Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>

Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>

Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries

Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes.

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having &lt;78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average &gt; 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

ABSTRACTFunctional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naïve and cultivation-independent discovery of novel genes from microbial communities. Preparation of these libraries is limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, METa assembly, for extremely efficient functional metagenomic library preparation. We apply tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergizes with homology-based assembly cloning to result in a 300-fold increase in library size compared to traditional blunt cloning based protocols. Compared to published libraries prepared by state-of-the-art protocols we show that METa assembly is on average 23- to 270-fold more efficient and can be effectively used to prepare gigabase-sized libraries with as little as 200 ng of input DNA. We demonstrate the utility of METa assembly to capture novel genes based on their function by discovering novel aminoglycoside (26% amino acid identity) and colistin (36% amino acid identity) resistance genes in soil and goose gut microbiomes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low biomass or scarce microbiomes.IMPORTANCEMedically and industrially important genes can be recovered from microbial communities by high-throughput sequencing but are limited to previously sequenced genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library is a sequence-naïve and cultivation-independent method to discover novel genes. This directly connects genes to functions, but the process of preparing these libraries is DNA greedy and inefficient. Here we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and from a goose fecal microbiome and allowed us to discover novel antibiotic resistance genes. This new library preparation method will potentially allow for the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such biomedical swabs or exotic samples.

Discovery of β-N-acetylglucosaminidases from screening metagenomic libraries and their use as thioglycoligase mutants

Screening of a human gut metagenomic library yielded a set of novel beta-N-acetyl-glucosaminidases. Mutation of active site residues yielded a thioglycoligase that efficiently S-GlcNAcylates protein targets as well as various thiosugar acceptors.