Periodic Reporting for period 4 - FattyCyanos (Fatty acid incorporation and modification in cyanobacterial natural products)
Período documentado: 2022-07-01 hasta 2023-12-31
At the onset of the FattyCyanos project, we were aware that cyanobacteria - a group of diverse photosynthetic bacteria that inhabit a large variety of environments - should be able to produce a large number of yet-unknown natural products. Natural products are small molecules that are often imbued with potent biological activity and have the potential to become drugs, research biochemicals, or, for example agrochemicals. We were also aware that, from looking at the available genomes of cyanobacteria, many of such novel natural products should present a fatty-acid derived moiety. These fatty acid moieties are very difficult to modify by traditional synthetic chemistry methods, but we expected that cyanobacteria would be able not only to incorporate this into their natural products, but also to modify them, through the action of enzymes that could find applications as biocatalysts, due to such demanding and exotic chemistry which they carry out. Therefore, the project FattyCyanos proposed to:
A) develop a method to swiftly uncover fatty acid incorporating natural products in cyanobacteria and apply it to large number of cyanobacterial strains. This method should work both with and without previous information on the genetic capacity of the target cyanobacterium to produce such compounds.
B) study the biosynthesis of selected fatty acid-incorporating and -modifying natural products, to reveal unusual biocatalytic transformations and the enzymes that catalyze them.
Over the course of the project, the two objectives were reached - a new method was developed in accordance with proposed obective A) and several new metabolites were revealed. The analysis of their structures and biosynthetic pathways revealed new enzymatic activities or paved the way to discover such new enzymes.
A) develop a method to swiftly uncover fatty acid incorporating natural products in cyanobacteria and apply it to large number of cyanobacterial strains. This method should work both with and without previous information on the genetic capacity of the target cyanobacterium to produce such compounds.
B) study the biosynthesis of selected fatty acid-incorporating and -modifying natural products, to reveal unusual biocatalytic transformations and the enzymes that catalyze them.
Over the course of the project, the two objectives were reached - a new method was developed in accordance with proposed obective A) and several new metabolites were revealed. The analysis of their structures and biosynthetic pathways revealed new enzymatic activities or paved the way to discover such new enzymes.
We have successfully developed the proposed method for fatty acid-incorporating metabolite discovery and have found several new natural products from only a couple of cyanobacteria. Some of these compounds are structurally unique, highlighting the potential of cyanobacteria for the discovery of novel natural products (Figueiredo et al. 2021).
Furthermore, we have studied the biosynthesis of the bartolosides and revealed a biocatalytic transformation, mediated by the enzyme BrtB, which was until then only known in synthetic chemistry. This enzymatic reaction is a new type of biological esterification in which the fatty acids are esterified to a halogenated carbon. Most strikingly, the fatty acids react as free carboxylates and therefore bypass what was thought was a universal requirement: fatty acid activation (Reis et al 2020).
We have critically reviewed the literature on the alkylresorcinol-type of fatty acid-incorporating metabolites. We looked into the entire diversity of these compounds in cyanobacteria and identified opportunities for discovery of new metabolites and enzymes (Martins et al 2019).
We disclosed the first natural biosynthetic pathway involved in the production of natural lactylates, which we envision will enable future biological production and engineering of industrially-relevant lactylates (Abt et al 2021).
We also showed the initial steps in the biosynthesis of the unusual natural product Noculin A, as well as the origin of all its carbon atoms, paving the way for uncovering key steps in the biosynthesis of its unique 1,2,3-oxadiazine core (Martins et al 2022).
With what we have learned during the course of the project, we summarised current knowledge on the incorporation, fate and turnover of free fatty acids in cyanobacteria in general, and also in the specific case of secondary metabolites with associated modifications of their alkyl chains, in two peer-reviewed publications (Kahn et al, 2023 and Leao et al, 2023).
Yet to be published, we have discovered the highly intriguing lipopeptides fischerazoles and that their biosynthesis involves an unprecedented mid-alkyl chain carbon rearrangement (Figueiredo et al, 2022 - preprint). We have also implicated NocC - an enzyme encoded in the nocuolactylates biosynthetic gene cluster - in the generation of beta, gamma-unsaturations in fatty acyl moieties. We have additionally further studied the biosynthesis of nocuolin A and uncovered the role of several enzymes in this pathway, in particular those of a C-oxygenase and an unusual aminotranferase that appears to generate an imino group of a ketone.
All of these findings have been communicated to scientific peers and stundents, in conferences and invited seminars.
The new method developed (Figueiredo et al 2021) and the discovery of the activity of BrtB (Reis et al 2020) were the subject of press releases and featured in news outlets of national (PT) reach.
Several MSc students, PhD students and postdocs were trained in multidisciplinary natural products research during the course of the project, which was also a major achievement of FattyCyanos.
Furthermore, we have studied the biosynthesis of the bartolosides and revealed a biocatalytic transformation, mediated by the enzyme BrtB, which was until then only known in synthetic chemistry. This enzymatic reaction is a new type of biological esterification in which the fatty acids are esterified to a halogenated carbon. Most strikingly, the fatty acids react as free carboxylates and therefore bypass what was thought was a universal requirement: fatty acid activation (Reis et al 2020).
We have critically reviewed the literature on the alkylresorcinol-type of fatty acid-incorporating metabolites. We looked into the entire diversity of these compounds in cyanobacteria and identified opportunities for discovery of new metabolites and enzymes (Martins et al 2019).
We disclosed the first natural biosynthetic pathway involved in the production of natural lactylates, which we envision will enable future biological production and engineering of industrially-relevant lactylates (Abt et al 2021).
We also showed the initial steps in the biosynthesis of the unusual natural product Noculin A, as well as the origin of all its carbon atoms, paving the way for uncovering key steps in the biosynthesis of its unique 1,2,3-oxadiazine core (Martins et al 2022).
With what we have learned during the course of the project, we summarised current knowledge on the incorporation, fate and turnover of free fatty acids in cyanobacteria in general, and also in the specific case of secondary metabolites with associated modifications of their alkyl chains, in two peer-reviewed publications (Kahn et al, 2023 and Leao et al, 2023).
Yet to be published, we have discovered the highly intriguing lipopeptides fischerazoles and that their biosynthesis involves an unprecedented mid-alkyl chain carbon rearrangement (Figueiredo et al, 2022 - preprint). We have also implicated NocC - an enzyme encoded in the nocuolactylates biosynthetic gene cluster - in the generation of beta, gamma-unsaturations in fatty acyl moieties. We have additionally further studied the biosynthesis of nocuolin A and uncovered the role of several enzymes in this pathway, in particular those of a C-oxygenase and an unusual aminotranferase that appears to generate an imino group of a ketone.
All of these findings have been communicated to scientific peers and stundents, in conferences and invited seminars.
The new method developed (Figueiredo et al 2021) and the discovery of the activity of BrtB (Reis et al 2020) were the subject of press releases and featured in news outlets of national (PT) reach.
Several MSc students, PhD students and postdocs were trained in multidisciplinary natural products research during the course of the project, which was also a major achievement of FattyCyanos.
The method developed in the FattyCyanos project extends the number of strategies for natural product discovery in the metabolically-rich cyanobacteria. With this method in hands, we will now be able to screen a large number of strains and gain access to target compounds for isolation. Judging from our progress during the course of the project, with three families of compounds uncovered by this method in just two tested cyanobacteria, the method will enable the discovery of multiple new-to-science natural products and their associated biological activities. The research group where this project took place at CIIMAR, led by the project's PI, now has a list of candidate molecules for isolation that were detected using this method, which will set the stage for natural products discovery efforts in the coming years, as was initially proposed in the grant agreement of this Starting Grant.
Our discovery of BrtB and of its unprecedented reactivity, has enabled additional explorations of the chemical and biosynthetic space associated with members of this fatty acid-acting enzyme class. These are underway, and we have already observed that BrtB is able, for example to generate C-C bonds, apart from its reported C-O bond formation activity.
Furthermore, our continuing biosynthesis studies on selected fatty acid incorporating metabolites is expected to reveal new biochemical transformations and new enzymes. An example is the ongoing work on nocuolin A and the fischerazoles, which are set to reveal completely unprecedented biochemistry.
Our findings to date and anticipated future discoveries (as the legacy of FattyCyanos) will overall lead to a better understanding of the utilization and modification of fatty acids in cyanobacterial secondary metabolism and facilitate natural products and enzyme discovery efforts in these organisms.
Therefore, and as proposed initially, we showed that fatty-acid incorporating secondary metabolites can be swiftly revealed using our newly-developed method, and that unusual enzymes that modify their alkyl changes can be discovered by studying the biosynthesis of several of these metabolites in detail. Some of these enzymes were revealed by this project and several others will be uncovered in the coming years.
Our discovery of BrtB and of its unprecedented reactivity, has enabled additional explorations of the chemical and biosynthetic space associated with members of this fatty acid-acting enzyme class. These are underway, and we have already observed that BrtB is able, for example to generate C-C bonds, apart from its reported C-O bond formation activity.
Furthermore, our continuing biosynthesis studies on selected fatty acid incorporating metabolites is expected to reveal new biochemical transformations and new enzymes. An example is the ongoing work on nocuolin A and the fischerazoles, which are set to reveal completely unprecedented biochemistry.
Our findings to date and anticipated future discoveries (as the legacy of FattyCyanos) will overall lead to a better understanding of the utilization and modification of fatty acids in cyanobacterial secondary metabolism and facilitate natural products and enzyme discovery efforts in these organisms.
Therefore, and as proposed initially, we showed that fatty-acid incorporating secondary metabolites can be swiftly revealed using our newly-developed method, and that unusual enzymes that modify their alkyl changes can be discovered by studying the biosynthesis of several of these metabolites in detail. Some of these enzymes were revealed by this project and several others will be uncovered in the coming years.