Exploring Amino Sugar and Phosphoenolpyruvate Metabolism to Improve <i>Escherichia coli</i> <i>N</i>-Acetylneuraminic Acid Production

Pang, Qingxiao; Han, Hui; Xu, Ya Ming; Liu, Xiaoqin; Qi, Qingsheng; Wang, Qian

doi:10.1021/acs.jafc.0c04725

Cited by 8 publications

(13 citation statements)

References 44 publications

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“…Increasing the intracellular concentration of PEP, a co‐precursor of NeuAc, is another potential way to improve NeuAc synthesis (Pang et al, 2020). The construction of dual‐carbon sources for the improved production of NeuAc has been investigated, for example, using glucose and malate to balance the key precursor supply in NeuAc biosynthesis (Zhang et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

Guo

Tian

Wang

et al. 2022

Food Bioengineering

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N-acetylneuraminic acid (NeuAc), which has been widely used as a nutraceutical and pharmaceutical intermediate, plays an important role in improving brain development and cognition while enhancing immunity. Bacillus subtilis, generally regarded as a food-safe microorganism, is suitable for developing as a chassis cell for efficient NeuAc synthesis. However, accumulated intermediates can lead to metabolic bottlenecks for NeuAc synthesis. To eliminate the accumulated byproduct N-acetylglucosamine (GlcNAc), the UDP-GlcNAc epimerase pathway without GlcNAc production was first reconstructed and optimized in B. subtilis, resulting in the NeuAc titer increase of 5.9 g/L with GlcNAc elimination. In addition, to reduce another accumulated byproduct N-acetylmannosamine (ManNAc), the directed evolution of N-acetylneuraminic acid synthase and the enhancement of phosphoenolpyruvate supply was implemented. Using this strategy, ManNAc decreased by 46.3%, and the NeuAc titer increased by 54.9%, reaching 7.9 g/L. Finally, the maximum titer of NeuAc in a 3-L fermenter reached 21.8 g/L with a productivity of 0.34 g/L/h.

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Section: Resultsmentioning

confidence: 99%

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

Guo

Tian

Wang

et al. 2022

Food Bioengineering

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“…By optimizing the amino sugar metabolism pathway consisting of NeuB from Campylobacter jejuni and AGE from Anabaena sp. CH1 and improving phosphoenolpyruvate (PEP) precursor supply, the NeuAc titer was further increased to 16.7 g/L in the shake flask . In Bacillus subtilis, based on the previous construction of the GlcNAc synthetic pathway and the expression optimization of the N-terminal coding sequence, , modular pathway engineering was used to balance the GlcNAc and PEP precursor supply by overexpressing AGE and NeuB with deletion of ptsG (glucose-specific PTS enzyme), which produced 2.75 g/L NeuAc .…”

Section: Introductionmentioning

confidence: 99%

Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid

Liu

et al. 2022

J. Agric. Food Chem.

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N-Acetylneuraminic acid (NeuAc) is widely used in the food and pharmaceutical industries. Therefore, it is important to develop an efficient and eco-friendly method for NeuAc production. Here, we achieved de novo biosynthesis of NeuAc in an engineered plasmid-free Escherichia coli strain, which efficiently synthesizes NeuAc using glycerol as the sole carbon source, via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-based genome editing. NeuAc key precursor, N-acetylmannosamine (ManNAc; 0.40 g/L), was produced by expressing UDP-N-acetylglucosamine-2-epimerase and glucosamine-6-phosphate synthase (GlmS) mutants and blocking the NeuAc catabolic pathway in E. coli BL21 (DE3). The expression levels of GlmM and GlmU-GlmS A metabolic modules were optimized, significantly increasing the ManNAc titer to 8.95 g/L. Next, the expression levels of NeuAc synthase from different microorganisms were optimized, leading to the production of 6.27 g/L of NeuAc. Blocking the competing pathway of NeuAc biosynthesis increased the NeuAc titer to 9.65 g/L. In fed-batch culture in a 3 L fermenter, NeuAc titer reached 23.46 g/L with productivity of 0.69 g/L/h, which is the highest level achieved by microbial synthesis using glycerol as the sole carbon source in E. coli. The strategies used in our study can aid in the efficient bioproduction of NeuAc and its derivatives.

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“…CH1. 6 Using Bacillus subtilis as a production host, 2. increasing PEP supply in the shake flask, showing the potential for de novo synthesis of NeuAc in a generally regarded as safe (GRAS) host. 7,8 Furthermore, the NeuAc titer increased to 9.71 g/L in B. subtilis grown in a 3 L fermenter using the same gene source through genetic code expansion-based cell growth and biosynthesis balance engineering.…”

Section: ■ Introductionmentioning

confidence: 99%

“…jejuni and AGE derived from Anabaena sp. CH1 . Using Bacillus subtilis as a production host, 2.75 g/L NeuAc titer was obtained through the modular pathway engineering and expression optimization based on N -terminal coding sequences by overexpression of AGE derived from Anabaena sp.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Synthetic Multiplexed Pathways for High-Level N-Acetylneuraminic Acid Bioproduction

Zhang

Wang

Liu

et al. 2021

J. Agric. Food Chem.

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N-Acetylneuraminic acid (NeuAc) is widely used as a supplement to promote brain health and enhance immunity. However, the low efficiency of de novo NeuAc synthesis limits its cost-efficient bioproduction. Herein, a synthetic multiplexed pathway engineering (SMPE) strategy is proposed to improve NeuAc synthesis. First, we compare the key enzyme sources and optimize the expression levels of three NeuAc synthesis pathways in Bacillus subtilis; the AGE, NeuC, and NanE pathways, for which NeuAc production reached 3.94, 5.67, and 0.19 g/L, respectively. Next, these synthesis pathways were combined and modularly optimized via the SMPE strategy, with production reaching 7.87 g/L. Finally, fed-batch fermentation in a 5 L fermenter reached 30.10 g/L NeuAc production, the highest reported production using glucose as the sole carbon source. Using a generally regarded as safe strain as a production host, the developed NeuAc-producing approach should be favorable for efficient bioproduction, without the need for plasmids, antibiotics, or chemical inducers.

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Exploring Amino Sugar and Phosphoenolpyruvate Metabolism to Improve Escherichia coli N-Acetylneuraminic Acid Production

Cited by 8 publications

References 44 publications

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid

Engineering of Synthetic Multiplexed Pathways for High-Level N-Acetylneuraminic Acid Bioproduction

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