Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo

Understanding the biosynthesis of novel specialized metabolites from Pseudomonas fluorescens

The rise of multidrug resistant bacteria and the gap in antibiotics development pose a serious threat to human health. Secondary metabolites (also termed specialized metabolites) have always been the main source of therapeutic antibiotics as well as molecules serving in various other pharmaceutical domains. They are compounds produced by bacteria, fungi or plants that are not essential for growth of the producing organism, but provide an extra advantage in difficult living conditions. As such, specialized metabolites can show antibiotic properties to combat natural enemies. As traditional screening of specialized metabolites for antibiotic properties does not yield sufficient new compounds, researchers are exploring new possibilities. Engineering of gene clusters to adapt existing metabolites has been popular, however researchers still hit the wall of nature’s ingenuity as the expected analogue is not produced or only in low amounts. In this dissertation, antibiotic research is continued in two ways. Firstly, the antibiotic activity of Pseudomonas fluorescens SWRI196 is investigated to elucidate the structure of the active compound and its biosynthetic pathway. Secondly, fundamental knowledge about gene cluster engineering is gathered, on the one hand by point mutagenesis of active sites and on the other hand by an interaction analysis, both in the kalimantacin biosynthesis cluster in P. fluorescens BCCM_ID9359. P. fluorescens SWRI196 was previously shown to exhibit antibacterial activity mainly against Gram-negative strains, and plasposon mutagenesis revealed a putative type II PKS cluster involved in the biosynthesis. In silico analysis showed the presence of a very similar cluster in two other P. fluorescens strains, a Xenorhabdus and a Pectobacterium strain. Transcription analysis showed that the cluster is transcribed as a single operon. Targeted gene deletions of eleven of the twelve individual genes in the cluster showed loss of bioactivity, indicating that these genes are essential for antibiotic production and/or activity. Only deletion of gene8 resulted in a reduced halo size, showing that the gene product is not essential for bioactivity. Gene deletion of a luxI homolog and consequent complementation by N-acyl homoserine lactones proved that transcription of the cluster is activated at least by N-decanoyl-homoserine lactone and N-dodecanoyl-homoserine lactone. Production parameters for structural elucidation were optimized and an extraction protocol was devised. Structural elucidation is still ongoing. The previously elucidated kalimantacin biosynthetic pathway was the target of point mutagenesis. Both inactivation of the ketosynthase domain KS4 and activation of the ketosynthase domain KS7b resulted in an enrichment of an unknown compound related to wildtype kalimantacin, however, none of the fermentation cultures showed the theoretically expected end product. Multiple acyl carrier proteins present in module 5 and module 9 of the assembly line appear to be redundant since inactivation of each of them individually did not result in significant loss of production. At last, the glycine-incorporating A domain of module 2 was mutated to incorporate other amino acids. An efficient cloning method was developed to introduce a genus-adapted specificity-conferring code. Predicted compounds could not be detected using LC-MS, confirming the need for extra fundamental knowledge of biosynthetic enzymes in such clusters. In the last part, a large-scale yeast two-hybrid interaction analysis was performed on all enzymes in the biosynthesis cluster. An initial interaction map showing 27 interacting partners was constructed and 17 of these interactions were confirmed in an independent yeast two-hybrid screen. This interaction map provides some first insight into the assembly of the biosynthetic pathway and can now be further investigated using other interaction techniques. To combat multidrug resistant bacteria the search for new antibacterials should be continued, as should the fundamental research on the assembly of the biosynthetic pathways. Filling the gap of knowledge on 3D structures will help targeted mutagenesis for the construction of hybrid biosynthetic clusters and might open a completely new platform for antibiotics development.nrpages: 144status: publishe

A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line

The antimicrobial secondary metabolite kalimantacin (also called batumin) is produced by a hybrid polyketide/non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites. This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.status: publishe

A protein interaction map of the kalimantacin biosynthesis assembly line

The antimicrobial secondary metabolite kalimantacin is produced by a hybrid polyketide/ non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites.This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters

Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

Kalimantacin is an antimicrobial compound with strong antistaphylococcal activity that is produced by a hybrid trans-acyltransferase polyketide synthase/nonribosomal peptide synthetase system in Pseudomonas fluorescens BCCM_ID9359. We here present a systematic analysis of the substrate specificity of the glycine-incorporating adenylation domain from the kalimantacin biosynthetic assembly line by a targeted mutagenesis approach. The specificity-conferring code was adapted for use in Pseudomonas and mutated adenylation domain active site sequences were introduced in the kalimantacin gene cluster, using a newly adapted ligation independent cloning method. Antimicrobial activity screens and LC-MS analyses revealed that the production of the kalimantacin analogues in the mutated strains was abolished. These results support the idea that further insight in the specificity of downstream domains in nonribosomal peptide synthetases and polyketide synthases is required to efficiently engineer these strains in vivo.status: publishe

A PKS/NRPS/FAS hybrid gene cluster from Serratia plymuthica RVH1 encoding the biosynthesis of three broad spectrum, Zeamine-related antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.status: publishe

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analyzed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labeling and pull down experiments. Yeast two-hybrid analysis gave clues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657 and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.status: publishe

A PKS/NRPS/FAS Hybrid Gene Cluster from <em>Serratia plymuthica</em> RVH1 Encoding the Biosynthesis of Three Broad Spectrum, Zeamine-Related Antibiotics

<div><p><em>Serratia plymuthica</em> strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.</p> </div

Overview of the zeamine antibiotics.

<p>Chemical structures of A) zeamine, B) zeamine I and C) zeamine II isolated from <i>S. plymuthica</i> strain RVH1. D) Proposed structure for the pre-zeamine antibiotics.</p

Specificity-conferring codes of the adenylation domains from Zmn16 and Zmn17 and their predicted building block specificity.

<p>Specificity-conferring codes of the adenylation domains from Zmn16 and Zmn17 and their predicted building block specificity.</p