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Pathway engineering of Escherichia coli for one-step fermentative production of L-theanine from sugars and ethylamine.

ABSTRACT: L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of ?-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-P _T7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 ?g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 ?g/g glucose and 2.72 ?g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future.

SUBMITTER: Fan X

PROVIDER: S-EPMC7677707 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Pathway engineering of <i>Escherichia coli</i> for one-step fermentative production of L-theanine from sugars and ethylamine.

Fan Xiaoguang X Zhang Tong T Ji Yuanqing Y Li Jie J Long Keyi K Yuan Yue Y Li Yanjun Y Xu Qingyang Q Chen Ning N Xie Xixian X

Metabolic engineering communications 20201106

L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type <i>Escherichia coli</i> for one-step fermentative production of theanine from ...[more]

PMID: 33251110

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Project description: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 a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number 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. Our study reports 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. In this study, 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.

Project description:Background(-)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (-)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock.ResultsHere, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%).ConclusionsThe engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale.

Project description:BACKGROUND: Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. RESULTS: The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. CONCLUSIONS: The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

Project description:UNLABELLED:Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. IMPORTANCE:The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.

Dataset Information

Pathway engineering of Escherichia coli for one-step fermentative production of L-theanine from sugars and ethylamine.

Publications

Pathway engineering of <i>Escherichia coli</i> for one-step fermentative production of L-theanine from sugars and ethylamine.

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