Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Abstract

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. -Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

“…To illustrate that this method was not only valid for NikP1, the substrate loading for the following systems were examined: the CloN5/CloN4 carrier/adenylation domain pair involved in the formation of clorobiocin (Table 1) (5), CouN5/CouN4 involved in the formation of the antibiotic coumermycin (5), EntB(ArCP)/EntE involved in the formation of the siderophore enterobactin (24), HMWP2, involved in the formation of the siderophore yersiniabactin (27), PchE/PchD involved in the biosynthesis of the siderophore pyochelin (38), JamC/JamA involved in the formation of the neurotoxin jamaicamide (39), and two NRPS modules from the orphan NRPS/PKS gene cluster on Bacillus subtilis for which the product is unknown (40,41,42). The adenylation-carrier di-domains analyzed are from PksN (BG12652) and the second NRPS module from PksJ (BG10929, gene annotation from http://genolist.pasteur.fr/SubtiList/.…”

Aromatic Prenylation in Phenazine Biosynthesis

“…Heterologous Expression of Cosmids ppzOS04 and ppzOS02-The plasmid pIJ787 (17) was first digested with DraI and BsaI, and the 4990-bp fragment containing the integrase cassette was used to replace the bla gene in the SuperCos 1 backbone in cosmids 11C7 and 18A9, using RED-mediated recombination (18,19), generating ppzOS02 and ppzOS04, respectively. Both cosmids were first transformed into the nonmethylating host E. coli ET12567, and the nonmethylated DNA was introduced into Streptomyces coelicolor M512 via triparental conjugation.…”

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

“…This could be explained by the determinant function of this A201A-resistance gene, since its disappearance would cause lethality. Because such genes are usually members of antibiotic biosynthetic clusters, 28 these findings indicate that ard1 is the terminal gene at the left-hand end of ata. Finally, it is noteworthy that a region close to 750 nucleotides with no apparent coding capacity flanks the 3ʹ region of the coding sequence of these two terminal genes, which contrasts with the compact organization of the DNA coding regions of streptomycetes.…”

“…A reasonable approach to overcome this problem is to express the antibiotic gene clusters in a heterologous host amenable to gene manipulation, like Streptomyces lividans or S. coelicolor. [26][27][28] In the case of A201A, many years ago we successfully identified two resistance determinants, ard1 16 and ard2, 19 as well as seven ORFs involved in the biosynthesis of the antibiotic. 12 Moreover, we next isolated a plasmid named pIES from a S. mutabilis subsp.…”