Enantioselective Synthesis of L-Phenylephrine by Ketone Reductase-Development of Novel Enzyme and Whole Cell Biotransformation Process

Abstract

(-)-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抵觸到國外之專利。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。

(-)-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.