Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives

The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin—a diterpene produced by Clitopilus passeckerianus—being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics.

As a geneticist, i would have found a schematic diagram of the biosynthetic genes very useful (although I realise this is also in the authors recent Scientific Reports paper) At the end of the discussion the authors talk about the potential to develop new semisynthetic pleuromutilins now that there is a full elucidation of the pathway, perhaps a little more detail on prospective molecules could be mentioned (unless of course this impinges on IP that is currently being assessed?).
I found the supplementary data extensive and well prepared.
Reviewer #2 (Remarks to the Author): The manuscript describes pleuromutilin biosynthetic pathway elucidation. The authors showed key pleuromutilin precursor is biosynthesized via two consecutive cyclization processes by a dualfunctional cyclase. The cyclized product structure, compound 2, was carefully examined using detailed NMR spectroscopic methods and the authentic standard was also prepared. After formation of 2, series of hydroxylation, oxidation and acetylation reactions occur to complete 1 production. The experiments were well planned and the intermediate as well as products characterization were carefully executed. However, it looks like the first chemical structure drawn in figure 1B has less carbons than other intermediates and product drawn in the same figure. This should be corrected. On the other hand, if it is on purpose, the mechanism of this specific reaction is a new discovery in the field and need to be addressed in the manuscript.
It would significantly improve the paper quality if the authors could test the proposed cyclization mechanism (Figure 1b). For example, the isotope tracer experiment to identify the origin of C6 and C10 protons by using regiospecific deuterium-enriched IPP and/or DMAPP for constructing GGPP or the deuterium solvent (D2O).
The overall pathway has been beautifully elucidated. It would also improve the paper quality if the author could discuss the possible biosynthetic rate limiting step(s) in the producing pleuromutilin in the system. Reviewer #3 (Remarks to the Author): The work by ALberti et al, describes a heterologous system to investigate the biosynthesis of pleuomutilin. Pleuomutiln is a gram positive acting antibiotic that works to inhibit protein synthesis acting through the peptidyl transferase centre. Amoung selective antibacterials is a fairly rare scaffold produced by-two basidiomycete fungi Pleurotus mutilis (synonymous to Clitopilus scyphoides f. mutilus) and Pleurotus passeckerianus (synonymous to Clitopilus passeckerianus). The authors contend there is much interest in this antibiotic as a lead molecule and a hinderance exist in developing synthetic analogs-as differential start materials are lacking but may be realized with establishment of a heterologous system of production -whereby analogs may be purposefully created (by removal of enzymes or feeding etc). The genetic cluster for the antibiotic has been established previously and the biosynthetic logic for the molecule suggested. In previous work also as the authors point out -total de novo biosynthesis was achieved through the expression of the entire gene cluster in the secondary host A. oryzae, proving that the seven genes isolated were sufficient for biosynthesis of the diterpene antibiotic. This missing pieces in that earlier work was to define exactly which genes of the seven were doing what -although it is a relatively small number of genes and many of which are bioinformatically & with biosynthetic logic decipherable. In the work here -the authors use a combination of biosynthetic engineering/heterologous express and some synthetic studies to build the authentic intermediates to proven the steps. In that regard there is not much but good science that they have done to delineate these steps-but in the opinion of this review nothing extraordinary was defined and much of the work was very predictable. In fact that de novo biosynthesis had been achieved previously. The kinetics of the reactions are not reported in any depth -and most of the work is end point assays and definition of the products (which is nice). The challenge is for Nature Communications this work does not rise to a standard that seizes on a new finding or a new technology and as such this is more in line with a work typically seen in the journal Biochemistry or ChemBioChem (although they will ask for kinetics).

Reply to Referee Comments
EDITOR COMMENTS : Editorially we feel that, given the novelty concerns raised by Referee #3, it would be important to show experimentally (and not only at a discussion level) the possibility of developing semisynthetic pleuromutilins (in response to Referee #1's concern). This, coupled with additional mechanistic and kinetic insights as suggested by Referees #2 and #3, would, in our view, greatly increase the impact of the paper. We therefore invite you to revise and resubmit your manuscript, taking into account the points raised. Please highlight all changes in the manuscript text file.

REPLY :
You very helpfully pull out a few possible threads for us to consider, as to which may add best to the paper. These are namely additional mechanistic and kinetic insights, as well as development of semisynthetic pleuromutilins.
We think the referees may be slightly off track on the first two points namely mechanistic and kinetics, and we will clarify below.
But we may be able to help with the additional, and perhaps the most significant point of the development of semisynthetic pleuromutilins, which we have made progress with since the submission of the manuscript. We will again outline this below, but first mechanistic and kinetic insights.
Kinetic insight: this is perhaps a slight misunderstanding, and would not be possible using this system directly. This is because each individual transformant is an independent transformation insertion event. Each transformant will therefore have positional effects based on localized insertion position. In addition, each gene within the gene cluster is not being expressed under its own endogenous promoter, but rather from Aspergillus promoters to allow this heterologous expression. Therefore any kinetic studies would be meaningless within this system, or indeed misleading if published. Also because this is a highly optimized system, there are no bottlenecks as can be seen from traces, with each additional gene leading to 100% conversion to the next, with no intermediates.
Mechanistic insight: the proposed cyclization mechanism is not from this work, but rather a starting point previously proposed within the literature, we will make this much clearer in the resubmission. We included this as historical starting point for the work that followed. Experiments that might elucidate such a mechanism cannot be carried out within Aspergillus.
Development of semisynthetic pleuromutilins: as both you and the referees realize this is probably the most exciting potential of this approach, and are probably right to raise it within the comments. Well, we think, with your agreement, we can add novelty by reporting our first proof of concept behind this work, and would be happy to add this work to this manuscript, as we strongly believe that Nature Communications is far and away the best place to publish this work, so that it can reach the largest most appropriate readership.
So, in brief, using our knowledge of the pathway we are able to generate transformants with particular gene combinations. These have then been fed with native and chemically modified intermediates, with the transformed genes providing the final tailoring steps to produce brand new novel compounds.
EDITOR REPLY : do agree that practically demonstrating the usefulness of your system for the development of novel pleuromutilins analogues would substantially raise both the novelty and the impact of the paper. These experiments would be crucial for us. However, I would not insist on having kinetic assays, especially considering your reasons, but I would suggest adding some explanatory text in the point-by-point letter and maybe in the paper as well, for the benefit of our readers. Some clarifications will also be needed regarding the cyclization mechanism reported in the paper.
Also, please note that in a couple of weeks I will move from Nature Communications to Nature nanotechnology, and therefore there is a high probability that I will not handle your revised version. However I will add a note about this conversation in your manuscript file, so that the person that will receive the manuscript is up-to-date.
Our Reply : as agreed with editor, we have now carried out the agreed expt and added it into the paper The abstract now also reflects this to state : A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a novel semi- The work is remarkable for several reasons -basidiomycete fungi are notoriously difficult to engineer and secondly from a synthetic biology perspective the production of pleuromutilin from a series of heterologously expressed constructs in Aspergillus has enabled a previously unknown, deep understanding of the biosynthetic route to pleuromutilin.

REPLY: Thank you
The manuscript is well written and easy to follow. I have a couple of relatively minor points for the authors to address, but I fully support the publication of this work.
As a geneticist, i would have found a schematic diagram of the biosynthetic genes very useful (although I realise this is also in the authors recent Scientific Reports paper) REPLY: We feel that it is not needed, but clearly if editor does think so we are happy to add it in Fig.3 -(Confession is I am not 1H-NMR expert) I found this figure difficult to interpretwhilst the structures were very useful, I wondered if some of the assignments that are in the text could be added to the spectra? REPLY: in response to the point about NMR assignments (reviewer 1) -all of the NMR assignments are listed in the supporting information and are also tabulated. In figure 3 the relevant signals are clearly labeled with appropriate numbering on the structures.
At the end of the discussion the authors talk about the potential to develop new semisynthetic pleuromutilins now that there is a full elucidation of the pathway, perhaps a little more detail on prospective molecules could be mentioned (unless of course this impinges on IP that is currently being assessed?). REPLY: This is part of the new information that is included in the paper. Using our knowledge of the pathway we are able to generate transformants with particular gene combinations. These have then been fed with native and chemically modified intermediates, with the transformed genes providing the final tailoring steps to produce brand new novel compounds.
I found the supplementary data extensive and well prepared.

REPLY: Thank you
Reviewer #2 (Remarks to the Author): The manuscript describes pleuromutilin biosynthetic pathway elucidation. The authors showed key pleuromutilin precursor is biosynthesized via two consecutive cyclization processes by a dual-functional cyclase. The cyclized product structure, compound 2, was carefully examined using detailed NMR spectroscopic methods and the authentic standard was also prepared. After formation of 2, series of hydroxylation, oxidation and acetylation reactions occur to complete 1 production. The experiments were well planned and the intermediate as well as products characterization were carefully executed.
However, it looks like the first chemical structure drawn in figure 1B has less carbons than other intermediates and product drawn in the same figure. This should be corrected. On the other hand, if it is on purpose, the mechanism of this specific reaction is a new discovery in the field and need to be addressed in the manuscript. REPLY: this has now been corrected.
It would significantly improve the paper quality if the authors could test the proposed cyclization mechanism (Figure 1b). For example, the isotope tracer experiment to identify the origin of C6 and C10 protons by using regiospecific deuterium-enriched IPP and/or DMAPP for constructing GGPP or the deuterium solvent (D2O). REPLY: the proposed cyclization mechanism is not from this work, but rather a starting point previously proposed within the literature, we will make this much clearer in the resubmission. We included this as historical starting point for the work that followed. Experiments that might elucidate such a mechanism cannot be carried out within Aspergillus.
The overall pathway has been beautifully elucidated. It would also improve the paper quality if the author could discuss the possible biosynthetic rate limiting step(s) in the producing pleuromutilin in the system. REPLY: This comment on kinetic insight is perhaps a slight misunderstanding, and would not be possible using this system directly. This is because each individual transformant is an independent transformation insertion event. Each transformant will therefore have positional effects based on localized insertion position. In addition, each gene within the gene cluster is not being expressed under its own endogenous promoter, but rather from Aspergillus promoters to allow this heterologous expression. Therefore any kinetic studies would be meaningless within this system, or indeed misleading if published. Also because this is a highly optimized system, there are no bottlenecks as can be seen from traces, with each additional gene leading to 100% conversion to the next, with no intermediates.
Reviewer #3 (Remarks to the Author): The work by ALberti et al, describes a heterologous system to investigate the biosynthesis of pleuomutilin. Pleuomutiln is a gram positive acting antibiotic that works to inhibit protein synthesis acting through the peptidyl transferase centre. Amoung selective antibacterials is a fairly rare scaffold produced by-two basidiomycete fungi Pleurotus mutilis (synonymous to Clitopilus scyphoides f. mutilus) and Pleurotus passeckerianus (synonymous to Clitopilus passeckerianus). The authors contend there is much interest in this antibiotic as a lead molecule and a hinderance exist in developing synthetic analogs-as differential start materials are lacking but may be realized with establishment of a heterologous system of production -whereby analogs may be purposefully created (by removal of enzymes or feeding etc). The genetic cluster for the antibiotic has been established previously and the biosynthetic logic for the molecule suggested. In previous work also as the authors point out -total de novo biosynthesis was achieved through the expression of the entire gene cluster in the secondary host A. oryzae, proving that the seven genes isolated were sufficient for biosynthesis of the diterpene antibiotic. This missing pieces in that earlier work was to define exactly which genes of the seven were doing what -although it is a relatively small number of genes and many of which are bioinformatically & with biosynthetic logic decipherable. In the work here -the authors use a combination of biosynthetic engineering/heterologous express and some synthetic studies to build the authentic intermediates to proven the steps. In that regard there is not much but good science that they have done to delineate these steps-but in the opinion of this review nothing extraordinary was defined and much of the work was very predictable. In fact that de novo biosynthesis had been achieved previously.
REPLY: as agreed with the Editor, we have added significant new data that addresses this general view, namely we can add novelty by reporting our first proof of concept behind this work. Using our knowledge of the pathway we are able to generate transformants with particular gene combinations. These have then been fed with native and chemically modified intermediates, with the transformed genes providing the final tailoring steps to produce brand new novel compounds. This has now been included in the paper/ The kinetics of the reactions are not reported in any depth -and most of the work is end point assays and definition of the products (which is nice). The challenge is for Nature Communications this work does not rise to a standard that seizes on a new finding or a new technology and as such this is more in line with a work typically seen in the journal Biochemistry or ChemBioChem (although they will ask for kinetics). REPLY: This comment on kinetic insight is perhaps a slight misunderstanding, and would not be possible using this system directly. This is because each individual transformant is an independent transformation insertion event. Each transformant will therefore have positional effects based on localized insertion position. In addition, each gene within the gene cluster is not being expressed under its own endogenous promoter, but rather from Aspergillus promoters to allow this heterologous expression. Therefore any kinetic studies would be meaningless within this system, or indeed misleading if published. Also because this is a highly optimized system, there are no bottlenecks as can be seen from traces, with each additional gene leading to 100% conversion to the next, with no intermediates.