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      Recent advances in applying cell-free systems for high-value and complex natural product biosynthesis

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      Current Opinion in Microbiology
      Elsevier BV

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          Abstract

          <p class="first" id="d4717896e75">Natural products have complex chemical structures and exhibit diverse bioactivities. To harness these high-value natural compounds, robust approaches are required for their rapid and sustainable production. Recently, cell-free systems as one promising solution have been applied to produce natural products by reconstitution of their entire biosynthetic pathways in vitro. Here, we highlight recent advances in cell-free biosynthesis of natural products and discuss future challenges and opportunities. We envision that cell-free technology holds tremendous potential for natural product research such as high-throughput screening of peptide-based natural product analogs/variants, expression of toxic metabolic pathways, characterization of enzyme functions, and activation of silent biosynthetic gene clusters. </p>

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          Most cited references52

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          Assembly-line enzymology for polyketide and nonribosomal Peptide antibiotics: logic, machinery, and mechanisms.

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            Cell-free translation reconstituted with purified components.

            We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 microg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).
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              New developments in RiPP discovery, enzymology and engineering

              This review provides a comprehensive update of the advances in discovery, biosynthesis, and engineering of ribosomally-synthesized and post-translationally modified peptides (RiPPs). Covering: up to June 2020 Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
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                Author and article information

                Contributors
                Journal
                Current Opinion in Microbiology
                Current Opinion in Microbiology
                Elsevier BV
                13695274
                June 2022
                June 2022
                : 67
                : 102142
                Article
                10.1016/j.mib.2022.102142
                35259662
                5ccea474-1040-4c22-83c5-2420a397f6e3
                © 2022

                https://www.elsevier.com/tdm/userlicense/1.0/

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