Efficient synthesis of γ-glutamyl compounds by co-expression of γ-glutamylmethylamide synthetase and polyphosphate kinase in engineered <i>Escherichia coli</i>

Pan, Xinru; Yu, Jie; Du, Qinglin; Zeng, Shuiyun; Liu, Junzhong; Jiao, Qingcai; Zhang, Hongjuan

doi:10.1007/s10295-020-02305-4

Cited by 19 publications

(18 citation statements)

References 34 publications

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“…[27] In this study, they also showed, that conversion of IPA and l-glutamate to GIPA is possible with a molar conversion of 85.5 % by lyophilized E. coli cells. [27] We used the GMAS enzyme from M. extorquens but also other methylotrophs harbor GMAS enzymes. By comparison of the enzymatic activities for conversion of l-glutamate and ethylamine to l-theanine of purified GMAS enzymes from different organisms, the GMAS from Methylovorus mays showed the highest activity, followed by M. extorquens and Methyloversatilis universalis.…”

Section: Resultssupporting

confidence: 59%

“…[26] Co-expression and purification of GMAS and polyphosphate kinase (PPK) were used to test conversion of 14 different amines yielding the respective γ-glutamyl compounds by lyophilized E. coli cells via enzymatic synthesis. The study revealed a broad substrate spectrum including, in particular, the short-chain nalkylamines MMA, monoethylamine (MEA), n-propylamine, isopropylamine (IPA) and n-butylamine [27] (Figure 1). The novelty of our study is the de novo fermentative production of GIPA by engineered P. putida strains from glucose, but also from the alternative carbon sources glycerol and xylose.…”

Section: Introductionmentioning

confidence: 99%

“…[18] GIPA was produced from the cheap and achiral IPA [34] and lglutamate by enzymatic synthesis using GMAS from Methylovorus mays [26] and PPK from Corynebacterium glutamicum [35] in a one-pot enzyme reaction with lyophilized E. coli cells. [27] While fermentative production of N-methylglutamate and ltheanine with addition of MMA and MEA during growth on glucose, xylose, or glycerol by P. putida strain Thea1 expressing GMAS from Methylorubrum extorquens DM4 has been established, [29,36] fermentative GIPA production has not yet described. Strain Thea1 is a KT2440 derivative, a model laboratory species due to its robustness, simple nutritional requirements and rapid growth on standard media.…”

Section: Introductionmentioning

confidence: 99%

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γ‐Glutamylation of Isopropylamine by Fermentation

Benninghaus,

Zagami,

Tassini

et al. 2023

ChemBioChem

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Glutamylation yields N‐functionalized amino acids in several natural pathways. γ‐Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ‐Glutamyl−isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l‐glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l‐alaninol (2‐amino‐1‐propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ‐glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed‐batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L−1 with a product yield of 0.11 g g−1 glycerol and a volumetric productivity of 0.24 g L−1 h−1. To the best of our knowledge, this is the first fermentative production of GIPA.

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Section: Resultssupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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γ‐Glutamylation of Isopropylamine by Fermentation

Benninghaus,

Zagami,

Tassini

et al. 2023

ChemBioChem

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“…A well-established approach to overcome the insufficient supply of cofactors in microbes is incorporation of extra metabolic pathways for cofactor regeneration ( 9, 10 ). For example, formate dehydrogenase (FDH) or glucose dehydrogenase (GDH) are incorporated into cells to enhance generation of NAD(P)H from NAD(P) + using formate and D-glucose as reductants ( 11, 12 ), while polyphosphate kinase (PPK) is used to regenerate ATP from ADP and polyphosphate ( 5, 13 ). Simple addition of substrates and cofactors (particularly charged molecules) is typically sub-optimal as they generally have low cell permeability and require further optimization such as adding a surfactant ( 5 ).…”

Section: Introductionmentioning

confidence: 99%

A versatilein situcofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Jaroensuk

Sutthaphirom

Phonbuppha

et al. 2023

Preprint

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Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

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“…To overcome this problem, an ATP-regeneration system using glucose and cells such as Saccharomyces cerevisiae is often applied (34). ATP regeneration using polyphosphate kinase (EC 2.7.4.1), which catalyzes the transfer of phosphates from polyphosphate to ADP, serves as an alternative approach (36).…”

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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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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Efficient synthesis of γ-glutamyl compounds by co-expression of γ-glutamylmethylamide synthetase and polyphosphate kinase in engineered Escherichia coli

Cited by 19 publications

References 34 publications

γ‐Glutamylation of Isopropylamine by Fermentation

γ‐Glutamylation of Isopropylamine by Fermentation

A versatilein situcofactor enhancing system for meeting cellular demands for engineered metabolic pathways

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

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