Heterologous production of thiostrepton A and biosynthetic engineering of thiostrepton analogs

Abstract

Thiostrepton A 1, produced by Streptomyces laurentii ATCC 31255 (S. laurentii), is one of the more well-recognized thiopeptide metabolites. Thiostrepton A 1 and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens. Although numerous lines of evidence have established that the thiopeptides arise from the post-translational modification of ribosomally-synthesized peptides, few details have been revealed concerning this elaborate process. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton post-translational machinery. Due to the difficulties in the genetic manipulation of S. laurentii, the heterologous production of thiostrepton A 1 from an alternate streptomycete host was sought to facilitate the biosynthetic investigations of the peptide metabolite. The production of thiostrepton A 1 from the non-cognate hosts did not lend itself to be as robust as S. laurentii-based production, therefore an alternate strategy was pursued for the production of thiostrepton variants. The introduction of a fosmid used in the heterologous production of thiostrepton A 1, harboring the entire thiostrepton biosynthetic gene cluster, into the tsrA deletion mutant permitted restoration of thiostrepton A 1 production near to that of the wild-type level. The fosmid was then engineered to enable the replacement of wild-type tsrA. Introduction of expression fosmids encoding alternate TsrA sequences into the S. laurentii tsrA deletion mutant led to the production of thiostrepton variants retaining antibacterial activity, demonstrating the utility of this expression platform toward thiopeptide engineering.

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

“…(20) Fosmid int-3A100 is derived from the single-copy fosmid pCC1FOS (Epicentre Biotechnologies) and was engineered to include an integrase and and attP sequence for integration into the S. laurentii chromosome. (20, 31) The entire tsr biosynthetic gene cluster is harbored within fosmid int-3A100, except that the TsrA core peptide-encoding region is replaced by a dual-marker selection cassette consisting of chl R (a chloramphenicol resistance gene) and a levansucrase-encoding sacB gene.…”

“…(20, 31) The entire tsr biosynthetic gene cluster is harbored within fosmid int-3A100, except that the TsrA core peptide-encoding region is replaced by a dual-marker selection cassette consisting of chl R (a chloramphenicol resistance gene) and a levansucrase-encoding sacB gene. (20) The tsrA genes varying at the position encoding the core peptide’s fourth residue, and codon-optimized for expression in Streptomyces , were incorporated into the fosmid by PCR-targeted gene replacement and the resulting fosmids were introduced into S. laurentii NDS1 by intergeneric conjugation. (32–34) By this method, 18 different S. laurentii NDS1/int-A4X (where “X” is the one-letter amino acid code for the introduced mutation) variants were successfully generated, in addition to the previously constructed S. laurentii NDS1/int-A4G (Figure 2A).…”

“…(20, 21) In this initial study, mutation of the fourth residue of the TsrA core peptide from alanine to glycine supported the production of a thiostrepton Ala4Gly variant that retained antibacterial activity, suggesting that this residue is amenable to substitution. (20) Herein, we thoroughly probe the range of proteinogenic amino acid residues tolerated at the fourth position of the TsrA core peptide by the saturation mutagenesis of Ala4. The newly generated thiostrepton variants are then interrogated for their antibacterial and proteasome inhibition properties to determine whether or not the identity of the fourth residue is critical for either activity.…”

Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans

“…Because Ile8 is found exclusively as mhP8 in GE37468, it is unknown whether replacement of mhP8 with an acyclic residue would impact antibiotic activity. Manipulation of the preprotein gene sequence and evaluation of the tailoring oxygenations would enable SAR studies in the 29-member subclass for comparison with preprotein gene replacement underway in genetically tractable hosts for ribosometargeting 26-member thiazolyl peptides (8,22). With this in mind, we now report the sequencing, stable incorporation, expression, and initial manipulation of the GE37468 cluster in Streptomyces lividans TK24.…”

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…Their ribosomal synthesis, however, makes them attractive targets for genetic engineering of both the core peptide and the biosynthetic enzymes to create diverse thiopeptide chemical libraries. Previous efforts to explore pathway promiscuity and define enzyme function in vivo have coupled expression of core-peptide variants and/or gene deletions with organic extraction of pathway products (4)(5)(6)(7)(8)(9)(10). These studies succeed in identifying extractable products and intermediates that have been prematurely cleaved from the leader peptide, but overlook peptide intermediates that are chemically very different from product molecules.…”