Cloning and Characterization of Novel D-Amino Acid Oxidase with High Activity Against Cephalosporin C Form Yeast

D型胺基酸氧化�(D-Amino Acid Oxidase,以下簡稱DAO �)屬Flavoenzyme,以FAD為輔成基,此種酵素能氧化D型 胺基酸,生成酮酸.DAO�廣泛存在於各種不同的微 生物及動物組織.截至目前為止,已發現Fusarium solani及Trigonopsis variabilis能很有效率的將Cephalosporin C(Ceph C)轉變成7-□-(5-Carboxy-5-oxo-pentanamido)-cephalosporinic Acid(keto-AD-7ACA),隨後再由H/ sub 2/O/sub 2/轉化成7-□-(4-Carboxybutanamido) cephalosporanic Acid(GL-7ACA), GL-7ACA可由GL-7ACA Acylase再 轉變成7-Amino-cephalosporanic Acid (7ACA).7ACA是Cephem抗 生素的原體(Starting Material).目前利用化學法生產 7ACA.過程太複雜,成本也高,因此製藥業一直想要 開發更有效的7ACA生產方法,酵素法將是最好的選 擇

Characterization of Alanine-Racemase-Transgenic Arabidopsis Plants and Elucidation of Mechanisms of D-Amino Acid Toxicity in Plants

植物基因轉殖需要適當的篩選標記，目前大多利用抗藥性與抗殺草劑基因作為基因重組的篩選標記，將這些標記基因使用於植物基因轉殖，可能會對人類或環境有不利的影響。最近，我們從土壤多元體基因庫中選殖出L-lysine racemase基因(Appl. Environ. Microbiol., 2009)，並發現此基因可以取代抗藥性基因，以L-lysine為篩選劑，作為植物基因轉殖時之篩選標記，可應用於菸草及阿拉伯芥這兩種模式植物的基因轉殖(Plant Mol. Biol., 2010)。然而，此種篩選系統僅適用於對L-lysine敏感的植物種類，因此必需再建立其他非抗藥性的篩選系統。上半年度的國科會計畫中，我們發現Corynebacteriumglutamicum NCHU 87078的alanine racemase基因(alr)可作為篩選標記，搭配D-alanine作為篩選劑，利用模式植物Arabidopsis為材料，已成功的發展出Alr篩選系統，或可作為其他植物基因轉殖之用。惟alr轉殖之阿拉伯芥的T1植株莖較為纖細，株高較低，花期早且花苞及莢果數目較少，種子產量較低，且具有早熟現象。由於D-amino acid造成植物生長抑制及其他生理上的失調之原因，尚未釐清。本研究的主要目的在於利用alr轉基因阿拉伯芥為材料，觀察野生型及alr轉基因阿拉伯芥經D-alanine處理後之農藝性狀變化，來釐清D-amino acid對植物生長具有毒性之可能機制。在上半年度的國科會計畫中，我們已由核酸微矩陣分析經D-alanine處理或未處理的模式植物-Arabidopsis幼苗，以了解其整體基因表現之差異。未來將以胺基酸轉運蛋白、胺基酸生合成蛋白、轉錄因子...等為標的，進行real-time RT-PCR及基因產物生化特性分析，來協助解析D-amino acid對植物的毒性機制。研發工作將在三年內完成，具體工作如下：第一年一、 阿拉伯芥同質alr 轉基因品系之遺傳、外表型、生化及分子特性的分析二、 轉基因阿拉伯芥alr 基因表現後的胺基酸組成份分析三、 利用具有毒性的D-alanine 來篩選gek1 突變株及gek1-overexpression 轉殖株，並觀察他們的胺基酸組成變化四、 根據核酸微矩陣分析結果及real-time RT-PCR，推測deacylase 酵素，胺基酸轉運蛋白，胺基酸的生合成基因及轉錄因子...等與D-amino acid 毒性之間的關係第二年一、 與D-alanine 毒性有關聯性的基因產物之生化特性分析二、 利用CaMV 35S 啟動子，在Arabidopsis 植物中大量表現與D-alanine 毒性有關的基因三、 觀察gene overexpression 之Arabidopsis T1 轉殖株在含有D-alanine 的培養基之生理反應與農藝性狀，並解析目標基因在D-amino acid 毒性上所扮演的角色四、 利用透射電子顯微鏡，觀察D-alanine 處理或未處理的alanine-racemase-transgenic 和野生型Arabidopsis 植株之根部細胞結構之差異第三年一、利用RNA interference 技術，降低與D-alanine 毒性有關的基因在Arabidopsis 植物之表現二、觀察gene knockdown 之 Arabidopsis T1 轉殖株在含有D-alanine 的培養基之生理反應與農藝性狀，並解析目標基因在D-amino acid 毒性上所扮演的角色Public concerns on the biosafety and regulatory issues over the use of antibiotic and herbicide-basedselectable markers have necessitated the scientists to develop alternative approaches for transgenic plantselection. Very recently, we have successfully isolated a lysine racemase gene from soil metagenomic library(Appl. Environ. Microbiol., 2009) and demonstrated the utilization of lysine racemase as a non-antibioticmarker using L-lysine as selection agent for the transformation of Arabidopsis and tobacco (Plant Mol. Biol.,2010). However, the use of our system is limited to lysine-sensitive plants. Therefore, it is imperative toestablish other non-antibiotic marker system for wider use in the development of transgenic plants. We havethen successfully used of alanine racemase (alr) gene from Corynebacterium glutamicum NCHU 87078 as aselectable marker and D-alanine as the selection agent for the plant transformation. However, the T1 plantsexhibit phenotypic variations including reduced number and small size of rosette leaves, decrease in seedyield, and early maturity. It has been known that plants are highly sensitive to exogenously applied D-alanineand D-serine, resulting in the growth inhibition and other physiological disorders, but the toxicity of D-aminoacids to plants remains to be elucidated. This proposal is primarily aimed at the characterization of wild-typeand alanine-racemase-transgenic Arabidopsis plants after treating D-alanine to study the underlyingmechanisms of D-amino acid toxicity in plants. The main thrust will be to gain new insights into themechanisms of toxicity based on the studies of amino acid transporters, amino acid biosynthetic genes, andtranscription factors using the methods of real-time RT-PCR and biochemical characterization. The researchwork in the coming three years is as follows:First grant year1. Genetic, phenotypic, biochemical and molecular characterization of selected homozygous alanine-racemase-transgenic lines.2. Analysis of endogenous amino acid composition in the alr-expression transgenic plants.3. Screening of gek1 mutants and gek1-overexpressing transgenic plants on toxic D-alanine and observationof differences in their amino acid profile.4. To correlate deacylase, amino acid transporters, amino acid biosynthetic genes and transcription factorsetc. to D-amino acid toxicity based on microarray data and real-time RT-PCR.Second grant year1. Biochemical characterization of the gene products involved in D-amino acid toxicity.2. Overexpression of the target genes related to D-amino acid toxicity in Arabidopsis plants using CaMV35S promoter.3. Determination of the phenotypic and agronomic traits of the Arabidopsis T1 transgenic plantsoverexpressing target genes in the medium containing D-alanine to elucidate the roles of target genes inD-amino acid toxicity.4. Examination of ultrastructural changes/differences in the root cells of control and D-alanine treatedalanine-racemase-transgenic and wild-type Arabidopsis plants using transmission electron microscopy.Third grant year1. Knockdown of the target genes related to D-amino acid toxicity in Arabidopsis plants using RNAinterference technique.2. Determination of the phenotypic and agronomic traits of the gene knockdown Arabidopsis T1 transgenicplants in the medium containing D-alanine to elucidate the roles of target genes in D-amino acid toxicity

Chitosanase基因之大量表現俾生產Chitosan oligosaccharide

Chitosan寡糖具有一些生物活性包括抗菌、免疫調節及抗癌，此種寡糖是以chitosan利用chitosanase水解而來，本研究將由Bacillus subtilis選殖endo-type chitosanase基因，並將此基因在E. coli及B. subtilis表現，我們將分析pre-peptide序列對基因表現能力的影響，也將分析chitosanase的生化性質，以評估所開發的chitosanase用於生產chitosan oligosaccharide的可行性。Chitosan oligosaccharides exhibit a variety of biological activities including antimicrobial activity, immunopotentiating effector and antitumor activity. This oligosaccharide is produced from the enzymatic process using chitosanase as biocatalyst. The gene encoding endo-type chitosanase will be cloned from Bacillus subtilis and expressed in Escherichia coli and B. subtilis. The effect of pre-peptide sequence on the expression level of chitosanase gene and the biochemical properties of chitosanase will be investigated to evaluate the feasibility of using our chitosanase in the producion of chitosan oligosaccharides

Biodegradation of Paclobutrazol, a Plant Growth Regulator, by Serratia marcescens

Paclobutrazol is a recalcitrant plant growth retardant that is used worldwide, for such purposesas increasing the yield of cereal crops and enhancing seed production in eucalypt orchards.Paclobutrazol has been widely used in wax-apple cultivation in Taiwan to depress new shootgrowing and promote early flowering. However, paclobutrazol is xenobiotic compound thatremains active in soil for several years and can severely affect the growth and development ofsubsequent crops, mainly by reducing vegetative vigor. This compound is quite resistant tobiodegradation by soil microbes. The use of recombinant microorganisms is expected to be aneffective tool for remediation of polluted environments. In previous work, we have established aquick method for the isolation of bacterium with capability of degrading the paclobutrazol. Serratiamarcescens strains NCHU 4-3 and NCHU 5-3 were isolated, to which about 9% of paclobutrazolsupplemented in the culture medium was degraded in 30 days. In this study, we are going to find outthe pathway for the catabolism of paclobutrazol by S. marcescens, clone the genes related to thedegradation of paclobutrazol, construct a soil bacterium capable of degrading the paclobutrazol, andevaluate the feasibility of using the recombinant soil bacteria in the remediation of the soils pollutedwith paclobutrazol.First grant year1. Determine the effect of cultural conditions on the biodegradation of paclobutrazol by S.marcescens NCHU 4-3 and S. marcescens NCHU 5-3.2. To predict the putative degradation pathway of paclobutrazol and the enzymes involvedin the pathway.3. Establishment of the methods for the extraction and chemical analysis of metabolitesfrom paclobutrazol.4. Cloning of the gene(s) involved in the first step of paclobutrazol biodegradation pathwayfrom S. marcescens and expression of cloned genes in E. coli.5. Create paclobutrazol-negative mutants (PBZ- strain) of S. marcescens using EZ-Tn5TM Tnp TransposomeTM Kit.Second grant year1. Isolation and identification of metabolites from paclobutrazol biodegradation by S.marcescens.2. Cloning and sequencing of the transposon-disrupted genes to elucidate the possible rolesof the genes in the paclobutrazol biodegradation.3. Cloning and expression of transposon-disrupted genes in E. coli and S. marcescens todetermine the possible roles of the genes in the paclobutrazol biodegradation.4. To find out the major microbial flora in the rhizosphere of wax-apple garden.Third grand year1. To develop a gene expression vector for the transformation of the major microbes in therhizosphere of wax-apple garden without the use of drug-resistant selective marker.2. To construct a soil bacterium capable of degrading the paclobutrazol by DNArecombination using 16S rDNA as homologous gene.3. To evaluate the feasibility of using recombinant paclobutrazol-degradative bacteria inthe bioremediation of paclobutrazol-polluted soils.4. Patent application.Paclobutrazol 為一種人工合成的植物生長調節劑，其主要作用方式為抑制植物的吉貝素(gibberellin)生合成，使得植株節間縮短達到矮化的效果；對於果樹也具有抑制新梢形成的能力，因此可以調整開花結果的時期。由於paclobutrazol 的使用劑量低且效果顯著，因此廣泛被農民使用於園藝作物之生產。然而paclobutrazol 在自然環境中非常穩定，半衰期超過兩年，水溶性非常差，因此施用後會長期累積於土壤中。如果農民不當超量使用，短期內雖然可以明顯的改進果樹生產，但是數年後常因為土壤中累積過多的paclobutrazol，使得植株根部生長被抑制而枯萎，改種其它果樹亦無法生長，造成農地廢耕。而這樣的問題確實已經出現在台灣南部地區的部分蓮霧果園中。因此，如何有效的處理土壤中殘留的paclobutrazol，恢復果園土地的生產能力，已成為一個相當重要的課題。Paclobutrazol 的穩定性極佳，一般微生物難以分解，截至目前為止，其代謝途徑也不清楚。四年前曾獲得農業國家型計畫的補助，當年曾經建立paclobutrazol 的分析方法及paclobutrazol 分解菌的大量篩選方法。由於研發主題不合農業國家型計畫之產業導向，隨後自行投入研究工作，很幸運的，由被paclobutrazol 汙染的土壤中分離到兩株Serratia marcescens菌株(NCHU 4-3 及NCHU 5-3)，對paclobutrazol 具有較強的分解能力，這是S. marcescens 首次被發現能分解paclobutrazol。因此，本研究的目的在於釐清S. marcescens 分解paclobutrazol的代謝途徑，選殖代謝paclobutrazol 之相關酵素基因，並將這些基因利用非抗藥性基因篩選標誌，選殖入非病源性的根圈細菌，利用具有新的代謝途徑的重組菌體，測試復育被paclobutrazol 汙染之土壤的可行性。本研究擬在三年內完成，研究工作如下：第一年一、分析S. marcescens NCHU 4-3 及S. marcescens NCHU 5-3 在不同培養條件下對paclobutrazol 之分解能力。二、預測可能參與paclobutrazol 生物分解之酵素及途徑。三、建立paclobutrazol 經菌株分解或修飾後之代謝產物之萃取(液相-液相萃取及固相萃取)與分析方法(包括氣相層析質譜、液相層析質譜及核磁共振分析)。四、從S. marcescens NCHU 4-3 及S. marcescens NCHU 5-3 選殖可能參與paclobutrazol 生物分解之第一個步驟的基因，並轉形至E. coli 中進行表現及分析。五、使用汎用型跳躍子(transposon)系統(EZ-Tn5TM Tnp TransposomeTM Kit)任意插入S. marcescens 菌株NCHU 4-3 或NCHU 5-3 的染色體中，篩選無paclobutrazol 分解能力的菌株(PBZ- 菌株)。第二年一、分離及純化paclobutrazol 之分解或修飾產物，並進行鑑定。二、針對被跳躍子破壞所產生的S. marcescens PBZ-菌株，利用跳躍子上之篩選標誌，分析插入位置的序列，並推斷其可能參與paclobutrazol 分解的角色。三、Paclobutrazol 分解或修飾途徑中，重要步驟的基因之選殖與表現，並分析其可能之角色。四、分析蓮霧根圈之主要微生物族群，俾建立環保微生物非抗藥性基因轉型系統。第三年一、使用非抗藥性基因篩選系統- lysine racemase，建立土壤微生物基因表現平台。二、以土壤細菌利用16S rDNA 之homologous recombintion 方式構築paclobutrazol 分解菌。三、構築完成之轉型菌株在實驗室內分別測試對培養基中之paclobutrazol 及土壤內外加paclobutrazol 之分解能力。四、申請專利

嘧啶還原代謝途徑 Brevibacillus agri 的生理角色及改變dihydropyrimidinase 之基質選擇性以應用於不對稱化合物之生產(I)

嘧啶還原代謝途徑包含了三個酵素的催化反應，第一個步驟是 dihydropyrimidine dehydrogenase 的催化反應，此反應在哺乳動物中是嘧啶降解過程中的一個速率決定步驟，利用 NADPH 為還原劑，將 uracil 及 thymine 分別水解成 dihydrouracil 及 dihydrothymine，再經由第二個酵素 dihydropyrimidinase 進行開環反應 將 dihydrouracil 及 dihydrothymine 水解成 N-carbamoyl-β-alanine 及 N-carbamoyl-β-aminoisobutyric acid，最後再經由 β-alanine synthase 的作用，生成 β-alanine 及 β-aminoisobutyric acid，ammonia 和二氧化碳。近年來在很多的研究中也證實了嘧啶的還原代謝過程在某些微生物及動物組織中是必須的，因為 uracil 經由此一還原代謝途徑所產生的終產物 β-alanine 是合成 pantothenic acid 及 coenzyme A 的主要成份，並且在哺乳動物中此一代謝途徑是合成神經傳導物質 β-alanine 及 β-aminoisobutyric acid 的唯一途徑。 本實驗室已由 B. agri 中選殖到一段約 8.2 kb 的 DNA 片段，經過序列比對及初步酵素的活性分析結果，顯示此序列上包含了可能參與嘧啶還原代謝途徑的三個基因，並分別命名為 pydA (dihydropyrimidine dehydrogenase)、pydB (dihydropyrimidinase) 及 pydC (β-alanine synthase)，其中，pydB 及 pydC 之基因產物的生化特性已確立，pydBC operon 的表現與調控也有初步的瞭解，這些研究成果已發表於 Biochemical and Biophysical Research Communications 303 (2003) 848-854。搜尋已發表的研究報告及微生物基因體序列，未曾發現其他微生物具有此基因串，尤其在同屬於 Bacillus 菌屬的 B. subtilis 及 B. halodurans 的基因體序列中，也不具有此.與嘧啶還原代謝基因的存在。因此，有必要對 B. agri 特有的嘧啶還原代謝基因串進行深入研究，以瞭解其真正的生理功能及角色。研究其基因的調控，有助於篩選系統的建立，俾進行以嘧啶類似物為主要結構的藥物開發，並可建立一個模式可利用生體外的分析，了解藥物的代謝命運及其解毒的功能。除此之外，在先前的研究中，發現 B. agri 的 dihydropyrimidinase 具有 D-hydantoinase 之活性，惟對於基質 (D,L-homophenylalanylhydantoin，以下簡稱 D,L-HPAH) 的催化是屬於非鏡面選擇性的。亦即，利用此 dihydropyrimidinase 搭配本研究室由 B. kaustophilus 選殖出來的耐熱性 L-N-carbamoylase 可將基質 D,L-HPAH 轉換成 L-homophenylalanine (L-HPA)。L-HPA 是合成許多血管緊縮素轉換酵素抑制劑的前驅物，在全世界的藥品市場佔有非常重要的地位。惟B. agri dihydropyrimidinase 的基質選擇性趨向 D-HPAH，轉換 L-HPAH 的速率相當差 (初步的實驗結果顯示，其效率只有5﹪)，若能加以改造使其偏向 L-HPAH，非常有工業化應用之可行性。特別是這兩個酵素都是熱穩定型的酵素，可以克服基質 (D,L-HPAH) 因溶解度低而必須在高溫下進行酵素轉換的問題。經過人工改造的 dihydropyrimidinase 配合 L-N-carbamoylase 的催化，可利用個別基因或融合兩酵素之基因的方式，在同一宿主內表現，以轉形細胞進行生物轉換生產 L-HPA，是值得嘗試的新方法。酵素的人工改造及生物轉換法生產不對稱化合物 HPA，在基礎及應用科技領域上都有相當大的價值及原創性。因此，未來三年，本計畫將進行下列工作： 第一年度 1. 完成 B. agri dihydropyrimidine dehydrogenase 酵素的純化及生化性質分析。 2. 分析 pydA 基因的啟動子以及在不同培養條件下基因的表現及調控。 3. 建立 B. agri 質體轉形系統。 4. 分析 dihydropyrimidinase 3-D 結構 (此部份將與清大生科系王雯靜教授合作執行)。 第二年度 1. 篩選高轉形效率的 B. agri 突變株並建立其轉形系統。 2. 以基因重組的方式破壞 pyd 基因，分析 pyd 基因在生理上所扮演的角色。 3. 利用 Error-prone PCR、DNA shuffling 及定點突變等方法改造 B. agri dihydropyrimidinase 的酵素性質，提升其對 L-HPAH 的基質選擇性及催化能力。 4. 分析 dihydropyrimidinase 的3-D 結構。 第三年度 1. 完成 dihydropyrimidinase 酵素的改造，使其更適合 L-HPAH 之轉換。 2. 分析變異 dihydropyrimidinase 的3-D結構。 3. 將改造的 dihydropyrimidinase 基因及 B. kaustophilus 的 L-N-carbamoylase 基因構築在同一宿主細胞，以個別基因或融合酵素基因的方式進行基因表現，利用轉形細胞進行生物轉換來生產 L-HPA

利用酮還原脢立體選擇性生合成L-Phenylephrine-新型酵素及全細胞生物轉換產程的開發

(-)-Ephedrine, phenylpropanolamine, (+)-pseudoephedrine and L- phenylephrine are effectivedecongestant and commonly incorporated into cold and allergy products. (-)-Ephedrine is derivedfrom several species of the genus Ephedra. Because of its indirect effect on neurotransmitterstores, long-term use of ephedrine can lead to tachyphylaxis. Increasing dosage can induce toxiceffects, including peripheral vasconstriction and cardiac stimulation, leading to increased bloodpressure and increased heart rate; adverse effects on the central nervous system include nervousness,anxiety, tremor, weakness, irritability and insomnia. These adverse effects decrease it used astherapeutic agent today. Phenylpropanolamine hydrochloride resembles ephedrine in its action,but it is somewhat more active as a vasoconstrictor and less active as a bronchodilator. Adverseeffects include hypertension, headache, jitteriness, irritability, insomnia, and cardiac rhythmirregularities. In November 2000, the FDA Food Advisory committee asked the restriction inusage of phenylpropanolamine due to the adverse effects associated with stroke. Moreover,pseudoephedrine can be used to make the illegal drug methamphetamine, also known as 「speed」and the access of pseudoephedrine is controlled at the retail level in many states in the USA.Therefore, the market for L-phenylephrine is increased due to less in risk and no new drugs withmajor the therapeutic benefits.The increased regulatory demands for optically pure drugs coupled with pressures to minimizethe environmental impacts of chemical processes makes enzyme-mediated processes logicalalternatives. In previous works, we have isolated two bacteria strains with capability to producephenylephrine from 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE). A ketonereductase gene was cloned from Rhodococcus erythcoplis and expressed in E. coli. We found that E. coli cells expressing ketone reductase gene can transform HPMAE to D-phenylephrine (>99％e.e.). This is fisrt enzymatic process for phenylephrine to be reported. However, the productobtained is D-form which has to be converted to L-form product by Walden inversion. Morevoer,recombinant cell expressing Rhodococcus ketone reductase exhibited low catalytic activity, andonly 50.6％ of conversion was obtained in a 8-h incubation. Therefore, recombinant DNA andprotein engineering techniques will be included in our further studies to change theenantioselectivity of the Rhodococcus ketone reductase to allow the novel ketone reductase withhigh efficiency in the bioconversion of HPMAE to L-phenylephrine. The research work in thecoming three years is as followings:First grant year1. Screen for microorganisms capable of converting HPMAE to L-phenylephrine.2. Overexpression of Rhodococcus ketone reductase gene in E. coli and determine the effects ofculture conditions on the bioconversion.3. Create a novel Rhodococcus ketone reductase with high catalytic activity using error-prone PCRtechnique and high-throughput screening method developed by our lab.4. Determine the 3-D structure of Rhodococcus ketone reductase (in collaboration with professorWen-ChingWang, Department of Life Science, National Tsing Hua University).5. Cloning and overexpression of genes involved in the recycle of NADPH in the recombinantcells.Second grant year1. To evaluate catalytic activity of ketone reductase and conversion of HPMAE to phenylephrineby the recombinant E. coli cells harboring NAD(P)H recycling genes.2. Improvement of the catalytic activity and enantioselevtivity of Rhodococcus ketone reductaseby site-directed mutagenesis, based on the 3-D structure of the enzyme.3. To determine the relationship between sustrate structure and enantioselectivity.4. Cloning and expression of ketone reductase gene with capability of converting HPMAE to L-phenylephrine.Third grant year1. Expression of ketone reductase gene in the bacteria cells other than E. coli for the conversion ofHPMAE to L-phenylephrine.2. Establishment of a whole cell process using enzyme variant with high enantioselectivity forL-phenylephrine production.3. Optimization of whole cell process with respect to conversion yield and productivity (gL-phenylephrine / l·h).In three grant years, key residues involved in the catalytic activity and enantioselectivity ofketone reductase will be elucidated by our proposed reseach works. Moreover, processes for theproduction of L-phenylephrine using novel ketone reductase will be developed, which do notinfringe the patents in the world.(-)-Ephedrine、phenylpropanolamine、(+)-pseudoephedrine 及L-phenylephrine 主要是用來鬆弛支氣管，治療鼻黏膜腫脹，因此廣泛被添加入傷風感冒及過敏藥物中。(-)-Ephedrine 由麻黃萃取，若長期使用，會有心悸、焦慮不安、高血壓、心律不整等副作用。Phenylpropanolamine(PPA)長期使用會引起嚴重高血壓異常、恐懼、焦慮不安、腦部出血及肺水腫，近年來，又發現有引起出血性中風的疑慮，美國FDA 在2000 年11 月，下令PPA 逐步下架。(+)-pseudoephedrine 可被用來生產迷幻藥methamphetamine，銷售上逐漸受限。在全球市場沒有更好替代品的情況下，L-phenylephrine 的市場再度受到重視，需求量往上提昇。L-phenylephrine 屬不對稱化合物(chiral compound)，由於國際上強烈要求醫藥品採用不對稱化合物以降低副作用，以及人類無法忍受化學合成對環境造成之衝擊，學術界及產業界已積極投入利用酵素法進行藥物之不對稱生合成。惟截至目前為止，有關此種藥物生合成之報告及專利非常少。經過兩年多來的努力， 我們篩選到兩株細菌， 有能力將1-(3-hydroxyphenyl)-2-(methylamino)ethanone(HPMAE)轉換成phenylephrine (>99％ e.e.)。我們將其中一株細菌的ketone reductase 基因選殖並表現於Escherichia coli，發現轉形株能夠專一地將HPMAE 還原成為phenylephrine (>99％ e.e.)，這是生物轉換生合成phenylephrine 的首次報導，惟轉換所得為D-form，不是本研究所需的L-form，必需再經由Walden inversion將其轉換成為具有藥效的L-phenylephrine，增加不少生產成本。除此之外，酵素催化能力不強，八小時轉換率只達到50.6％。因此，本研究將針對所選殖到的ketone reductase 進行蛋白質工程，探討如何改變酵素之enantioselectivity，使其能將HPMAE 直接轉換成L-phenylephrine之可能性，也將提昇菌體對NAD(P)H 的再生能力，以加速生物轉換之進行，並進行產程之最適化。除此之外，也將設法再篩選具有將HPMAE 轉換成L-phenylephrine 能力之生產菌，所開發的新方法不會牴觸到國外之專利。我們的研發工作將在三年內完成，具體內容如下：第一年一、繼續篩選L-phenylephrine 生產菌。二、Rhodococcus ketone reductase 基因的大量表現及培養條件對E. coli 轉形株轉換能力的影響。三、Rhodococcus ketone reductase 的error-prone PCR 突變及高轉換能力之酵素的高通量篩選。四、Rhodococcus ketone reductase 之3-D 結構分析(此部份將與清華大學生科系王雯靜教授合作執行)。五、NADPH 生合成相關基因的選殖及大量表現。第二年一、測試E. coli NAD(P)H 生合成基因大量表現對菌體ketone reductase 活性及phenylephrine生產能力之影響。二、根據Rhodococcus ketone reductase 之3-D 結構及功能之關係，進行選位變異，並分析變異對酵素之enantioselectivity 的影響。三、分析substrate modification 對ketone reductase 的enantioselectivity 之影響。四、其他L-phenylephrine 生產菌之ketone reductase 基因之選殖及表現。第三年一、測試ketone reductase 基因在其他微生物宿主之表現能力及轉形株對phenylephrine 之生產能力。二、利用變異酵素建立HPMAE 轉換成L-phenylephrine 之全細胞產程。三、全細胞產程之最適化：包括cell enzyme activity、carbon source 及其他環境條件等等，並以conversion yield 及productivity (g L-phenylephrine / l·h)為指標。若一切順利，在基礎科學上，將釐清影響ketone reductase 之enantioselectivity 之residues，並提出其反應機制，此部分目前尚無任何學術報告；應用科技上，將首度成功建立L-phenylephrine 之生物轉換法，並提出專利申請