Cloning and characterization of the Rhizopus niveus leu1 gene and its use for homologous transformation.

The Rhizopus niveus leul gene was cloned using a 2-kb HindIII fragment of the leuA gene of Mucor circinelloides as a hybridization probe. The coding region of this gene comprises 1890 bp that encode a putative protein of 630 amino acids. The predicted amino acid sequence is similar to those of other alpha-isopropylmalate isomerases (alpha-IPMIs), showing 79.4%, 71.7%, and 60.6% identity with the Mucor circinelloides LeuA, Phycomyces blaskesleeanus Leu1, and Ustilago maydis Leu1, respectively. The vector pRN1, which contains a 3.7-kb BamHI-SacI fragment of the R. niveus leul gene, could complement leucine auxotrophy of R. niveus M-37 (leu1-), suggesting that the gene codes for alpha-IPMI of R. niveus. Both the transformation frequency and mitotic stability with pRN1 were higher than those with leuA gene-containing vectors. It was found by using the leul gene that the autonomously replicating sequence (ARS) of P. blakesleeanus partially functions as an ARS in R. niveus.