The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants

Doroshenko, V. G.; Lobanov, A. O.; Fedorina, E. A.

doi:10.1134/s0003683813020026

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…Histidine biosynthesis is mainly regulated by the histidine-mediated feedback inhibition of HisG that catalyzes the first step of the pathway. Residues in HisG that are responsible for this feedback inhibition have been identified, and mutants have been determined that result in the deregulation of the enzymes. ,,, To relieve the feedback inhibition, we tested expression of five variant HisG enzymes from E. coli (HisG eco ) or C. glutamicum (HisG cgl ) in HW1, including HisG eco E271 K , HisG cgl N215 K/L231F/T235A , HisG cgl N215I , HisG cgl G233H/T235Q , and HisG cgl S143F (HisG cgl with S143F mutation and C-terminal regulatory domain deletion). It has been demonstrated that the native HisG from E. coli is in equilibrium between a dimeric and hexameric form .…”

Section: Resultsmentioning

confidence: 99%

“…26 However, the modification of a single enzyme had limited influence on histidine production. Therefore, rational metabolic engineering strategies were applied in the construction of histidine producers, including promoter exchange of the hisD operon in C. glutamicum, 27 disruption of the attenuator region and the purine biosynthesis regulator gene in E. coli, 28 and improving the recycling of AICAR to AMP in E. coli. 29 Despite all these efforts, the fermentation capacity of the engineered strains remains far from meeting the industrial demand because these engineering strategies are confined to local metabolic pathways rather than global metabolic and regulatory networks.…”

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confidence: 99%

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Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli

Tian

Fan

et al. 2020

ACS Synth. Biol.

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l-Histidine is a functional amino acid with numerous therapeutic and ergogenic properties. It is one of the few amino acids that is not produced on a large scale by microbial fermentation due to the lack of an efficient microbial cell factory. In this study, we demonstrated the engineering of wild-type Escherichia coli to overproduce histidine from glucose. First, removal of transcription attenuation and histidine-mediated feedback inhibition resulted in 0.8 g/L histidine accumulation. Second, chromosome-based optimization of the expression levels of histidine biosynthesis genes led to a 4.75-fold increase in histidine titer. Third, strengthening phosphoribosyl pyrophosphate supply and rerouting the purine nucleotide biosynthetic pathway improved the histidine production to 8.2 g/L. Fourth, introduction of the NADH-dependent glutamate dehydrogenase from Bacillus subtilis and the lysine exporter from Corynebacterium glutamicum enabled the final strain HW6-3 to produce 11.8 g/L histidine. Finally, 66.5 g/L histidine was produced under fed-batch fermentation, with a yield of 0.23 g/g glucose and a productivity of 1.5 g/L/h. This is the highest titer and productivity of histidine ever reported from an engineered strain. Additionally, the metabolic strategies utilized here can be applied to engineering other microorganisms for the industrial production of histidine and related bioproducts.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli

Tian

Fan

et al. 2020

ACS Synth. Biol.

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“…Using a scarless CRISPR method ( Reisch and Prather, 2015 ), we introduced point mutations into genes encoding the allosteric enzyme that catalyzes the committed reaction in each pathway ( argA , hisG , trpE , leuA , ilvA , thrA , and proB ). These mutations have been shown previously to abolish the allosteric interaction while not affecting enzyme activity, thereby allowing us to study regulation of the pathway in the absence of allosteric feedback ( Caligiuri and Bauerle, 1991 , Csonka et al., 1988 , Doroshenko et al., 2013 , Gusyatiner et al., 2005 , LaRossa et al., 1987 , Lee et al., 2003 , Rajagopal et al., 1998 ). For N-acetylglutamate synthase (ArgA), we confirmed with in vitro assays that the mutation does not affect enzymatic activity and abolishes inhibition by arginine ( Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

Allosteric Feedback Inhibition Enables Robust Amino Acid Biosynthesis in E. coli by Enforcing Enzyme Overabundance

et al. 2019

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SummaryMicrobes must ensure robust amino acid metabolism in the face of external and internal perturbations. This robustness is thought to emerge from regulatory interactions in metabolic and genetic networks. Here, we explored the consequences of removing allosteric feedback inhibition in seven amino acid biosynthesis pathways in Escherichia coli (arginine, histidine, tryptophan, leucine, isoleucine, threonine, and proline). Proteome data revealed that enzyme levels decreased in five of the seven dysregulated pathways. Despite that, flux through the dysregulated pathways was not limited, indicating that enzyme levels are higher than absolutely needed in wild-type cells. We showed that such enzyme overabundance renders the arginine, histidine, and tryptophan pathways robust against perturbations of gene expression, using a metabolic model and CRISPR interference experiments. The results suggested a sensitive interaction between allosteric feedback inhibition and enzyme-level regulation that ensures robust yet efficient biosynthesis of histidine, arginine, and tryptophan in E. coli.

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“…The transcriptional elongation of the his operon is attenuated by tRNA when L-histidine is overproduced. 25 Then, we employed an artificial open reading frame hisL′, which was reported to enhance the translation of HisG, 26 to replace hisL along with the downstream noncoding region to generate strain BHS2 (Figure 5B). The chromosomal modification dramatically increases the L-histidine titer to 347.53 mg/L in strain BHS2, a 4.4-fold increase compared to strain BHS1.…”

Section: Journal Ofmentioning

confidence: 99%

Metabolic Engineering of Escherichia coli for de Novo Production of Betaxanthins

Hou

et al. 2020

J. Agric. Food Chem.

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Betalains are emerging natural pigments with high tinctorial strength and stability, physiological activities, and fluorescent properties for potential application in food, cosmetic, and pharmaceutical industries. Betalains including yellow betaxanthins and red betacyanins are mainly restricted in the Caryophyllales plants. To expand the availability of individual betaxanthins, here, we constructed an Escherichia coli BTA6 for de novo biosynthesis of betalamic acid. Using this strain as a monoculture platform, 14 yellow and 2 red betaxanthins were produced by feeding amino acids and amines. Furthermore, we constructed an l-histidine overproducing strain using chromosome engineering to deattenuate regulation and established a coculture system. After optimization of the initial inoculation ratios and fermentation conditions, the compatible and robust coculture system produced 287.69 mg/L of histidine-betaxanthin. This is the first report on de novo production of betaxanthins in engineered E. coli using glucose as a carbon source. Our work highlights the feasibility of microbial cell factories to produce individual betalains.

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The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants

Cited by 5 publications

References 15 publications

Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli

Highly Efficient Production of l-Histidine from Glucose by Metabolically Engineered Escherichia coli

Allosteric Feedback Inhibition Enables Robust Amino Acid Biosynthesis in E. coli by Enforcing Enzyme Overabundance

Metabolic Engineering of Escherichia coli for de Novo Production of Betaxanthins

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