Development of a highly efficient and specific l-theanine synthase

Yao, Jun; Li, Jing; Xiong, Dandan; Qiu, Yuanyuan; Shi, Guizhi; Jin, Jian‐Ming; Tao, Yong; Tang, Shuang-Yan

doi:10.1007/s00253-020-10482-6

Cited by 16 publications

(15 citation statements)

References 58 publications

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“…3). GshA (g-GCS) from E. coli W3110 served as a positive control (30). Each extract synthesized L-theanine, and the activities of PsGMAS and SmGMAS were highest.…”

Section: Resultsmentioning

confidence: 99%

“…g-GCS, which catalyzes the first step in glutathione biosynthesis in Escherichia coli, synthesizes low yields of L-theanine (29). This problem was addressed by the conversion of g-GCS to an L-theanine synthase through directed evolution, whereas the original catalytic activity remained (30). GS derived from Corynebacterium glutamicum ATCC 13032 was first reported to synthesize L-theanine from Glu and ethylamine in the presence of ATP (31).…”

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confidence: 99%

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Production of l -Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

Hagihara

Ohno

Hayashi

et al. 2021

Appl Environ Microbiol

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l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected the novel transaminase AAN70747 from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase AAY37316 from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt, which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. To our knowledge, this is the first report of efficient methods to produce l-theanine in the absence of supplemental ethylamine. IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. Here we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine.

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“…3). GshA (g-GCS) from E. coli W3110 served as a positive control (30). Each extract synthesized L-theanine, and the activities of PsGMAS and SmGMAS were highest.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Production of l -Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

Hagihara

Ohno

Hayashi

et al. 2021

Appl Environ Microbiol

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“…Notably, lyophilised cell‐free extracts were employed for the synthesis of 336 mM of L‐theanine 21 from glutamate, ethylamine and 5 mM ATP (STY of 2.4 g L −1 h −1 , entry 8, Table 3). [55] Starting from the same precursors, the EcPPK1‐powered L‐theanine 21 synthesis yielded 164.7 mM product in a 1 L‐scale reaction starting from 5 mM ATP [56] . The latter ATP‐recycling enzyme was also implemented in the PLP‐dependent decarboxylation of lysine 18 to cadaverine 19 (entry 7, Table 3), but in that case EcPPK1 was adopted to enhance cadaverine 19 production through in situ synthesis of millimolar amounts of PLP from pyridoxal (PL), rather than being directly involved in the generation of considerable amounts of product [57–58] …”

Section: Application Of Polyphosphate Kinasesmentioning

confidence: 99%

“…These two rather contradictory facets of polyP‐driven ATP regeneration were addressed through the addition of polyP in fed‐batch mode and optimisation of Mg 2+ concentration [41,54–56,58] . For example, fed‐batch addition of polyP during glycerol 3‐phosphate 7 production boosted final product concentration from 30 mM to 81 mM (entry 3, Table 3), [34] whereas L‐glutathione 15 yield went from 15.2 mM to 28.5 mM with stepwise addition of polyP (entry 5, Table 3).…”

Section: Application Of Polyphosphate Kinasesmentioning

confidence: 99%

“…The intensification of ATP‐dependent processes with fully in vitro polyP‐driven cofactor recycling is not often carried out with pH monitoring. However, continuous base additions were needed to maintain pH to the desired value during L‐theanine 21 synthesis [55–56] and an excessive amount of base was required to maintain pH close to neutrality during carboxylate reduction with integrated ATP‐recycling [36] . The reasons behind this phenomenon were not investigated, but it was speculated that polyP hydrolysis and/or acid‐generating host background activities might contribute to the observed acidification [36,55–56,77−78] …”

Section: Application Of Polyphosphate Kinasesmentioning

confidence: 99%

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Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

et al. 2021

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Polyphosphate kinases (PPKs) have emerged as valuable candidates to address the unmet need for scalable recycling of the common enzyme cofactor adenosine‐5’‐triphosphate (ATP) because they use cheap, freely available and stable polyphosphate (polyP) salts as the phosphate donor. The aim of this review is not only to present recent efforts in the characterisation of PPKs but also to provide an overview of the challenges associated with their implementation in chemical manufacturing. In assessing the current status of polyP‐driven biocatalysts, we identify gaps that need to be filled for successful adoption of these biocatalysts in industry. Guidelines for a wider adoption of PPKs are also provided.

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Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering

Zhang

Long

Zheng

et al. 2023

Appl Microbiol Biotechnol

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Development of a highly efficient and specific l-theanine synthase

Cited by 16 publications

References 58 publications

Production of l -Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

Production of l -Theanine by Escherichia coli in the Absence of Supplemental Ethylamine

Recent Developments and Challenges for the Industrial Implementation of Polyphosphate Kinases

Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering

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