請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21523
標題: Characterization and Regulation of the Putative Pyrimidine Reductive Catabolism Pathway in Brevibacillus agri NCHU1002 and the Use of Dihydropyrimidinase for L-Homophenylalanine Production
Brevibacillus agri NCHU1002 嘧啶還原代謝途徑的調控及 Dihydropyrimidinase 應用於生產 L-Homophenylalanine
作者: 高肇鴻
Kao, Chao-Hung
關鍵字: Brevibacillus agri
Brevibacillus agri
Pyrimidine reductive catabolism
L-Homophenylalanine
Dihydropyrimidinase
嘧啶還原代謝
L-Homophenylalanine
Dihydropyrimidinase
出版社: 分子生物學研究所
摘要: 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.
本研究中所篩選到的中高溫菌,菌株 NCHU1002,經由型態觀察、基本生理試驗、16S rRNA 基因序列分析及脂肪酸組成分析的結果可以判定此菌株之學名應為 Brevibacillus agri。從 B. agri NCHU1002 選殖出 8,159 bp 的 DNA 片段,經由 DNA 序列分析發現,在此 DNA 片段上有九個可能的 ORF,其中 pyd 基因串上有三個相鄰的基因,分別為 pydA , pydB 及 pydC,此三個基因有相同的轉錄方向。在先前的研究中已證實 PydB 是参與嘧啶還原代謝的酵素 dihydropyrimidinase。分析 PydC 的酵素活性,發現此酵素有很嚴格的基質專一性,只對 N-carbamoyl-b-alanine 具有催化能力,因此可確定 PydC 是 b-alanine synthase。pydA 基因在 E. coli 中表現都形成不可溶的內涵體,因此無法分析其酵素活性,惟根據其胺基酸序列的比對分析及基因的組成,我們推測 PydA 應該是 dihydropyrimidine dehydrogenase。分析 pyd 基因的表現,發現基因表現與菌體生長時期有相關性,PydB 酵素活性在菌體生長對數期末期時達到最高。根據北方轉漬雜交法分析的結果顯示,pydBC 基因之轉錄方式為 polycistronic,可藉由嘧啶還原代謝的中間產物 dihydrouracil 所誘導,且基因的表現是在轉錄層次上的調控。藉由引子延伸的實驗尋找 pydBC 操作子轉錄起始點,結果顯示,轉錄起始於 pydB 基因轉譯起始點上游的第 106 個核苷酸殘基 A。在轉錄起始點上游 -12 及 -24 的序列附近發現有一個可能的啟動子序列,此啟動子序列為 d54 因子所識別的專一性序列。所有的結果均顯示, pydABC 基因參與嘧啶還原代謝途徑,此 pyd 基因串在微生物中也是首次被報導。 先前的研究中已由中高溫菌 Brevibacillus agri NCHU1002 中選殖出熱穩定性的 dihydropyrimidinase 基因 (pydB)。分析酵素對基質的光學選擇性,顯示 PydB 對於基質 D,L-p-hydroxyphenylhydantoin 及 D,L-5-[2- (methylthio)ethyl]hydantoin 的催化是絕對的 D-選擇性,而對於 D,L-homophenylalanylhydantoin (D,L-HPAH) 則是非選擇性。因此,將 pydB 基因以及由 Bacillus kaustophilus 中選殖出來的 L-N-carbamoylase (lnc) 基因,以融合基因或兩個基因同在一個菌體內表現的方式,來進行 D,L-HPAH 的生物轉換。利用載體 pQE-30 上誘導型的 T5 啟動子來帶動 pydB 及 lnc 基因之表現,兩種基因共存於單一宿主細胞 E. coli 中。SDS-PAGE 分析結果顯示,pydB 及 lnc 基因可同時在單一宿主細胞中大量表現,且可溶性蛋白的量約佔了總蛋白質量的 20 %,然而融合基因的表現都是形成不可溶的內涵體。因此,以個別基因在 E. coli 宿主細胞中同時表現的全細胞 來進行 HPAH 的轉換。在 pH 7.0 的反應條件下,HPAH 的消旋速率較慢,所以轉換生產率較高,以 L-HPAH 或 D,L-HPAH 為基質,L-HPA 轉換生產率可分別達到 98 % 及 49 %,且中間產物 NC-D- homophenylalanine 的累積較少,以 chiral HPLC 分析所生產的 L-HPA,其光學純度可大於 99 % ee。結果顯示,pydB 及 lnc 個別基因在 E. coli 同時表現而成的全細胞生物催化劑系統,具有轉換 D,L-HPAH 生產 L-HPA 的潛力。
URI: http://hdl.handle.net/11455/21523
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