Origin and diversification of polycyclic triterpene and carotenoid metabolisms in the Tree of Life
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AuthorshipSantana Molina, Carlos
Palabras claveBiología molecular de microorganismos
Árbol de la Vida (Biología)
DirectionDevos, Damien Paul; Canosa Pérez-Fragero, Inés
Fecha de lectura2021-11-25
Cell membranes are key components of life, delimiting the cells, performing essential physiological functions and having important roles in environmental adaptations. Membranes have therefore, a crucial influence in the Tree of Life and resolving their evolution is a fundamental issue in Biology. Membranes have evolved heterogeneously between Bacteria, Archaea and Eukaryotes due to the prevalence of specific metabolisms that produce diverse components. Understanding the origin and evolution of membrane components could shed light into the ancestral relationship between the three domains of life as well as the mode of diversification of metabolisms. In this work, we analyzed the origin and evolution of two kind of isoprenoid derivatives (terpenoids) that form different components of the membranes: polycyclic triterpenes and carotenoids. Polycyclic triterpenes are derived from the cyclization of the squalene precursor, a terpene backbone with 30 carbon atoms (C30), including mole ...
Cell membranes are key components of life, delimiting the cells, performing essential physiological functions and having important roles in environmental adaptations. Membranes have therefore, a crucial influence in the Tree of Life and resolving their evolution is a fundamental issue in Biology. Membranes have evolved heterogeneously between Bacteria, Archaea and Eukaryotes due to the prevalence of specific metabolisms that produce diverse components. Understanding the origin and evolution of membrane components could shed light into the ancestral relationship between the three domains of life as well as the mode of diversification of metabolisms. In this work, we analyzed the origin and evolution of two kind of isoprenoid derivatives (terpenoids) that form different components of the membranes: polycyclic triterpenes and carotenoids. Polycyclic triterpenes are derived from the cyclization of the squalene precursor, a terpene backbone with 30 carbon atoms (C30), including molecules such as hopanoids that are mostly found in Bacteria and are associated to environmental adaptations, or sterols, whose biosynthesis is almost omnipresent in Eukaryotes, and thus, represents a key component of the process of eukaryogenesis. Carotenoids in contrast, are mostly linear terpenoids, mostly classified in C30, C40 and C50 backbones, whose biosynthetic pathways show a more restricted distribution in the Tree of Life as they are mostly associated to photosynthetic organisms. So carotenoids could be one of the many components that contributed to the photosynthesis metabolism. On the other hand, carotenoids are also present in non-photosynthetic organisms (mostly prokaryotes and fungi) in which in addition to the antioxidant and light absorbency properties, carotenoids are also involved in environmental adaptations modulating membrane properties, similarly to hopanoids. Therefore, both kind of molecules have similar functions at the level of membranes. Another important feature of polycyclic triterpenes and carotenoids is their capacity to fossilize through diagenetic process, which is perfect to use them as biomarkers in the fossil record. Biomarkers are remains of biological compounds that are stable over geological timescales and thus, terpene fossils provide a window to understand the lifestyles through the Earth's history. The homologies between the enzymes that form the biosynthetic pathways of carotenoids and polycyclic triterpenes demonstrate that the biosynthesis of both is evolutionarily related. Although their evolution have been previously addressed independently, their specific relationships remain less understood. Elucidating the origin and evolution of the polycyclic triterpene and carotenoid biosynthetic pathways is important to understand the role of these compounds in the tree of life as well as their geobiological value as biomarkers in the fossil record. In this study we address two main question regarding these molecules: first, how did eukaryotes obtained the sterol synthesis genes, and second, what are the biosynthetic and evolutionary relationships between polycyclic triterpenes and carotenoids. While the first questions resolves questions about eukaryogenesis, the second resolves the evolutionary relationship between polycyclic triterpenes and carotenoids. Through phylogenetic and syntenic analyses, and using more than 20,000 prokaryotic and 36 representative eukaryotic genomes, we reconstructed the evolutionary history of the squalene (HpnCDE or Sqs), hopanoid and sterol (through Shc and Osc respectively) and the diverse C30, C40 or C50 (through CrtI-CrtD, CrtN-CrtP, CrtI-50) biosynthetic pathways. Squalene is biosynthesized by two distantly related pathways, HpnCDE and Sqs. The HpnCDE is mostly found bacteria and it is closely related to carotenoid enzymes. This pathway seems to have assembled relatively early in bacteria evolution as the phylogeny of the three enzymes shows a common evolutionary history of the monophyletic groups and form a conserved operon between distant organisms. Sqs in contrast, is the most divergent subfamily of its protein family that also includes HpnC, HpnD or carotenoid synthases like CrtB or CrtM. Sqs is present in the three domains of life, although the taxonomic distribution combined with the phylogeny suggest that Sqs had a bacterial origin and was later transferred to eukaryotes and archaea independently. Another relevant fact, is that when we combined the evolution of HpnCDE and Sqs, we observed that Sqs has been more prone to LGT and in some cases, has displaced the function of HpnCDE through gene loss, which could be in agreement with the fact that it is easier and more efficient to conserved one gene rather than three. The evolution of hopanoids biosynthesis through Shc, shows a most likely vertical evolution in the Gracilicutes bacterial supergroup, but we suspected that it has been transferred through LGT to different Terrabacteria clades. Thus, hopanoid biosynthesis seems to be ancestral but with a heterogenous evolution in the Bacteria domain. Sterol biosynthesis in bacteria shows a limited distribution although number of bacteria bearing sterol genes seem to increase as more amount of genomic information we have. Phylogenetic reconstructions of sterol enzymes, Osc and Sqmo, shows that few bacteria have obtained the sterol genes from eukaryotes, while other bacteria have sterol genes that are possibly unrelated to eukaryotic sources. Bacterial Osc and Sqmo have similar evolutionary histories because they are usually contiguously in the genomes and therefore, are transferred together. Indeed, the osc gene is also associated to other sterol-related genes in the genomes, showing that bacteria have developed gene clusters for sterol metabolism. Another important fact from these results is that we did not found any triterpene cyclase in Archaea, invoking a potential scenario in which eukaryotic genes for sterol biosynthesis assembled from ancestral bacterial contributions in early eukaryotic lineages. One of the major bacterial contributions during the eukaryogenesis was the mitochondria. Due to the scarcity of sterol genes in Alphaproteobacteria, it is unlikely that sterol genes have mitochondrial origin. However, according to recent discoveries placing the origin of mitochondria outside Alphaprotebacteria, it is uncertain whether sterol genes assembled from mitochondrial contributions. On the other hand, due to the existence of a specific sterol pathway in bacteria, and the fact that sterol can be also essential for some bacteria, like for the Plactomycete Gemmata obscuriglobus, we also consider, and indeed favor, the possibility that sterol genes originated in bacteria independently of the eukaryotes. We then combined the evolution of squalene with the one of polycyclic triterpenes. HpnCDE-Shc have more similar evolutionary patterns than Sqs-SHC, which suggests that HpnCDE could have been the primitive pathway for hopanoids. On the other hand, our result also argues against a concurrent origin of Sqs and Osc/Sqmo. In cases like Deltaprotebacteria or Planctomycetes, the production of polycyclic triterpenes could be independent of squalene precursors, as could be the case of Firmicutes and their biosynthesis of sporulenol (a very specific polycyclic triterpene). Another important observation was the lack of carotenoid synthases (CrtB or CrtM) in bacteria that are known to produce carotenoids, such as the Planctomycetes. Taking into account that by genetic engineer it has been demonstrated that squalene is a potential precursor for C30 carotenoids, we suspected that Planctomycetes make use of this route to produce carotenoids. This prompted us to understand the evolution of carotenoid biosynthesis mainly focusing on the C30 pathway, which could represent an interesting connection between hopanoids and carotenoids evolution. The evolution of the carotenoid pathways (C30, C40 and C50) shows more scattered distributions than the one of hopanoids. There are two C40 pathways, one typically from Proteobacteria and the other from Cyanobacteria, and both have common origin but probably emerged sepparately. In contrast, we observed evidence of direct evolution between C40 and C30 pathways, which could be explained by the duplication and neofunctionalization of a entire operon containing the respective C30 or C40 genes. Focusing on the C30 pathway, we observe that its evolution is heterogenous because it has been transferred through LGT multiple times between bacteria. In addition, C30 carotenoid pathway is associated with CrtM only in Firmicutes (the canonical pathway) while in other organisms like in Planctomycetes, it is usually associated to the squalene pathways, either to HpnCDE or Sqs, showing that the potential source of precursor have changed between bacteria, and supporting the idea that C30 carotenoids are synthesized from squalene. We then demonstrated experimentally the natural production of carotenoids through squalene in Planctomycetes (work by Elena Rivas-Marin, Valentina Henriques and Damaso Hornero). Through random transposon insertion in the Planctomycete Planctopirus limnophila, heterologous expression in Escherichia coli, and high performance liquid chromatography analyses (HPLC), we confirm the production of C30 carotenoids via squalene in Planctomycetes being the first description of such pathway in nature. Finally, we integrated the evolutionary history of polycyclic triterpenes and carotenoids to try to determine how the biosynthesis of both originated. Which metabolism is more ancestral? Did ancestral organisms share the same precursor of carotenoid and polycyclic triterpene (squalene) and through the time the metabolic pathways separated and diversified (into C40 pathways for example)? Or did C30 carotenoids and hopanoids originate independently, and coincided just by metabolic compatibility? According to our results, it is difficult to infer a consecutive evolution of carotenoids and polycyclic triterpenes and instead, they could have originated independently. In addition, hopanoids display a more ancestral and/or conserved evolution than carotenoids, which could argue against the classical view of carotenoids as more ancestral than polycyclic triterpenes. In conclusion this is the first work that integrate the evolution of polycyclic triterpenes and carotenoids in the context of the Tree of Life, and altogether, provides novel insights into the evolution cell membranes through the history of life.
Programa de Doctorado en Biotecnología, Ingeniería y Tecnología Química
Línea de Investigación: Biotecnología en la Agricultura, el Medioambiente, la Industria y la Alimentación
Clave Programa: DBI
Código Línea: 109
- Tesis Doctorales