrgA can perf
