Characterization and Regulation of the Putative Pyrimidine Reductive Catabolism Pathway in Brevibacillus agri NCHU1002 and the Use of Dihydropyrimidinase for L-Homophenylalanine Production

• Main Authors: Chao-Hung Kao, 高肇鴻
• Other Authors: Wen-Hwei Hsu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/70166610304612561917

博士 === 國立中興大學 === 分子生物學研究所 === 91 === The taxonomical characteristics of the moderate thermophile, strain NCHU1002, were examined and it was identified as Brevibacillus agri. Genes involved in pyrimidine reductive catabolism (pyd) were isolated, and nine ORFs in an 8.2-kb DNA fragment were identified by DNA sequence analysis. The pyd gene cluster included three closely spaced ORFs, designated pydA, pydB and pydC, transcribed in the same orientation. PydB has been identified as dihydropyrimidinase in our previously study, and the PydC exhibited strict substrate specificity to N-carbamoyl-b-alanine, implying that it is a b-alanine synthase. Although the PydA protein was not identified by enzyme assay due to the formation of inclusion bodies in the induced E. coli cells harboring pydA gene, based on its amino acid sequence identity and genetic organization, we proposed that PydA is the dihydropyrimidine dehydrogenase. Expression of pyd genes seemed to be correlated with the growth phase of the bacterial culture and the PydB activity was maximum at the late exponential phase. Northern blot analysis revealed that the pydBC genes are transcribed as a polycistronic mRNA, induced by dihydrouracil, and regulated at transcriptional level. Primer extension experiment was performed to locate the transcription start site of the pydBC operon. Transcription starts at an A residue, 106 nucleotides upstream from the initiation codon of the pydB gene. Close inspection of the nucleotide sequences upstream of the transcription start site revealed a potential promoter with -12 and -24 sites (TTGGCAN4GGGGCA) specific for the alternative sigma factor s54. Our results indicated that the pydABC genes participate in the pathway of the pyrimidine reductive catabolism. This is the first bacterial pyd gene cluster to be reported. A thermostable dihydropyrimdinase gene (pydB) has been cloned and characterized from the moderate thermophilic Brevibacillus agri NCHU1002. This enzyme is strictly D-selective for the cleavage of D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, while non-stereoselective to D,L-homophenylalanylhydantoin (D,L-HPAH). Due to these findings, we developed a whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by either fusion or coexpressing the genes encoding the PydB and a thermostable L-N-carbamoylase (Lnc) from Bacillus kaustophilus CCRC 11223 in the single Escherichia coli host cell. The gene was expressed under the control of T5 promoter on pQE-30 vector. The expression level of coexpressed enzymes in E. coli (pDNc) was estimated as a 20 % of the whole soluble proteins, however, all expressed fusion proteins were found to be insoluble form. Individual pydB and lnc gene co-expressed in E. coli were soluble form and therefore used as biocatalyst for L-HPA production. After incubated at 50 ℃ for 6 h, highest conversion yield of 98 % was achieved from L-HPAH. When D,L-HPAH was used as substrate, a 49 % conversion yield was obtained by 16 h incubation at pH 7.0. The enantiomeric excess of the L-HPA is over 99 %. Our results indicated that pydB and lnc gene coexpressed in E. coli might be a potential biocatalyst for the production of L-homophenylalanine from D,L-HPAH.