Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products

Abstract

Combining biosynthetic enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features; however, progress has been hampered by the incompatibility of enzymes from unrelated pathways and intolerance toward alternative substrates. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a diverse natural product class that employs a biosynthetic logic that is highly amenable to engineering new compounds. RiPP biosynthetic proteins modify their substrates by binding to a motif typically located in the N-terminal leader region of the precursor peptide. Here, we exploit this feature by designing leader peptides that enable recognition and processing by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a thiazoline-forming cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs. We also provide insight into design features that enable control over the hybrid biosynthesis to optimize enzyme compatibility and establish a general platform for engineering additional hybrid RiPPs.

Figure 1

Overview of RiPP biosynthesis. (a) A generic RiPP gene cluster and the key maturation steps. (b) Chimeric leader peptide strategy for combining modifying enzymes from unrelated pathways. Examples of primary RiPP modification enzymes combined in this work include (c) Cα-cross-linked sactipeptides, (d) azoline-containing RiPPs, and (e) Cβ-cross-linked lanthipeptides. (f) Examples of secondary RiPP modification enzymes used in this work to install d-Ala residues and decarboxylate C-terminal Cys residues.

Figure 2

Production of a thiazoline–lanthipeptide (class I) hybrid. (a) Design of the Hyp1.1 sequence from HcaA and NisA precursor peptides. Portions of the leader peptides that were combined are colored. The recognition sequences (RSs) bound by the RREs of the cognate enzymes are indicated with a pink box. The orange underlined regions were combined to generate the chimeric core region. (b) Overview of experimental procedure. (c) Structure of thiazoline–lanthipeptide (class I) hybrid Hyp1.1a upon AspN digestion. Thiazolines are blue while dehydrations and (Me)Lan are red. Numbering is based on the core position, not peptide length after digestion. (d) The mass of Hyp1.1a (MALDI-TOF-MS) is consistent with one thiazoline, two dehydrations, and two (Me)Lan. These modifications are supported by acid hydrolysis (blue arrow, +18 Da) and by lack of labeling by iodoacetamide (green arrow, +57 Da).

Figure 3

Production of a thiazoline–sactipeptide hybrid. (a) Design of hybrid peptides. Orange underlining indicates edited sequences. (b) Overview of the experimental procedure for combination of HcaD/F and AlbA. (c) Structure of thiazoline–sactipeptide hybrid (modified Hyp2.2). Numbering is based on the core position, not peptide length after digestion. (d) The mass of the modified peptide is consistent with two thiazolines and two sactionine linkages. The two sets of peaks correspond to protease digestion at different sites (gray font). The structure is supported by acid hydrolysis, lack of labeling by iodoacetamide, and resistance to proteolytic digestion (see also Figure S6). Arrows and triangles represent +18 and −2 Da, respectively. Red coloring indicates positions resistant to trypsin digestion.

Figure 4

Production of hybrid thiazoline–lanthionine (class II) hybrids. (a) Design of hybrid peptides. Orange underlining indicates edited positions of the core peptide. The dashed line indicates the leader peptide of the ProcA2.8 and Proc3.3. (b) Experimental overview for combination of HcaD/F and ProcM. (c) Deduced structure and MALDI-TOF-MS support of posttranslational modifications for Hyp4.3. Numbering is based on the core position, not peptide length after digestion. (d) Same as in panel c but for Hyp3.3.

Figure 5

Combination of two primary and one secondary RiPP modifications. (a) Design of hybrid peptide. Orange underlining highlights changed residues. (b) Experimental overview for combining HcaF/D, ProcM, and NpnJ_A. (c) Deduced structure of Hyp4.4b. (d) MALDI-TOF-MS analysis of Hyp4.4 products. The initially formed hybrid has a Dha, thiazoline (arrow indicates hydrolysis), and MeLan. The formation of d-Ala from Dha is indicated by the +2 Da shift (triangle) of the Hyp4.4a species upon addition of NpnJ_A. The weak ion labeled as −72 Da is likely a peptide that underwent two cyclodehydrations and two dehydrations.