Enzyme Assembly for Compartmentalized Metabolic Flux Control

Cui, Shumao; Gu, Yang; Li, Jianghua; Du, Guocheng; Liu, Long

doi:10.3390/metabo10040125

Cited by 19 publications

(17 citation statements)

References 174 publications

(243 reference statements)

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“…Moreover, the method of scaffold‐free enzyme assemblies for the construction of enzyme complex is an effective strategy to prevent intermediate diffusion, improve product yield and control the flux of metabolites in vivo and in vitro 54 . Some pairs of short peptide tags from the dock‐and‐lock peptide interacting family, such as RIAD and RIDD, SpyTag and SpyCatcher, could be used to create scaffold‐free enzyme complexes 55 . By scaffold‐free assembling the key enzyme IPS with the cascade enzymes in the myo ‐inositol biosynthetic pathway, it will lead to the formation of a pathway node and the improvement of myo‐inositol production.…”

Section: Discussionmentioning

confidence: 99%

Production of myo‐inositol: Recent advance and prospective

Han

Wang

et al. 2021

Biotech and App Biochem

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Myo‐inositol and its derivatives have been extensively used in the pharmaceutics, cosmetics, and food and feed industries. In recent years, compared with traditional chemical acid hydrolysis, biological methods have been taken as viable and cost‐effective ways to myo‐inositol production from cheap raw materials. In this review, we provide a thorough overview of the development, progress, current status, and future direction of myo‐inositol production (e.g., chemical acid hydrolysis, microbial fermentation, and in vitro enzymatic biocatalysis). The chemical acid hydrolysis of phytate suffers from serious phosphorous pollution and intricate product separation, resulting in myo‐inositol production at a high cost. For microbial fermentation, creative strategies have been provided for the efficient myo‐inositol biosynthesis by synergetic utilization of glucose and glycerol in Escherichia coli. in vitro cascade enzymatic biocatalysis is a multienzymatic transformation of various substrates to myo‐inositol. Here, the different in vitro pathways design, the source of selected enzymes, and the catalytic condition optimization have been summarized and analyzed. Also, we discuss some important existing challenges and suggest several viewpoints. The development of in vitro enzymatic biosystems featuring low cost, high volumetric productivity, flexible compatibility, and great robustness could be one of the promising strategies for future myo‐inositol industrial biomanufacturing.

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Section: Discussionmentioning

confidence: 99%

Production of myo‐inositol: Recent advance and prospective

Han

Wang

et al. 2021

Biotech and App Biochem

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“…The system allows tuneable control of multiple parameters and is suitable for high throughput analysis of combinatorial cargo protein designs in vivo . The orientation of enzyme fusions are known to impact activity [30]; the relative timing of the expression of enzyme cargo and coat protein can be an important factor in enzyme maturation before encapsulation in VLPs [12]; and the loading density is expected to be influenced by both the timing of expression and the level of cargo enzyme induction. The system presented here allows for facile testing of these parameters to optimise the influence of loading density on enzyme activities within P22-based biocatalytic nanoreactors.…”

Section: Introductionmentioning

confidence: 99%

Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

Esquirol

McNeale

Douglas

et al. 2022

Preprint

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Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons, and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside virus-like particles can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here we present a set of acceptor vectors that provide a quick and efficient way to build, test and optimise cargo loading inside P22 virus-like particles. We prototyped the system using yellow fluorescent protein then applied it to mevalonate kinases, a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different mevalonate kinases required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimise cargo enzyme behaviour in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behaviour.

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“…Interestingly, attention is currently being focused on metabolic regulation via the supramolecular assembly or high-order structure formation of metabolic enzymes into micron-sized structures within living cells, as has been revealed in several species [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. The highly dynamic and reversible assembly and disassembly of metabolic enzymes has been suggested as an intracellular mechanism to either stimulate or suppress the activity of the target metabolic enzymes, allowing the modulation of their cognate metabolic flux to reach the desired metabolic state and cellular outcome [ 10 , 11 , 14 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase

Surasiang

Noree

2021

Biology

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Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.

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Enzyme Assembly for Compartmentalized Metabolic Flux Control

Cited by 19 publications

References 174 publications

Production of myo‐inositol: Recent advance and prospective

Production of myo‐inositol: Recent advance and prospective

Rapid design and prototyping of biocatalytic virus-like particle nanoreactors

Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase

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