Efficient fermentative production of l-theanine by Corynebacterium glutamicum

Ma, Hongkun; Fan, Xiaoguang; Cai, Ningyun; Zhang, Dezhi; Zhao, Genhai; Wang, Ting; Su, Rui; Yuan, Meng; Ma, Qian; Zhang, Chenglin; Xu, Qingyang; Xie, Xiulan; Chen, Ning; Li, Yanjun

doi:10.1007/s00253-019-10255-w

Cited by 33 publications

(39 citation statements)

References 30 publications

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“…All experiments were performed at least in triplicate, and statistical significance was determined using one-way analysis of variance followed by Dunnett’s multiple comparisons test. P < 0.05 was considered statistically significant [ 16 ].…”

Section: Methodsmentioning

confidence: 99%

Enhancing the efficiency of L-tyrosine by repeated batch fermentation

Chen

et al. 2020

Bioengineered

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L-tyrosine is a widely used aromatic amino acid with an increasing market demand. Improving the parameters of L-tyrosine production results in a more cost-effective process that is of great interest for industrial applications. E. coli GHLTYR-168 was used to ferment L-tyrosine, with a productivity of 1.73 g/(L•h) and a yield of 17.6%. To further increase its production efficiency, repeated batch fermentation was applied to L-tyrosine, in which both replacement time points and ratios were studied during different fermentations. The broth substitution time point had no significant effect on L-tyrosine subjected to repeated batch fermentation, and 70% broth replacement ratio was the best choice. Repeated batch fermentation was performed in 5 batches within 100 h, among which the efficiency of the third batch fermentation was the highest. In the third batch fermentation, the productivity and yield were 2.53 g/(L•h) and 30.1%, respectively. Compared with that during fedbatch fermentation, the productivity and yield of L-tyrosine increased by 43.8% and 74.0%, respectively, during repeated batch fermentation. This is the highest level of L-tyrosine fermentation reported so far. Thus, repeated batch fermentation of L-tyrosine can improve its production efficiency.

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Section: Methodsmentioning

confidence: 99%

Enhancing the efficiency of L-tyrosine by repeated batch fermentation

Chen

et al. 2020

Bioengineered

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“…Biomass was expressed in terms of cell dry weight (cell dry weight = 0.364 × OD 600 ), where OD 600 refers to the absorbance of the fermentation broth measured at 600 nm, as determined using a spectrophotometer [ 7 ]. Glucose in the fermented broth was measured using a bioanalyzer [ 8 ], and amino acid concentrations were measured using an automatic amino acid analyzer [ 7 ]. The acetic acid in the fermented broth was measured using high-performance liquid chromatography [ 5 ].…”

Section: Methodsmentioning

confidence: 99%

A new method to recover L-tyrosine from E. coli fermentation broth

JingYang

Chen

et al. 2020

Bioengineered

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Although the production of L-tyrosine by recombinant Escherichia coli has been widely reported, L-tyrosine recovery from the fermentation broth has been rarely reported. Methods to recover L-tyrosine from the broth after alkaline lysis of the bacterial cells have been described. However, the broth becomes viscous and dark following cell lysis, making further extraction and purification difficult. Here, a new method for L-tyrosine extraction and purification from the fermentation broth without the lysis of bacteria is reported. First, acids, rather than bases, were used to dissolve L-tyrosine in the broth without causing lysis of the bacterial cells. E. coli cells in the broth were then removed through centrifugation. Activated carbon was then used to decolorize the supernatant containing L-tyrosine. Finally, sodium hydroxide was added to the clarified L-tyrosine solution for isoelectrocrystallization. L-tyrosine was obtained after filtration and drying. The recovery yield of L-tyrosine was 92%, and the purity was >98.5%, indicating high efficiency of the new method of L-tyrosine recovery from fermented broth. Furthermore, the method provides a reference for the extraction of guanosine, inosine, hypoxanthine, and other biological fermentation products.

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“…Therefore, the fermentative production of L-theanine was accomplished using a C. glutamicum strain that expresses recombinant GMAS derived from M. mays No. 9 (MmGMAS), supplemented with ethylamine (12). Under these conditions, the enzymes and substrates, except ethylamine, are simultaneously produced.…”

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confidence: 99%

“…To address this problem, use of alanine decarboxylase (AlaDC) in a fermentation method was attempted (12). AlaDC decarboxylates L-alanine (Ala) to produce ethylamine, which plays an important role in L-theanine biosynthesis (38).…”

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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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Efficient fermentative production of l-theanine by Corynebacterium glutamicum

Cited by 33 publications

References 30 publications

Enhancing the efficiency of L-tyrosine by repeated batch fermentation

Enhancing the efficiency of L-tyrosine by repeated batch fermentation

A new method to recover L-tyrosine from E. coli fermentation broth

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

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