Weber Nora, Gorwa-Grauslund Marie, Carlquist Magnus
Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, PO Box 124, 221 00, Lund, Sweden.
Evolva SA, Duggingerstrasse 23, 4153, Reinach, Switzerland.
Microb Cell Fact. 2017 Jan 3;16(1):3. doi: 10.1186/s12934-016-0615-3.
Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels.
Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions.
Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.
基于具有工程化转氨活性的代谢活跃面包酵母的全细胞生物催化可用于生成带有手性胺部分的分子。然而，一个先决条件是表达高效的ω-转氨酶并达到足够的细胞内前体水平。
本文比较了源自辣椒、紫色色杆菌和嗜人寡养单胞菌的三种不同ω-转氨酶对消旋1-苯乙胺进行全细胞催化动力学拆分生成（R）-1-苯乙胺的效率。最有前景的候选基因——紫色色杆菌ω-转氨酶（CV-TA）的基因，在缺乏丙酮酸脱羧酶活性的菌株中表达，该菌株在葡萄糖同化过程中会积累共底物丙酮酸。然而，在所应用的反应条件下，转化率仅略有提高。同时，研究了在培养过程中通过省略硫胺素增加细胞内磷酸吡哆醛（PLP）水平的效果。发现没有硫胺素时，补充PLP对于维持高体内转氨活性是多余的。此外，使用含有多个CV-TA基因拷贝的菌株可实现更高的反应速率，突出了提高细胞内转氨酶水平的必要性。最后，还研究了该菌株以L-丙氨酸作为胺供体将苯乙酮不对称全细胞生物转化为（S）-1-苯乙胺的情况。虽然可以证明其功能性，但活性极低，表明天然的副产物去除系统在应用的反应条件下无法驱动反应生成胺。
总之，我们的结果表明，当结合适当的基因和工艺工程方法时，通过动力学拆分，以葡萄糖作为唯一共底物，在浓度高于25 mM的外消旋底物条件下，可以获得对映体过量（ee）>99%的（R）-1-苯乙胺。此外，具有最高转氨酶活性的工程酵母菌株也被证明可作为全细胞催化剂，通过苯乙酮的不对称转氨作用生产（S）-1-苯乙胺，尽管转化率非常低。
