Investigation of class v lanthipeptide biosynthetic pathway

Liang, Haoqian

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https://hdl.handle.net/2142/124684 Copy

Description

Title

Investigation of class v lanthipeptide biosynthetic pathway

Author(s)

Liang, Haoqian

Issue Date

2024-04-26

Director of Research (if dissertation) or Advisor (if thesis)

van der Donk, Wilfred A

Doctoral Committee Chair(s)

van der Donk, Wilfred A

Committee Member(s)

• Nair, Satish K
• Cronan, John E
• Stadtmueller, Beth M

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Lanthipeptide
• RiPPs
• Biosynthesis
• Bioengineering
• Enzymology

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding class of natural products. The largest class of RiPPs is the lanthipeptides, which contain the β-thioether crosslinked bis amino acids lanthionine (Lan) and methyllanthionine (MeLan). Four classes of enzymes have been identified to install such motifs in a substrate peptide. Recently, a novel group of (Me)Lan containing RiPPs were discovered for which the biosynthetic gene cluster (BGC) lacked genes encoding well-defined class I−IV (Me)Lan synthase homologues, suggesting the existence of an unknown synthase. This new group of lanthipeptides was termed class V. In Chapter 2, the dehydration of Ser/Thr during the biosynthesis of the first discovered class V lanthipeptide cacaoidin was reconstituted in vitro. The amino-glycoside phosphotransferase-like enzyme CaoK phosphorylates Ser/Thr on the precursor peptide CaoA, followed by phosphate elimination catalyzed by the HopA1 effector-like protein CaoY to achieve eight dehydrations. CaoK and CaoY form a dehydratase complex. The mechanistic similarity between CaoKY and dehydratases from class III-IV lanthipeptides, and leader peptide-dependent substrate recognition of the dehydratase complex were uncovered through study of predicted structures coupled with site-directed mutagenesis. In Chapter 3, a methyltransferase CaoSC that installs dimethylation on the cacaoidin N-terminal Lan was identified from the cao BGC. In vitro methylation catalyzed by CaoSC on heterologously expressed peptides revealed the promiscuity of CaoSC towards substrate with the requirement of amino acids that have a planar hybridization (sp2) of the a-carbon such as dehydroalanine (Dha) or dehydrobutyrine (Dhb) at the second position in the substrates. Bioactivity changes due to the methylation of these lanthipeptides were also evaluated, which paves the way for the future application of CaoSC in lantibiotics bioengineering. Chapter 4 describes the establishment of a lanthipeptide expression platform using cell-free gene expression. Novel class V lanthipeptide precursors discovered through CaoKY co-occurrence guided genome mining that were unable to be produced in E. coli were successfully produced through this platform. This cell-free expression platform was also assessed in its ability of functional reconstitution of peptide-amino acyl tRNA ligases (PEARLs), and investigation of the substrate requirement of the PEARL enzyme BhaB-C-Trp.

Graduation Semester

2024-05

Type of Resource

Thesis

Copyright and License Information

I will need 2-year embargo, as the Chapter 3 in my thesis was submitted for publication and is currently under revision.

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