Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

Tian, Xiong; Liu, Wanqiu; Xu, Huiling; Ji, Xiangyang; Liu, Yushi; Li, Jian

doi:10.1016/j.synbio.2022.03.006

Cited by 20 publications

(35 citation statements)

References 49 publications

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“…37 Interestingly, Streptomyces CFE systems can improve the solubility of proteins originated from Streptomyces when compared to E. coli-based CFE. 34,39,47 This could be attributed to the presence of some yet unknown accessory proteins (e.g., molecular chaperones) or metabolite factors in Streptomyces cell extracts. Another reason could be that a strain-specic physicochemical environment (e.g., metabolites) that mimics the native Streptomyces cytoplasm may play a role in protein folding and solubility.…”

Section: Natural Product Biosynthetic Enzymes and Pathwaysmentioning

confidence: 99%

Section: Natural Product Biosynthetic Enzymes and Pathwaysmentioning

confidence: 99%

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Streptomyces cell-free systems for natural product discovery and engineering

Lai

Freemont

2023

Nat. Prod. Rep.

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Section: Natural Product Biosynthetic Enzymes and Pathwaysmentioning

confidence: 99%

Section: Natural Product Biosynthetic Enzymes and Pathwaysmentioning

confidence: 99%

Streptomyces cell-free systems for natural product discovery and engineering

Lai

Freemont

2023

Nat. Prod. Rep.

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“…Recently, CFPS has been used for various protein production such as antibodies [ 20 ], metalloproteins [ 21 ], large proteins [ 22 , 23 ], membrane proteins [ 21 , 24 ], and virus-like particles [ 25 ]. In addition, CFPS-based systems have been used for gene circuit construction [ 26 , 27 ], metabolic pathway prototyping [ 28 , 29 , 30 ], natural product biosynthesis [ 31 , 32 , 33 ], and medical diagnosis [ 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Expression of a Therapeutic Protein Serratiopeptidase

Yang

Liu

et al. 2023

Molecules

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Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca2+ ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications.

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“…Cell-free protein synthesis (CFPS) systems, which use crude cellular extracts instead of intact cells to complete in vitro transcription and translation, are robust platforms for protein synthesis and biological applications [ 1 , 2 , 3 ]. Because of the open nature of cell-free systems, CFPS reactions have many advantages such as easy manipulation, high productivity, and tolerance of toxic products [ 4 , 5 , 6 , 7 , 8 ]. Using the CFPS technology, various proteins have been produced, including membrane proteins, therapeutic proteins, metalloenzymes, and unnatural amino acid-modified proteins [ 9 , 10 , 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System

Yang

Zhao

et al. 2022

Molecules

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Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 μg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.

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Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

Cited by 20 publications

References 49 publications

Streptomyces cell-free systems for natural product discovery and engineering

Streptomyces cell-free systems for natural product discovery and engineering

Cell-Free Expression of a Therapeutic Protein Serratiopeptidase

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System

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