New Gene Cluster from Thermophile Bacillus fordii MH602 for Conversion of DL-5-Substituted Hydantoins to L-Amino Acids

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction occurs at higher temperature, the advantages for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile is firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2-kb DNA fragment by restriction site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein gene hyuP, hyperprotein gene hyuHP, and L-Carbamoylase gene hyuC The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 compared respectively with reported hydantoinase and carbamoylase showed the highest identities of 71%, and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.