Functional analysis of MbtH-like proteins in the biosynthesis of thaxtomin A in Streptomyces scabiei 87.22

Li, Yuting (2021) Functional analysis of MbtH-like proteins in the biosynthesis of thaxtomin A in Streptomyces scabiei 87.22. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Streptomyces scabiei is an important causative agent of potato common scab disease. The main pathogenicity factor produced by this organism is thaxtomin A, a phytotoxin that is also a promising bioherbicide for agricultural applications. The biosynthesis of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A-) domain that recruits and activates the amino acid substrate to be integrated into the product. A small gene, txtH, encoding a predicted member of the MbtH-like protein (MLP) family, is also present in the thaxtomin (Txt) biosynthetic gene cluster. MLPs are normally required for proper folding of the A-domain(s) and/or for stimulating the enzymatic activity of the domain. In addition, some MLPs can interact with NRPSs from different biosynthetic pathways, though the mechanism behind this is not fully understood. Here, I investigated the role of MLPs during thaxtomin A biosynthesis in S. scabiei. The results showed that TxtH likely functions as a chaperone protein for ensuring the proper folding of the TxtA and TxtB A-domains, and that this function is essential for thaxtomin biosynthesis in S. scabiei. Using site-directed mutagenesis, I identified amino acid residues within TxtH that are important for the function of the protein. I also showed that two other MLPs encoded in the S. scabiei genome can promote thaxtomin production in the absence of TxtH, indicating that they can exhibit functional cross-talk with TxtH. A survey of various MLPs from diverse phylogenetic lineages revealed that most of these MLPs can exhibit functional cross-talk with TxtH to varying degrees, though two MLPs were identified that could not replace TxtH in the assays performed. In silico analysis revealed that the conservation of key residues at the Txt MLP-NRPS interacting interface may determine the ability of an MLP to interact with the Txt NRPSs. I additionally attempted to assess the impact of TxtH and other MLPs on the enzymology of the Txt A-domains in vitro; however, my assays were unsuccessful despite testing different conditions. Overall, this study is the first to investigate the function of MLPs during thaxtomin A biosynthesis in S. scabiei.

Item Type: Thesis (Doctoral (PhD))
Item ID: 15399
Additional Information: Includes bibliographical references.
Keywords: Plant pathology, Streptomyces, specialized metabolism, non-ribosomal peptides, thaxtomin
Department(s): Science, Faculty of > Biology
Date: March 2021
Date Type: Submission
Digital Object Identifier (DOI):
Library of Congress Subject Heading: Plant diseases; Streptomyces scabies; Metabolism; Peptides; Biosynthesis; Potato scab; Phytotoxins.

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