Periodic Reporting for period 4 - FattyCyanos (Fatty acid incorporation and modification in cyanobacterial natural products)
Berichtszeitraum: 2022-07-01 bis 2023-12-31
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.
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.
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.