scholarly journals Production of L-theanine by Escherichia coli in the absence of supplemental ethylamine

2021 ◽  
Author(s):  
Ryota Hagihara ◽  
Shoto Ohno ◽  
Mikiro Hayashi ◽  
Kazuhiko Tabata ◽  
Hirofumi Endo
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Industrial Production ◽  
Large Scale ◽  
Food Additives ◽  
Production Capacity ◽  
Production Costs ◽  
Scale Production ◽  
Large Scale Production ◽  
Tea Plants

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.

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

Production ofBacillus amyloliquefaciensOG and its metabolites in renewable media: valorisation for biodiesel production andp-xylene decontamination

2017 ◽  
Vol 63 (1) ◽  
pp. 46-60 ◽  
Author(s):  
Augusto Etchegaray ◽  
François Coutte ◽  
Gabrielle Chataigné ◽  
Max Béchet ◽  
Ramon H.Z. dos Santos ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Large Scale ◽  
Production Costs ◽  
Biodiesel Production ◽  
Scale Production ◽  
Specific Production ◽  
Primary And Secondary Metabolites ◽  
Large Scale Production ◽  
Promote Plant Growth ◽  
Acetoin Production

Biosurfactants are important in many areas; however, costs impede large-scale production. This work aimed to develop a global sustainable strategy for the production of biosurfactants by a novel strain of Bacillus amyloliquefaciens. Initially, Bacillus sp. strain 0G was renamed B. amyloliquefaciens subsp. plantarum (syn. Bacillus velezensis) after analysis of the gyrA and gyrB DNA sequences. Growth in modified Landy’s medium produced 3 main recoverable metabolites: surfactin, fengycin, and acetoin, which promote plant growth. Cultivation was studied in the presence of renewable carbon (as glycerol) and nitrogen (as arginine) sources. While diverse kinetics of acetoin production were observed in different media, similar yields (6–8 g·L–1) were obtained after 72 h of growth. Glycerol increased surfactin-specific production, while arginine increased the yields of surfactin and fengycin and increased biomass significantly. The specific production of fengycin increased ∼10 times, possibly due to a connecting pathway involving arginine and ornithine. Adding value to crude extracts and biomass, both were shown to be useful, respectively, for the removal of p-xylene from contaminated water and for biodiesel production, yielding ∼70 mg·g–1cells and glycerol, which could be recycled in novel media. This is the first study considering circular bioeconomy to lower the production costs of biosurfactants by valorisation of both microbial cells and their primary and secondary metabolites.

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