RESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.
Assuntos
Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ensaios Enzimáticos , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Covering: Up to mid-2019 Cyclodipeptide synthases (CDPSs) catalyse the formation of cyclodipeptides using aminoacylated-tRNA as substrates. The recent characterization of large sets of CDPSs has revealed that they can produce highly diverse products, and therefore have great potential for use in the production of different 2,5-diketopiperazines (2,5-DKPs). Sequence similarity networks (SSNs) are presented as a new, efficient way of classifying CDPSs by specificity and identifying new CDPS likely to display novel specificities. Several strategies for further increasing the diversity accessible with these enzymes are discussed here, including the incorporation of non-canonical amino acids by CDPSs and use of the remarkable diversity of 2,5-DKP-tailoring enzymes discovered in recent years.
Assuntos
Biotecnologia/métodos , Dicetopiperazinas/síntese química , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Engenharia de Proteínas/métodos , Aminoácidos/química , Dicetopiperazinas/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cß dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. RESULTS: We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. CONCLUSIONS: Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.
Assuntos
Biotecnologia/métodos , Dicetopiperazinas/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Peptídeo Sintases/metabolismo , Vias Biossintéticas/genética , Dicetopiperazinas/química , Escherichia coli/genética , Oxirredutases/genética , Peptídeo Sintases/genética , FilogeniaRESUMO
The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5-diketopiperazines (2,5-DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an inâ vivo method for incorporating non-canonical amino acids (ncAAs) into 2,5-DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5-DKP biosynthetic pathways. CDPSs use aminoacyl-tRNAs as substrates. We exploited the natural ability of aminoacyl-tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non-canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5-DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide-tailoring enzymes found in 2,5-DKP biosynthetic pathways.
Assuntos
Aminoácidos/metabolismo , Dicetopiperazinas/metabolismo , Peptídeo Sintases/metabolismo , Aminoácidos/química , Dicetopiperazinas/química , Conformação MolecularRESUMO
Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an "IPP-bypass" isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either kcat or Ki of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.
Assuntos
Carboxiliases , Proteínas de Escherichia coli , Escherichia coli , Ácido Mevalônico/metabolismo , Pentanóis/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2-26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.
Assuntos
Dicetopiperazinas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , PrenilaçãoRESUMO
Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored. Here, we describe the activity of 32 new CDPSs, bringing the number of experimentally characterized CDPSs to about 100. We detect 16 new cyclodipeptides, one of which containing an arginine which has never been observed previously. This brings to 75 the number of cyclodipeptides formed by CDPSs out of the possible 210 natural ones. We also identify several consensus sequences related to the synthesis of a specific cyclodipeptide, improving the predictive model of CDPS specificity. The improved prediction method enables to propose the main product synthesized for about 80% of the CDPS sequences available in databases and opens the way for the deciphering of CDPS-dependent pathways. Analysis of phylum distribution and predicted activity for all CDPSs identified in databases shows that the experimentally characterized set is representative of the whole family. Our work also demonstrates that some cyclodipeptides, precursors of diketopiperazines with interesting pharmacological properties and previously described as being synthesized by fungal non-ribosomal peptide synthetases, can also be produced by CDPSs in bacteria.