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

Abstract

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 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 Escherichia coli 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 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 μM. 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.

Designing Modular Cell-free Systems for Tunable Biotransformation of l-phenylalanine to Aromatic Compounds

“…Recently, crude extract based cell-free protein synthesis (CFPS) systems were well developed and their applications have been expanded from single protein synthesis to multiple enzyme coexpression ( Kwon et al, 2013 ; Li et al, 2016 ; Goering et al, 2017 ; Moore et al, 2021 ). The CFPS-expressed enzymes without purification can carry out in situ cascade biotransformation to synthesize a wide variety of molecules, such as n -butanol ( Karim and Jewett, 2016 ), diketopiperazine ( Goering et al, 2017 ), polyhydroxyalkanoate ( Kelwick et al, 2018 ), styrene ( Grubbe et al, 2020 ), 3-hydroxybutyrate ( Karim et al, 2020 ), limonene ( Dudley et al, 2020 ), l -theanine ( Feng et al, 2021 ), and the complex natural product valinomycin ( Zhuang et al, 2020 ). Notably, the yields of the CFPS-based production platform are often higher than in vivo production.…”

Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

“…With the development of cell-free biotechnology, cell-free systems are emerging as powerful platforms not only for protein synthesis but also for many synthetic biology applications. In this context, cell-free strategies are creating a new frontier for next-generation biosynthesis through the rapid and rational construction of in vitro metabolic pathways [ [25] , [26] , [27] , [28] , [29] , [30] , [31] ]. To further expand the scope (type) of proteins and small high-value compounds that can be produced by cell-free systems, here we demonstrated cell-free biosynthesis of a nitro-containing compound L-4-nitro-Trp by in vitro reconstitution of the native biosynthetic pathway.…”

“…After optimization, the cell-free system was able to synthesize about 360 μM of L-4-nitro-Trp, which is a >10-fold improvement as compared to the initial system without optimization. Looking forward, we anticipate that cell-free biosynthetic platforms will play an important role in the production of various valuable compounds as showcased recently, including, for example, fine chemicals, pharmaceutical intermediates, food-related amino acids, and complex natural products [ [25] , [26] , [27] , [28] , [29] , [30] , [31] ]. In addition, sustainable production of such compounds in large amounts might also be possible in the near future as the E. coli CFPS system has been shown to scale up linearly from 250 μL to 100 L reaction volumes [ 48 ].…”

“…Now, the CFPS systems have been evolved into promising and powerful platforms for biomanufacturing by reconstitution of cell-free expressed enzymes. For example, several studies have demonstrated the rapid, high-yield synthesis of valuable chemicals relevant to energy, food, pharmaceuticals, and materials [ [25] , [26] , [27] , [28] ]. Because of the absence of cell walls, cell-free reactions are open that enables easy and flexible manipulation, monitoring, and optimization.…”

Biotechnology Applications of Cell-Free Expression Systems

“…Presently, natural ingredients that are used in a variety of food productions are produced via cell-based bacterial and yeast production platforms [125]. Microbial fermentation systems suffer from a myriad of disadvantages, such as low yields and the inability to produce many complex ingredients found in nature [126,127]. As a result, many ingredients must be extracted from natural sources, namely from plants and insects, which is often expensive and time-consuming.…”