Research Center for Translational Medicine at Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
ACS Synth Biol. 2021 Mar 19;10(3):620-631. doi: 10.1021/acssynbio.0c00618. Epub 2021 Mar 10.
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 (CsTS2) could produce l-theanine at a concentration of 11.31 μM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from could produce l-theanine at the highest concentration of 302.96 μM 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 (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from (PtGS) could synthesize l-theanine at the highest concentration of 13.42 μM. Then, we constructed an engineered strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered 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.
L-茶氨酸是茶叶叶片中的一种活性成分，具有多种健康益处和广泛的应用。L-茶氨酸可以通过化学合成得到，但是这种方法会生成手性化合物，需要进一步分离纯的 L-异构体。异源生物合成是一种替代策略，但是一个主要的限制因素是微生物宿主细胞对底物乙胺的毒性。在本研究中，我们引入了一种无细胞蛋白合成（CFPS）系统来生产 L-茶氨酸。CFPS 表达的来自 （CsTS2）的 L-茶氨酸合成酶 2 在合成反应 32 小时后可以产生 11.31 μM 的 L-茶氨酸。此外，我们在 CFPS 中表达了来自微生物的三种同工酶来进行 L-茶氨酸生物合成。来自 （E. coli）的 γ-谷氨酰半胱氨酸合成酶可以在反应 24 小时后产生最高浓度为 302.96 μM 的 L-茶氨酸。此外，我们还使用 CFPS 验证了一个由 （CsAD）和多个 L-茶氨酸合成酶组成的假设的两步 L-茶氨酸生物合成途径。其中，CsAD 和来自 （PtGS）的 L-谷氨酰胺合成酶的组合可以在最高浓度 13.42 μM 下合成 L-茶氨酸。然后，我们构建了一个过表达 CsAD 和 PtGS 的工程 菌株，以进一步确认在活细胞中进行 L-茶氨酸生物合成的能力。该工程 菌株可以在 72 小时的发酵后将培养基中的 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 系统可以通过两步 L-茶氨酸生物合成途径来生产 L-茶氨酸，这表明 CFPS 系统在其他活性化合物生物合成方面具有潜在的应用前景。
