Design of an <i>in vitro</i> multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide

Zhou, Changyu; Jiao, Feng; Wang, Jing; Hao, Ning; Wang, Xin; Chen, Kequan

doi:10.1039/d1cy01798e

Cited by 20 publications

(19 citation statements)

References 41 publications

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“…Therefore, the biocascade reaction is promising to be further scaled up with the same yield. Recently, an in vitro multienzyme cascade system for NMN synthesis via adenosine phosphate hydrolysis pathway was developed to generate 9 mM NMN, but the final condition requires 60 mM AMP as the starting material with the addition of 20 mM ATP and 60 mM NAM [45] . According to the initial AMP concentration, its yield is only 15 %.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, an in vitro multienzyme cascade system for NMN synthesis via adenosine phosphate hydrolysis pathway was developed to generate 9 mM NMN, but the final condition requires 60 mM AMP as the starting material with the addition of 20 mM ATP and 60 mM NAM. [45] According to the initial AMP concentration, its yield is only 15 %. In addition, AMP is a more expensive precursor for PRPP synthesis than xylose.…”

Section: Supernatant Of the Recombinant Strain Culture Applied In The...mentioning

confidence: 99%

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Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

et al. 2022

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β‐Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D‐ribose in the medium. The supernatant containing D‐ribose was applied in the second module which is composed of EcRbsK‐EcPRPS‐CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D‐ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Supernatant Of the Recombinant Strain Culture Applied In The...mentioning

confidence: 99%

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

et al. 2022

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“…The results showed that the optimal concentration of EcPPase was 0.5 µM (Supporting Information: Figure S6), and under this condition, the final titer of NMN was increased to 1213 mg/L, with a yield of 90.8% (Figure 7d, Supporting Information: Figure S6), which was a more than 12‐fold improvement of NMN titer over the initial setup (Figure 2c). Recently, several in vitro synthetic pathways used for NMN synthesis have been reported (Ngivprom et al, 2022; Qian et al, 2022; Zhou et al, 2022). Among them, an impressively high NMN titer of 93.5 g/L was achieved by enzymatic phosphorylation of nicotinamide riboside to NMN using a new nicotinamide riboside kinase.…”

Section: Resultsmentioning

confidence: 99%

“…However, the current high price of NMN hampers the widespread use and practical implementation of this molecule. While there have been many efforts to improve the production of NMN by engineering biosynthetic pathways in vivo (Marinescu et al, 2018; Shoji et al, 2021) or in vitro (Qian et al, 2022; Zhou et al, 2022), these efforts have typically explored only a small set of enzyme homologs in their optimization strategies. Hence, productive enzyme homologs and combinations for efficient synthesis of NMN are still required.…”

Section: Introductionmentioning

confidence: 99%

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

Yuan

Liao

et al. 2023

Biotech & Bioengineering

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Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cellfree protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

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“…However, the current high price of NMN hampers the widespread use and practical implementation of this molecule. While there have been many efforts to improve the production of NMN by engineering biosynthetic pathways in vivo (Marinescu et al, 2018;Shoji et al, 2021) or in vitro (Qian et al, 2022;Zhou et al, 2022), these efforts have typically explored only a small set of enzyme homologs in their optimization strategies. Hence, productive enzyme homologs and combinations for efficient synthesis of NMN are still required.…”

Section: Introductionmentioning

confidence: 99%

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP

Yuan

Liao

et al. 2022

Preprint

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Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a novel strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split GFP. This strategy featured two main advantages: 1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and 2) the expression level and activity of each homolog was determined [simultaneously](javascript:;) by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

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Design of an in vitro multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide

Abstract: Design the adenosine phosphate hydrolysis (APH) pathway multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide (NMN) in vitro.

Cited by 20 publications

References 41 publications

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP

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