Application of Cell-Free Protein Synthesis System for the Biosynthesis of L-Theanine

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.

Automated summary

L-Theanine, an active component in tea leaves, can be synthesized using a cell-free protein synthesis system. Results show that CFPS can efficiently produce L-theanine, highlighting its potential application for the biosynthesis of other active compounds.