Establishing Modular Cell-Free Expression System for the Biosynthesis of Bicyclomycin from a Chemically Synthesized Cyclodipeptide

Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.

Dihydrofolate reductase-like protein inactivates hemiaminal pharmacophore for self-resistance in safracin biosynthesis

Dihydrofolate reductase(DHFR),a housekeeping enzyme in primary metabolism,has been extensively studied as a model of acid-base catalysis and a clinic drug target.Herein,we investigated the enzymology of a DHFR-like protein SacH in safracin(SAC)biosynthesis,which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance.Furthermore,based on the crystal structure of SacH−NADPH−SAC-A ternary complexes and mutagenesis,we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore.These findings expand the functions of DHFR family proteins,reveal that the common reaction can be catalyzed by distinct family of enzymes,and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.

Deciphering the Origin and Formation of Aminopyrrole Moiety in Kosinostatin Biosynthesis

Kosinostatin(KST)contains an uncommon aminopyrrole moiety,whose biosynthesis has remained elusive.Herein,aminopyrrolinic acid,which was generated by an L-ectoine synthase-like enzyme KstB3 via cyclization of L-glutamine,was identified to be the real substrate of adenylation enzyme KstB1.Subsequently,a FAD-dependent dehydrogenase KstB4 along with a transglutaminase-like enzyme KstB6 were also involved in formation of aminopyrrole.These results provided an unusual pathway for 2-aminopyrrole formation in KST biosynthesis.

Characterization of a Membrane-Bound O-Acetyltransferase Involved in Trioxacarcin Biosynthesis Offers Insights into Its Catalytic Mechanism

of main observation and conclusion Trioxacarcin A(TXN-A)is a polycyclic aromatic natural product with remarkable biological activity.TxnB11,a membrane-bound O-acetyltransferase involved in TXN-A biosynthesis that is difficult for protein manipulation,is biochemically characterized here for its acetylation function on C4-sugar.A proposed catalytic mechanism is supported by transmembrane helix analysis and site-directed mutagenesis,in which four conserved amino acid residues His35,Ser71,His1OO and His317 are essential for enzyme activity.We tested the substrate tolerance of TxnB11 to acyl donors in vitro and found that TxnB11 can also utilize propionyl-CoA and glycolyl-CoA,resulting in two new TXN analogs with anti-tumor activity.This work provides a first understanding of the catalytic mechanism of this unusual acyltransferase family and presents good candidates for creating structural diversity for natural products.

Characterizing Post-PKS Modifications of 16-Demethyl-rifamycin Revealed Two Dehydrogenases Diverting the Aromatization Mode of Naphthalenic Ring in Ansamycin Biosynthesis

The anti-tuberculous rifamycins belong to naphthalenic ansamycin based on the structure of aromatic chromophore.Herein,we explored the post-polyketide synthase(PkS)modifications in the biosynthesis of 16-demethyl-rifamycins via gene knockout,complementation and in vitro enzyme assays.The collective evidences showed that i)the aromatization of 8-hydroxyl-7,8-dihydronaphtoquinone was accomplished by the combined action of two dehydrogenases,Rifs and Rif;i)the acetylation and methylation of the macrocycle was carried out on naphthoquinone intermediates in preference to naphthol,by Rif-Orf20 and Rif-Orf14,respectively ii)the presence of Rifs/T homologs in ansamycin biosynthetic gene clusters corresponds to the dehydrogenation aromatization mode of dihydronaphthalene.These findings cast new insights into the naphthalene formation and post-PkS modification of ansamycins.