Metagenomic domain substitution for the high-throughput modification of nonribosomal peptides.

The modular nature of nonribosomal peptide biosynthesis has driven efforts to generate peptide analogs by substituting amino acid-specifying domains within nonribosomal peptide synthetase (NRPS) enzymes. Rational NRPS engineering has increasingly focused on finding evolutionarily favored recombination sites for domain substitution. Here we present an alternative evolution-inspired approach that involves large-scale diversification and screening. By amplifying amino acid-specifying domains en masse from soil metagenomic DNA, we substitute more than 1,000 unique domains into a pyoverdine NRPS. Initial fluorescence and mass spectrometry screens followed by sequencing reveal more than 100 functional domain substitutions, collectively yielding 16 distinct pyoverdines as major products. This metagenomic approach does not require the high success rates demanded by rational NRPS engineering but instead enables the exploration of large numbers of substitutions in parallel. This opens possibilities for the discovery and production of nonribosomal peptides with diverse biological activities.

Cyclotheonellazoles D-I, Potent Elastase Inhibitory Thiazole-Containing Cyclic Peptides from Theonella sp. (2131).

Six new thiazole-containing cyclic peptides, the cyclotheonellazoles D-I (1-6), were isolated from the Australian marine sponge Theonella sp. (2131) with their structures assigned by comprehensive 1D and 2D NMR spectroscopic and MS spectrometric analyses, Marfey's derivatization studies, and comparison with time-dependent density functional theory (TDDFT) calculated ECD data. The Type 2 azole-homologated peptides herein comprise up to five nonproteinogenic amino acids, including the protease transition state mimic α-keto-ß-amino acid residue 3-amino-4-methyl-2-oxohexanoic acid (Amoha), while 1-3 also contain a terminal hydantoin residue not previously found in cyclotheonellazoles. The keramamides A (7) and L (8) were reisolated affording expanded exploration of their biological activities. The peptides were examined for protease inhibitory activities against two mammalian serine proteases (elastase and chymotrypsin) and SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), a validated antiviral therapeutic target for COVID-19. Peptides 1-6 and keramamide A (7) displayed potent nanomolar inhibition of elastase (IC50 16.0 to 61.8 nM), while 7 also contained modest inhibition of chymotrypsin and SARS-CoV-2 3CLpro (IC50 0.73 and 1.1 µM, respectively). The cyclotheonellazoles D-E (1-3) do not affect the viability of human breast, ovarian, and colon cancer cells (>100 µM), with the cytotoxicity previously reported for keramamide L (8) not replicated (inactive >20 µM).

A Genomic Survey of the Natural Product Biosynthetic Potential of Actinomycetes Isolated from New Zealand Lichens.

Actinomycetes are prolific producers of industrially valuable and medically important compounds. Historically, the most efficient method of obtaining compounds has been bioactivity-guided isolation and characterization of drug-like molecules from culturable soil actinomycetes. Unfortunately, this pipeline has been met with an increasing number of rediscoveries, to the point where it is no longer considered an attractive approach for drug discovery. To address this challenge and to continue finding new compounds, researchers have increasingly focused on alternative environmental niches and screening methods. Here, we report the genetic investigation of actinomycetes from an underexplored source, New Zealand lichens. In this work, we obtain draft genome sequences for 322 lichen-associated actinomycetes. We then explore this genetic resource with an emphasis on biosynthetic potential. By enumerating biosynthetic gene clusters (BGCs) in our data sets and comparing these to various reference collections, we demonstrate that actinomycetes sourced from New Zealand lichens have the genetic capacity to produce large numbers of natural products, many of which are expected to be broadly different from those identified in previous efforts predominantly based on soil samples. Our data shed light on the actinomycete assemblage in New Zealand lichens and demonstrate that lichen-sourced actinobacteria could serve as reservoirs for discovering new secondary metabolites. IMPORTANCE Lichens are home to complex and distinctive microbial cohorts that have not been extensively explored for the ability to produce novel secondary metabolites. Here, we isolate and obtain genome sequence data for 322 actinomycetes from New Zealand lichens. In doing so, we delineate at least 85 potentially undescribed species, and show that lichen associated actinomycetes have the potential to yield many new secondary metabolites, and as such, might serve as a productive starting point for drug discovery efforts.

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues.

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

In the cell, most tasks are performed by big molecules called proteins, which behave like molecular machines. Although proteins are often described as having one job each, this is not always true, and many proteins can perform different roles. Enzymes are a type of protein that facilitate chemical reactions. They are often specialised to one reaction, but they can also accelerate other side-reactions. During evolution, these side-reactions can become more useful and, as a result, the role of the enzyme may change over time. The main role of the enzyme called NfsA in Escherichia coli bacteria is thought to be to convert molecules called quinones into hydroquinones, which can protect the cell from toxic molecules produced in oxidation reactions. As a side-reaction, NfsA has the potential to protect bacteria from an antibiotic called chloramphenicol, but it generally does this with such low efficacy that the effects are negligible. Producing hydroquinones is helpful to the cell in some situations, but if bacteria are regularly exposed to chloramphenicol, NfsA's role aiding antibiotic resistance could become more important. Over time, the enzyme could evolve to become better at neutralising chloramphenicol. Therefore, NfsA provides an opportunity to study the evolution of proteins and how bacteria adapt to antibiotics. To see how evolution might affect the activity of NfsA, Hall et al. generated 250 million E. coli with either random or targeted changes to the gene that codes for the NfsA enzyme. The resulting variants of NfsA that were most effective against chloramphenicol all had a change that eliminated the enzyme's ability to convert quinones. This result demonstrates a key trade-off between roles for NfsA, where one must be lost for the other to improve. These results demonstrate the interplay between a protein's different roles and provide insight into bacterial drug resistance. Additionally, the experiments showed that the bacteria with improved resistance to chloramphenicol also became more sensitive to another antibiotic, metronidazole. These findings could inform the fight against drug-resistant bacterial infections and may also be helpful in guiding the design of proteins with different roles.

High-Throughput Screening for Inhibitors of the SARS-CoV-2 Protease Using a FRET-Biosensor.

The global SARS-CoV-2 pandemic started late 2019 and currently continues unabated. The lag-time for developing vaccines means it is of paramount importance to be able to quickly develop and repurpose therapeutic drugs. Protein-based biosensors allow screening to be performed using routine molecular laboratory equipment without a need for expensive chemical reagents. Here we present a biosensor for the 3-chymotrypsin-like cysteine protease from SARS-CoV-2, comprising a FRET-capable pair of fluorescent proteins held in proximity by a protease cleavable linker. We demonstrate the utility of this biosensor for inhibitor discovery by screening 1280 compounds from the Library of Pharmaceutically Active Compounds collection. The screening identified 65 inhibitors, with the 20 most active exhibiting sub-micromolar inhibition of 3CLpro in follow-up EC50 assays. The top hits included several compounds not previously identified as 3CLpro inhibitors, in particular five members of a family of aporphine alkaloids that offer promise as new antiviral drug leads.

Efficient rational modification of non-ribosomal peptides by adenylation domain substitution.

Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.

The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes.

OBJECTIVES: To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes. RESULTS: BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate. CONCLUSIONS: The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.

Structural, functional and evolutionary perspectives on effective re-engineering of non-ribosomal peptide synthetase assembly lines.

Covering: up to May 2018 Non-ribosomal peptide synthetases (NRPSs) are mega-enzymes that form modular templates to assemble specific peptide products, independent of the ribosome. The autonomous nature of the modules in the template offers prospects for re-engineering NRPS enzymes to generate modified peptide products. Although this has clearly been a primary mechanism of natural product diversification throughout evolution, equivalent strategies have proven challenging to implement in the laboratory. In this review we examine key examples of successful and less-successful re-engineering of NRPS templates to generate novel peptides, with the aim of extracting practical guidelines to inform future efforts. We emphasise the importance of maintaining effective protein-protein interactions in recombinant NRPS templates, and identify strengths and limitations of diverse strategies for achieving different engineering outcomes.

Secondary metabolism in the lichen symbiosis.

Lichens, which are defined by a core symbiosis between a mycobiont (fungal partner) and a photobiont (photoautotrophic partner), are in fact complex assemblages of microorganisms that constitute a largely untapped source of bioactive secondary metabolites. Historically, compounds isolated from lichens have predominantly been those produced by the dominant fungal partner, and these continue to be of great interest for their unique chemistry and biotechnological potential. In recent years it has become apparent that many photobionts and lichen-associated bacteria also produce a range of potentially valuable molecules. There is evidence to suggest that the unique nature of the symbiosis has played a substantial role in shaping many aspects of lichen chemistry, for example driving bacteria to produce metabolites that do not bring them direct benefit but are useful to the lichen as a whole. This is most evident in studies of cyanobacterial photobionts, which produce compounds that differ from free living cyanobacteria and are unique to symbiotic organisms. The roles that these and other lichen-derived molecules may play in communication and maintaining the symbiosis are poorly understood at present. Nonetheless, advances in genomics, mass spectrometry and other analytical technologies are continuing to illuminate the wealth of biological and chemical diversity present within the lichen holobiome. Implementation of novel biodiscovery strategies such as metagenomic screening, coupled with synthetic biology approaches to reconstitute, re-engineer and heterologously express lichen-derived biosynthetic gene clusters in a cultivable host, offer a promising means for tapping into this hitherto inaccessible wealth of natural products.

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

Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases.

BACKGROUND: Non-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD. RESULTS: To test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context. CONCLUSIONS: Our data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.

Genetic manipulation of non-ribosomal peptide synthetases to generate novel bioactive peptide products.

Non-ribosomal peptide synthetases (NRPS) are large modular enzymes that govern the synthesis of numerous biotechnologically relevant products. Their mode of action is frequently compared to an assembly line, in which each module acts in a semi-autonomous but coordinated manner to add a specific monomer to a growing peptide chain, unfettered by ribosomal constraints. The modular nature of these systems offers tantalising prospects for synthetic biology, wherein the assembly line is re-engineered at a genetic level to generate a specific or combinatorial modified product. However, despite some success stories, a "one size fits all" approach to NRPS synthetic biology remains elusive. This review examines both rational and random mutagenesis strategies that have been employed to modify NRPS function, in an attempt to highlight key points that should be considered when seeking to re-engineer an NRPS biosynthetic template.

Biosynthesis of novel Pyoverdines by domain substitution in a nonribosomal peptide synthetase of Pseudomonas aeruginosa.

Pyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an l-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine-at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.