Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin

Zhuang, Lei; Huang, San Yuan; Liu, Wan Qiu; Karim, Ashty S.; Jewett, Michael C.; Li, Jian

doi:10.1016/j.ymben.2020.03.009

Cited by 64 publications

(81 citation statements)

References 50 publications

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“…Bioprocess optimization has led to the production of 3.26 g/L mycophenolic acid in Penicillium brevicompactum by feeding sorbitol and controlling the cultivation pH at 6 [ 58 ]. Remarkably, a cell-free system has been recently optimized to improve the production of cyclic polyketides valinomycin [ 59 ], which has been proven effective to eradicate SARS-CoV virus. Cell free system and synthetic enzyme cascades have also been employed to synthesize the anti-HIV drug islatravir, which is a nucleotide analog and functions as a nucleoside reverse transcriptase translocation inhibitor [ 60 ].…”

Section: Microbial Production Of Natural Products With Antiviral Actimentioning

confidence: 99%

A roadmap to engineering antiviral natural products synthesis in microbes

2020

Current Opinion in Biotechnology

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Section: Microbial Production Of Natural Products With Antiviral Actimentioning

confidence: 99%

A roadmap to engineering antiviral natural products synthesis in microbes

2020

Current Opinion in Biotechnology

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“…Therefore, CFPS systems have recently attracted considerable attention as a robust approach for the production of various proteins, for example, membrane proteins (Henrich et al, 2015;Sonnabend et al, 2017), therapeutic proteins (Min et al, 2016;Wilding et al, 2019), unnatural amino acid modified proteins (Martin et al, 2018;Gao et al, 2019), and difficult-to-express proteins (Li et al, 2016;Jin and Hong, 2018). With the advances of synthetic biology, CFPS technology has also been used to construct protein-based biosensors (Pardee et al, 2016;Thavarajah et al, 2020), metabolic pathways (Goering et al, 2017;Zhuang et al, 2020), high-throughput screening platforms (Sawasaki et al, 2002;Swank et al, 2019), bottom-up synthetic cells (Karzbrun et al, 2014;van Nies et al, 2018), and classroom education kits (Huang et al, 2018;Stark et al, 2018), among others.…”

Section: Introductionmentioning

confidence: 99%

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

Zhang

Liu

2020

Front. Bioeng. Biotechnol.

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In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 µg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.

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“…Prokaryotic cell-free coupled transcription-translation systems are emerging as a new tool for studying natural product biosynthesis (2)(3)(4)(5)(6)(7)(8). Cell-free transcription-translation uses a crude cell-extract or purified ribosomes and translation factors -the PURE system -in a 'one-pot' reaction (9,10).…”

Section: Introductionmentioning

confidence: 99%

AStreptomyces venezuelaeCell-Free Toolkit for Synthetic Biology

Lai

Chee

Toh

et al. 2020

Preprint

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Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single ‘one-pot’ reaction.

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Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin

Cited by 64 publications

References 50 publications

A roadmap to engineering antiviral natural products synthesis in microbes

A roadmap to engineering antiviral natural products synthesis in microbes

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

AStreptomyces venezuelaeCell-Free Toolkit for Synthetic Biology

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