Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange

Abstract

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.

Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering

“…BpsA and homologous indigoidine synthetases such as IndC (Reverchon et al, 2002) are the only single-module NRPSs presently known to be autonomously capable of generating a pigmented product. We and others have shown that these features can be exploited to provide a portable and flexible template for PCP-domain substitutions (Beer et al, 2014;Owen et al, 2012). We now demonstrate that they also confer an effective high-throughput screening capability for recovery of improved indigoidine-synthesizing variants at a single-colony level.…”

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

“…A recent study swapped various PCP domains in the single module IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. [40] Replacing the PCP of IndC with that of BpsA, a closely related indigoidine synthetase from Streptomyces lavebdulae disrupted pigment production. Surprised by this the authors turned to the PCP linkers.…”

L 1 regularization facilitates detection of cell type-specific parameters in dynamical systems

“…Data2Dynamics is a state-of-the-art software package that has been used in a variety of applications (Bachmann et al , 2011; Becker et al , 2010; Beer et al , 2014) to perform parameter estimation, uncertainty analysis, and experimental design of partially observed nonlinear ODE systems. In the following, we summarize the L 1 specific enhancements in addition to the established modeling routine as implemented in our approach for discovering cell type-specific parameters.…”