Multiplex modification of Escherichia coli for enhanced β-alanine biosynthesis through metabolic engineering

Wang, Pei; Zhou, Haïyan; Ли, Бо; Ding, Wenqing; Liu, Zhiqiang; Zheng, Yu‐Guo

doi:10.1016/j.biortech.2021.126050

Cited by 24 publications

(7 citation statements)

References 45 publications

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“…23 Therefore, many studies have explored strategies to import glucose without using PEP as a phosphate donor to reduce carbon loss at the Pyr node. 10,19,21 As an alternative strategy, a non-PTS using ATP as a phosphate donor has been most widely applied to construct strains for β-alanine synthesis in previous studies (Figure 2A), 19,21 whereas metabolic burden from glucose uptake will be increased. 22 By this route, one glucose can theoretically biosynthesize two β-alanine.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…However, as a phosphate donor for the PTS, a partial PEP is consumed in the PTS to form pyruvate (Pyr), which subsequently enters into the oxidative branch of the TCA cycle and degrades to organic acids when glucose is used as a carbon source . Therefore, many studies have explored strategies to import glucose without using PEP as a phosphate donor to reduce carbon loss at the Pyr node. ,, As an alternative strategy, a non-PTS using ATP as a phosphate donor has been most widely applied to construct strains for β-alanine synthesis in previous studies (Figure A), , whereas metabolic burden from glucose uptake will be increased . By this route, one glucose can theoretically biosynthesize two β-alanine.…”

Section: Resultsmentioning

confidence: 99%

“…19 Subsequently, a similar route was also used to engineer E. coli which accumulated 43.94 g/L βalanine in fed-batch fermentation. 21 However, the engineered strains with this modification use ATP as a phosphate donor instead of PEP in glucose uptake and extract OAA in the TCA cycle, which may increase the metabolic burden in the cell. 22 In addition, the specific rate of acetate production is obviously increased when replacing a non-PTS with a PTS.…”

Section: ■ Introductionmentioning

confidence: 99%

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Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

Zhang

Wang

et al. 2022

ACS Synth. Biol.

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β-Alanine, with the amino group at the β-position, is an important platform chemical that has been widely applied in pharmaceuticals and feed and food additives. However, the current modest titer and productivity, increased fermentation cost, and complicated operation are the challenges for producing β-alanine by microbial fermentation. In this study, a high-yield β-alanineproducing strain was constructed by combining metabolic engineering, protein engineering, and fed-batch bioprocess optimization strategies. First, an aspartate-α-decarboxylase from Bacillus subtilis was introduced in Escherichia coli W3110 to construct an initial β-alanine-producing strain. Production of βalanine was obviously increased to 4.36 g/L via improving the metabolic flux and reducing carbon loss by rerouting fluxes of the central carbon metabolism. To further increase β-alanine production, mechanism-based inactivation of aspartate-α-decarboxylase was relieved by rational design to maintain the productivity at a high level in β-alanine fed-batch fermentation. Finally, fed-batch bioprocess optimization strategies were used to improve β-alanine production to 85.18 g/L with 0.24 g/g glucose yield and 1.05 g/ L/h productivity in fed-batch fermentation. These strategies can be effectively used in the construction of engineered strains for βalanine and production of its derivatives, and the final engineered strain was a valuable microbial cell factory that can be used for the industrial production of β-alanine.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

Zhang

Wang

et al. 2022

ACS Synth. Biol.

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“…Generally, AFB1 and CPA had a greater effect on amino acid metabolism in hepatocyte spheroids. Among these, β-alanine metabolism was the most affected metabolic pathway in all six groups, with enrichment P values around 0.4. β-Alanine is the only naturally occurring β-amino acid that plays an essential role in developing all organisms, affecting glucose production and metabolism in the liver. , In addition, the metabolism of a variety of amino acids such as glycine, serine, threonine, and aspartate was affected. As a substrate for protein synthesis, amino acids play an important role in cell proliferation and differentiation by regulating energy production while driving the synthesis of nucleosides and maintaining cellular redox homeostasis.…”

Section: Resultsmentioning

confidence: 99%

Coexposure of Cyclopiazonic Acid with Aflatoxin B1 Involved in Disrupting Amino Acid Metabolism and Redox Homeostasis Causing Synergistic Toxic Effects in Hepatocyte Spheroids

Sun

et al. 2022

J. Agric. Food Chem.

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Cyclopiazonic acid (CPA), an emerging toxin, has been found in various foods such as corn, peanuts, and figs.Aspergillus flavus can produce CPA, leading to coexposure with highly toxic aflatoxin B1 (AFB1), but the mechanism of their combined action is not clear. In this study, cocultured hepatocyte spheroids were used as the evaluation model, and two concentration settings of isotoxicity and different toxicity ratios were used to investigate the combined toxic effects. Metabolomics was subsequently used to analyze the potential mechanisms underlying the effects of their exposure. AFB1 and CPA might exhibit stronger cytotoxicity, with significant combined effects on mitochondrial morphology, activity, and reactive oxygen levels. The gene expression analysis revealed that the overexpression of AKT genes could mitigate the combined effects of AFB1 and CPA to some extent. Metabolomics analysis indicated that AFB1 and CPA significantly downregulated the metabolism of L-aspartate and antioxidant substances (e.g., penicillamine, myricetin, and ethanolamine). The pathway enrichment analysis also revealed a large impact on amino acid metabolism, likely affecting intracellular redox homeostasis. In addition, the presence of CPA affects intracellular glucose metabolism and lipid metabolism pathways. This study suggested a direction for future research on relevant toxic pathways and provided possible ideas for inhibiting or mitigating toxicity.

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“…High-level expression of rate-limiting enzymes to drive the metabolic flux of the target product biosynthetic pathway is a quick and straightforward strategy to increase the synthesis of precursors. In Escherichia coli, overexpression and knockout techniques are used to up- or downregulate gene expression to promote natural and unnatural products. , When introducing natural and unnatural product metabolic pathways into engineered strains, multiple enzymes are often required to participate. For example, the biosynthetic pathway of opioids thebaine and hydrocodone contains 21 and 23 enzyme reactions, respectively, from plants, mammals, yeast, and bacteria .…”

Section: Introductionmentioning

confidence: 99%

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis

You

Pan

et al. 2022

ACS Synth. Biol.

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Riboflavin is an essential vitamin widely used in the food, pharmaceutical, and feed industries. However, the insufficient supply of precursors caused by the imbalance of intracellular metabolic flow limits the riboflavin synthesis by industrial strains. Here, we increase riboflavin production by tuning multiple gene expression to balance intracellular metabolic flow. First, we tuned the expression of mCherry and egfp genes within operons by generating libraries of tunable intergenic regions (TIGRs) and confirmed the relative expression of the two reporter genes. The TIGR library can coordinate the expression ratio of reporter genes more than 180 times in Escherichia coli and more than 70 times in Bacillus subtilis. Next, we used this strategy to tune the expression of zwf, ribBA, and ywlf genes within operons through the TIGR library to increase the intracellular precursor pool for riboflavin biosynthesis. Based on the fluorescence characteristics of riboflavin, 96-well plates were used to screen the optimal combination mutants quickly. The best-engineered strain was selected from the library, which produced 2.7 g/L riboflavin, increasing by 64.35% in the shake flask. Finally, the riboflavin titer increased by 59.27% to 11.77 g/L in fed-batch fermentation. The strategy described here will contribute to the industrial production of riboflavin and related products by B. subtilis.

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Multiplex modification of Escherichia coli for enhanced β-alanine biosynthesis through metabolic engineering

Cited by 24 publications

References 45 publications

Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli

Coexposure of Cyclopiazonic Acid with Aflatoxin B1 Involved in Disrupting Amino Acid Metabolism and Redox Homeostasis Causing Synergistic Toxic Effects in Hepatocyte Spheroids

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis

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