Recombineering enables genome mining of novel siderophores in a non-model Burkholderiales strain

Iron is essential for bacterial survival,and most bacteria capture iron by producing siderophores.Burkholde-riales bacteria produce various types of bioactive secondary metabolites,such as ornibactin and malleobactin siderophores.In this study,the genome analysis of Burkholderiales genomes showed a putative novel siderophore gene cluster crb,which is highly similar to the ornibactin and malleobactin gene clusters but does not have pvdF,a gene encoding a formyltransferase for N-δ-hydroxy-ornithine formylation.Establishing the bacteriophage recom-binase Redγ-Redδβ7029 mediated genome editing system in a non-model Burkholderiales strain Paraburkholderia caribensis CICC 10960 allowed the rapid identification of the products of crb gene cluster,caribactins A-F(1-6).Caribactins contain a special amino acid residue N-δ-hydroxy-N-δ-acetylornithine(haOrn),which differs from the counterpart N-δ-hydroxy-N-δ-formylornithine(hOrn)in ornibactin and malleobactin,owing to the absence of pvdF.Gene inactivation showed that the acetylation of hOrn is catalyzed by CrbK,whose homologs proba-bly not be involved in the biosynthesis of ornibactin and malleobactin,showing possible evolutionary clues of these siderophore biosynthetic pathways from different genera.Caribactins promote biofilm production and en-hance swarming and swimming abilities,suggesting that they may play crucial roles in biofilm formation.This study also revealed that recombineering has the capability to mine novel secondary metabolites from non-model Burkholderiales species.

Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution

Polyethylene terephthalate(PET),one of the most widely used plastics in the world,causes serious environmental pollution.Recently,researchers have focused their efforts on enzymatic degradation of PET,which is an attractive way of degrading and recycling PET.In this work,PET hydrolase Sb PETase from Schlegelella brevitalea sp.nov.was biochemically characterized,and rational design was performed based on its sequence similarity with the previ-ously reported Is PETase from Ideonella sakaiensis,resulting in a triple mutant with increased activity.Furthermore,using a sec-dependent signal peptide PeIB and colicin release protein Kil,we set up a high-efficiency secretion system of PETase in Escherichia coli BL21(DE3),enabling higher PETase secretion.Utilizing this secretion system,we established a high-throughput screening method named SecHTS(sec retion-based h igh-throughput s creening)and performed directed evolution of Is PETase and Sb PETase through DNA shuffling.Finally,we generated a mutant Is PETase S139T with increased activity from the mutant library.

Cascade-Cas3 facilitates high-accuracy genome engineering in Pseudomona s using phage-encoded homologous recombination

Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas genome editing,false positives can be a problem.In this study,we combined a compact Cascade-Cas3 system from P.aeruginosa(PaeCas3c)with a Pseudomonas-specific PEHR system,and the results of our recom-bineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.

In vitro characterization of a nitro-forming oxygenase involved in 3-(trans-2’-aminocyclopropyl)alanine biosynthesis

In vitro characterization experiments revealed the formations of 3-(trans-2’-aminocyclopropyl)alanine((3-Acp)Ala)and 3-(trans-2’-nitrocyclopropyl)alanine((3-Ncp)Ala)are originated via two homologous proteins,BelK and HrmI,which regioselectively catalyze the N𝜀-oxygenation of l-lysine.The two enzymes belong to the emerg-ing heme-oxygenase-like diiron oxidase and oxygenase(HDO)superfamily and the catalytic center of BelK is validated by homology modeling and site-directed mutations.Based on the in vitro characterization,the biosyn-thetic pathways of(3-Acp)Ala and(3-Ncp)Ala are proposed.

Recent developments in the identification and biosynthesis of antitumor drugs derived from microorganisms

Secondary metabolites in microorganisms represent a resource for drug discovery and development.In particular,microbial-derived antitumor agents are in clinical use worldwide.Herein,we provide an overview of the devel-opment of classical antitumor drugs derived from microorganisms.Currently used drugs and drug candidates are comprehensively described in terms of pharmacological activities,mechanisms of action,microbial sources,and biosynthesis.We further discuss recent studies that have demonstrated the utility of gene-editing technologies and synthetic biology tools for the identification of new gene clusters,expansion of natural products,and eluci-dation of biosynthetic pathways.This review summarizes recent progress in the discovery and development of microbial-derived anticancer compounds with emphasis on biosynthesis.