Application of Cell-Free Protein Synthesis System for the Biosynthesis of L-Theanine
Application of Cell-Free Protein Synthesis System for the Biosynthesis of L-Theanine
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无细胞蛋白质合成系统在L-茶氨酸生物合成中的应用
DOI：
10.1021/acssynbio.0c00618
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发表时间：
2021-03-10
影响因子：
4.7
通讯作者：
Li, Ping
中科院分区：
文献类型：
--
作者：
Feng, Junchen;Yang, Chen;Li, Ping
茶氨酸是茶树叶片中的一种活性成分，具有多种保健功效，应用广泛。L-茶氨酸的化学合成是可能的;然而，这种方法产生手性化合物，需要进一步分离纯L-异构体。杂环戊烯生物合成是一种替代策略，但一个主要限制是底物乙胺对微生物宿主细胞的毒性。在这项研究中，我们介绍了一个无细胞蛋白质合成（CFPS）系统生产L-茶氨酸。CFPS表达的茶树L-茶氨酸合成酶2（CsTS 2）在合成反应32 h后可产生浓度为11.31 μ M的L-茶氨酸。此外，从微生物的三种同工酶表达在CFPS的L-茶氨酸的生物合成。大肠杆菌的γ-谷氨酰半胱氨酸合成酶在反应24小时后可产生最高浓度为302.96 μ M的L-茶氨酸。此外，CFPS被用来验证一个假设的两步L-茶氨酸生物合成途径组成的L-丙氨酸脱羧酶从C。CsAD和多种L-茶氨酸脱氢酶。其中，CsAD与产于铜绿假单胞菌的L-谷氨酰胺合成酶（PtGS）的组合可以合成L-茶氨酸，最高浓度为13.42 μ M。然后，我们构建了一个工程E。coli菌株过量表达CsAD和PtGS，以进一步证实活细胞中L-茶氨酸的生物合成能力。这个工程E.大肠杆菌菌株发酵72 h后可将培养基中的L-丙氨酸和L-谷氨酸转化为浓度为3.82 mM的L-茶氨酸。综上所述，这些结果表明CFPS系统可以用于通过两步L-茶氨酸生物合成途径生产L-茶氨酸，表明CFPS在其他活性化合物的生物合成中具有潜在的应用前景。
L-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of L-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure L-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for L-theanine production. The CFPS expressed L-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce L-theanine at a concentration of 11.31 mu M after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for L-theanine biosynthesis. The gamma-glutamylcysteine synthetase from Escherichia coli could produce L-theanine at the highest concentration of 302.96 mu M after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step L-theanine biosynthetic pathway consisting of the L-alanine decarboxylase from C. sinensis (CsAD) and multiple L-theanine synthases. Among them, the combination of CsAD and the L-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize L-theanine at the highest concentration of 13.42 mu M. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the L-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert L-alanine and L-glutamate in the medium to L-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the L-theanine through the two-step L-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.