Engineering of prodigiosin ligase pigC towards production of short-chain prodiginines

Brands, Stefanie; Schwaneberg, Ulrich (Thesis advisor); Jaeger, Karl-Erich (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Prodigiosin synthetase PigC is an ATP-dependent membrane-associated ligase that catalyzes the final condensation step in the biosynthesis of prodigiosin in Serratia marcescens. Two pyrrolic precursor molecules, 4-methoxy-2,2’-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-amyl-pyrrole (MAP) are linearly combined to form the deeply red tripyrrole pigment prodigiosin. As secondary metabolites, prodiginines provide a wide spectrum of bioactivities like antibiotic, antifungal, nematicidal, antimalaria as well as anticancer effects that have attracted the interest of agrobusiness, pharma and food industries. Interestingly, the side chains that decorate the tripyrrolic prodiginine scaffold modulate the bioactivity of prodiginines. The anticancer effect was especially pronounced in prodiginines with short aliphatic side chains. Prodiginine precursors with short side chains are, however, badly accepted by prodiginine ligases, which are bottleneck to biocatalytic prodiginine production. In this work, the engineering of prodigiosin ligase PigC towards acceptance of short-chain pyrroles opens up a promising biosynthetic route to short-chain prodiginines and provides a first molecular understanding on substrate binding in the active pocket of PigC. Mutagenesis libraries of pigC were heterologously generated in Pseudomonas putida KT2440, which has proved remarkably tolerant towards antibiotic prodiginine compounds. The establishment of a high-throughput screening system with low product detection limits (0.5 µM), wide linear product detection ranges (0.5-250 µM) and low standard deviations <15% with different pyrrolic substrates allowed for the first directed evolution campaign on PigC. In one round of KnowVolution, the PigC catalytic activity was increased up to 10.7-times compared to the PigC wild type (kcat = 0.7 min-1) with short-chain substrates in the final variant S1 (I365G/M671V; kcat = 7.5 min-1), resulting in a nearly 40-times increased catalytic efficiency (kcat/KM = 23.9 mM-1 s-1 compared to 0.6 mM-1 s-1 for the PigC wild type). In a consecutive semi-rational approach, the PigC active pocket and access tunnels were engineered by site-saturation of medium-conserved residues. Three beneficial substitutions (V333A, T334A and R674Q) enhanced the substrate binding mode by enabling a preferred docking pose more than 3 Å closer to the active site H840 (from 6.9 to 3.5 Å distance), as well as the ligand transport by widening an access tunnel (radius cross section area increased by 4 Å2), increasing the wild type kcat after recombination up to 3.5-times from 0.9 to 3.1 min-1. In conclusion, PigC has been successfully engineered by both directed evolution and semi-rational design approaches, opening access to short-chain prodiginines with enhanced anticancer bioactivity.