Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

Autor:	Siebels I; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany., Nowak S; Fachbereich Biowissenschaften, Molecular Biotechnology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany., Heil CS; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany., Tufar P; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany., Cortina NS; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany., Bode HB; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Fachbereich Biowissenschaften, Molecular Biotechnology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Senckenberg Gesellschaft für Naturforschung, Frankfurt am Main, 60325, Germany., Grininger M; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.
Jazyk:	angličtina
Zdroj:	ACS synthetic biology [ACS Synth Biol] 2020 Sep 18; Vol. 9 (9), pp. 2418-2426. Date of Electronic Publication: 2020 Sep 03.
DOI:	10.1021/acssynbio.0c00186
Abstrakt:	A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.
Databáze:	MEDLINE
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