Compositions and methods for the synthesis of APPA-containing peptides

Inventors

Metcalf, William W. • van der Donk, Wilfred A. • Zhang, Junkal • Circello, Benjamin T. • Borisova, Svetlana A.

Assignees

University of Illinois at Urbana Champaign

Abstract

The disclosure of the present application provides polypeptide sequences and nucleotide sequences coding for the polypeptide sequences of proteins used in the production of APPA-containing peptides. In at least one embodiment of the present disclosure, an isolated nucleic acid is disclosed which comprises a nucleotide sequence encoding a polypeptide having a sequence identity of 60 percent or greater to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2-13, and 15-23.

Core Innovation

The disclosure identifies biosynthetic gene operons for APPA-containing peptides, including the rhi operon in B. subtilis ATCC6633 and the plu operon in S. plumbeus. It implicates specific enzymes within these operons, including PEP mutase RhiH and aldolase RhiG, together with other pathway components described as RhiA, RhiB, RhiC, RhiD, RhiE/RhiF, RhiI, RhiJ, RhiK, RhiL, RhiM, and RhiN. This provides a defined set of proteins associated with APPA-containing peptide biosynthesis.

The disclosure defines isolated nucleic acids and vectors encoding APPA-containing peptide biosynthesis proteins using sequence-identity-based relationships to specified SEQ ID NOs, including amino acid sequence identity thresholds of at least 70 percent and at least 71 percent to particular SEQ ID reference sequences. It further discloses encoded nucleic acids that can include additional nucleotide sequences encoding additional polypeptides with their own sequence identity requirements to specified SEQ ID reference sequences.

The disclosure supports transformed-cell production of APPA-containing peptides, including rhizocticin B, by introducing the rhi gene cluster into B. subtilis 168 or by using engineered/heterologous production contexts described in the disclosure. It also supports RhiG function via in vitro reconstitution yielding APPA pathway intermediates characterized as Ia/Ib, with characterization using 31P NMR and LC-MS.

Claims Coverage

The partial claim set includes four independent claims. Across these, inventive coverage centers on isolated nucleic acids, vectors, transformed cells, and methods for producing APPA-containing peptides, all defined by sequence identity to specified SEQ ID reference amino acid sequences.

Across the independent claims, protection is directed to APPA-containing peptide biosynthesis through sequence-identity-defined nucleic acids and vectors, and to transformed cells and methods that use those constructs to produce an APPA-containing peptide.

Stated Advantages

Documented Applications