Metabolic Engineering and Fermentation Process Strategies for L-Tryptophan Production by Escherichia coli

Liu, Lina; Bilal, Muhammad; Luo, Hongzhen; Zhao, Yuping; Iqbal, Hafiz M.N.

doi:10.3390/pr7040213

Cited by 25 publications

(14 citation statements)

References 70 publications

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“…To optimise producer strains it is aimed to circumvent these regulatory mechanisms, and a multitude of possible targets can be developed for rational design. Possible modifications to improve L-tryptophan producer strains have been previously summarised by [18,20,[22][23][24][25][26]. High L-tryptophan concentrations of 48.68 g L −1 with glucose as carbon source were reported with rationally engineered E. coli strains [23], and increased titres of 52.57 g L −1 were reached in combination with adapted pH and dissolved oxygen control strategies in the fed-batch process [27].…”

Section: Introductionmentioning

confidence: 99%

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate

Schoppel

Trachtmann

Mittermeier

et al. 2021

Bioprocess Biosyst Eng

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L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H+-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error. Metabolic analyses and subsequent extensive data evaluation were performed based on the data of the parallel analysis reactors and the production process. Extracellular rates and intracellular metabolite concentrations displayed evident deflections in cell metabolism and particularly in chorismate biosynthesis due to the perturbations with shikimate. Intracellular flux distributions were estimated using a thermodynamics-based flux analysis method, which integrates thermodynamic constraints and intracellular metabolite concentrations to restrain the solution space. Feasible flux distributions, Gibbs reaction energies and concentration ranges were computed simultaneously for the genome-wide metabolic model, with minimum bias in relation to the direction of metabolic reactions. Metabolic control analysis was applied to estimate elasticities and flux control coefficients, predicting controlling sites for L-tryptophan biosynthesis. The addition of shikimate led to enhanced deviations in chorismate biosynthesis, revealing a so far not observed control of 3-dehydroquinate synthase on L-tryptophan formation. The relative expression of the identified target genes was analysed with RT-qPCR. Transcriptome analysis revealed disparities in gene expression and the localisation of target genes to further improve the microbial L-tryptophan producer by metabolic engineering.

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Section: Introductionmentioning

confidence: 99%

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate

Schoppel

Trachtmann

Mittermeier

et al. 2021

Bioprocess Biosyst Eng

View full text Add to dashboard Cite

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“…L-Tryptophan (Trp), one of the high-value aromatic amino acids, is widely used in feed additive and as intermediate for pharmaceutics such as neurotransmitters ( Mateos et al., 2009 ; Williams et al., 2014 ) and antitumor drugs ( Fang et al., 2016 ; Rodrigues et al., 2013 ). Escherichia coli has been extensively exploited for effective bioproduction of Trp from renewable feedstock, e.g., glucose, and various rational strategies have been applied in order to achieve a high Trp productivity ( Liu et al., 2019 ; Li et al., 2020 ), including (i) alleviation of restrictive regulations ( Chen et al., 2018 ; Ikeda, 2003 ; Oldiges et al., 2004 ); (ii) deletion of competing pathways; (iii) enhancement and balancing of precursor supplements ( Ikeda, 2006 ); and (iv) removal of degradation pathways ( Aiba et al., 1980 ). For instance, Zhao et al.…”

Section: Introductionmentioning

confidence: 99%

Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis

Chen

et al. 2021

Metabolic Engineering Communications

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L-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered Escherichia coli has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.e. the galactose permease/glucokinase (GalP/Glk) system, can theoretically double the Trp yield from glucose. To explore this possibility, a PTS − and GalP/Glk-dependent E. coli strain was constructed from a previously rationally developed Trp producer strain S028. However, the growth rate of the S028 mutant was severely impaired. To overcome this problem, promoter screening for modulated gene expression of GalP/Glk was carried out, following by a batch mode of adaptive laboratory evolution (ALE) which resulted in a strain K3 with a similar Trp yield and concentration as S028. In order to obtain a more efficient Trp producer, a novel continuous ALE system was developed by combining CRISPR/Cas9-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of Trp-mediated fluorescence intensity. With the aid of this automatic system (auto-CGSS), a promising strain T5 was obtained and fed-batch fermentations showed an increase of Trp yield by 19.71% with this strain compared with that obtained by the strain K3 (0.164 vs. 0.137 g/g). At the same time, the specific production rate was increased by 52.93% (25.28 vs. 16.53 mg/g DCW /h). Two previously engineered enzyme variants AroG D6G−D7A and An TrpC R378F were integrated into the strain T5, resulting in a highly productive strain T5AA with a Trp yield of 0.195 g/g and a specific production rate of 28.83 mg/g DCW /h.

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“…Generally speaking, microbial degradation of xenobiotics involves the utilization of microbes with specific enzyme systems responsible for the degradation, mineralization, transformation or detoxification of pollutants [100]. Nevertheless, under certain growth conditions, composition, type and concentration of the pollutant, effective degradation is not expected even with the availability of microbes with degradation potentials.…”

Section: Genetically Modified Microorganism For Enhanced Eco-recoverymentioning

confidence: 99%

Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment

Ehis-Eriakha¹,

Akemu²,

Otumala³

et al. 2022

Crude Oil - New Technologies and Recent Approaches

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Globally, the environment is facing a very challenging situation with constant influx of crude oil and its derivatives due to rapid urbanization and industrialization. The release of this essential energy source has caused tremendous consequences on land, water, groundwater, air and biodiversity. Crude oil is a very complex and variable mixture of thousands of individual compounds that can be degraded with microbes with corresponding enzymatic systems harboring the genes. With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds and industrial solvents. Different remediation methods have been introduced and applied with varied degrees of success in terms of reduction in contamination concentration without considering ecotoxicity and restoration of biodiversity. Researchers have now developed methods that consider ecotoxicology, environmental sustainability and ecorestoration in remediation of crude oil impacted sites and they are categorized as biotechnological tools such as bioremediation. The approach involves a natural process of microorganisms with inherent genetic capabilities completely mineralizing/degrading contaminants into innocuous substances. Progressive advances in bioremediation such as the use of genetically engineered microbes have become an improved system for empowering microbes to degrade very complex recalcitrant substances through the modification of rate-limiting steps in the metabolic pathway of hydrocarbon degrading microbes to yield increase in mineralization rates or the development of completely new metabolic pathways incorporated into the bacterial strains for the degradation of highly persistent compounds. Other areas discussed in this chapter include the biosurfactant-enhanced bioremediation, microbial and plant bioremediation (phytoremediation), their mechanism of action and the environmental factors influencing the processes.

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Metabolic Engineering and Fermentation Process Strategies for L-Tryptophan Production by Escherichia coli

Cited by 25 publications

References 70 publications

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate

Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis

Biotechnological Potentials of Microbe Assisted Eco-Recovery of Crude Oil Impacted Environment

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