Discovery and characterization of a terpene biosynthetic pathway featuring a norbornene-forming Diels-Alderase

Pericyclases, enzymes that catalyze pericyclic reactions, form an expanding family of enzymes that have biocatalytic utility. Despite the increasing number of pericyclases discovered, the Diels-Alder cyclization between a cyclopentadiene and an olefinic dienophile to form norbornene, which is among the best-studied cycloadditions in synthetic chemistry, has surprisingly no enzymatic counterpart to date. Here we report the discovery of a pathway featuring a norbornene synthase SdnG for the biosynthesis of sordaricin-the terpene precursor of antifungal natural product sordarin. Full reconstitution of sordaricin biosynthesis reveals a concise oxidative strategy used by Nature to transform an entirely hydrocarbon precursor into the highly functionalized substrate of SdnG for intramolecular Diels-Alder cycloaddition. SdnG generates the norbornene core of sordaricin and accelerates this reaction to suppress host-mediated redox modifications of the activated dienophile. Findings from this work expand the scopes of pericyclase-catalyzed reactions and P450-mediated terpene maturation.

It is also important to note the quality of the supplementary material. However, authors are advised to include enlargements of the two-dimensional spectra,making it easier for readers to use them.
The high quality of the manuscript allows to recommend it be published in its current state.
Reviewer #3 (Remarks to the Author): This manuscript describes comprehensive elucidation of the core sordaricin biosynthetic pathway, with particularly intriguing discovery of a novel pericyclase catalyzing the heretofore unknown Diels-Alder cyclization between a cyclopentadiene and dienophile to form a norborene ring structure. As prominently noted here, this is a commonly utilized and studied cycloaddition in synthetic chemistry, but had previously not been seen in a biosynthetic pathway. While the relevant diterpene cyclase SdnA had been previously identified, along with an extended biosynthetic gene cluster (BGC) associated with production of the extensively more elaborated hypoxysordarin, here the subsequently acting cytochromes P450 SdnB, E, F and H, as well as novel pericyclase SdnG were identified. This was accomplished by discovery of more compact BGCs associated with production of the simpler sordarins, which enabled more focused studies of the above-mentioned enzymes. Notably, SdnB was found to play multiple roles, both serving to di-hydroxylate neighboring carbons in the tricyclic cycloaraneosene diterpene produced by SdnA, but also latter scission between these hydroxylated carbons, with this oxidative cleavage generating a dialdehyde derivative. Interestingly, this only occurs following an intervening dehydrogenation reaction catalyzed by SdnH. Although a ring cleavage product is observed in the absence of SdnH, data is shown indicating that such cleavage is more readily accomplished following formation of the cyclopentadiene by SdnH. This oxidative ring cleavage is followed by further oxidation of the aldehyde substituent of the cyclopentadiene moiety to a carboxylate, which enables the Diels-Alder cycloaddition to form the norborene ring structure. Although this can occur spontaneously, with subsequent hydroxylation catalyzed by SdnE then producing sordaricin, observation of significant amounts of shunt products led to further investigation of SdnG, which was found to act as a pericyclase increasing the Diels-Alder cycloaddition reaction (>500-fold). Intriguingly, SdnG is a novel sequence, representing a new class of pericyclases, discovery of which adds further significance to the reported studies. Indeed, given the noted concise oxidative strategy and novel pericyclase revealed here, in conjunction with the long-sought nature of the catalyzed Diels-Alder cyclization between a cyclopentadiene and dienophile to form a norborene, this study should be of reasonably wide-spread interest.
Line 136 -Extended data fig. 3a does not demonstrate oxidative cleavage of 2 to 4, as only 3 is observed. Instead, it is a combination of that experiment and the one shown in extended data fig. 3d, where feeding of 4 is shown result in efficient conversion to 3. Thus, both should be cited here.
c.f. Line 146 & Fig. 2b -Please discuss how 6 is generated in the absence of SdnF.
Reviewer #4 (Remarks to the Author): The Authors present an investigation concerning the discovery and characterization of a terpene biosynthetic pathway featuring a Norbornene-forming Diels-Alderase.
I cannot comment on the biological and biochemical relevance of the manuscript since my expertise is in computational organic chemistry and NMR spectroscopy. Therefore my review below is limited to the technical aspects of these two techniques.
Concerning the DFT calculations, these appears to be run at a rather high level of theory. The functional used, w-B97XD, is a modern functional which includes a treatment of dispersive interactions and long range corrections -necessary when dealing with molecules with pi-electrons and where van der Waals interactions are expected to play a significant role. The basis set is also sufficiently large. The level of theory used for the calculation is indeed adequate.
However the Authors do not comment on the method used to calculate the transition states. In Figure 3 they report transition states, how these stationary points were calculated? Did the Authors check that the TS was actually connecting reactants and products by following the reaction path?
Concerning the NMR part, the intermediates have been thoroughly characterized by 1H, 13C, COSY, HSQC and HMBC NMR (as well as HR mass) with a high resolution 500 MHz instrument. When necessary, a NOESY spectrum is also included (please check the spelling in Supplementary Material, it is reported as NOSEY instead of NOESY).
In short, the computational and NMR parts are very well executed, except for some minor issues mentioned above.
We thank the reviewers for their comments and suggestions. We have provided a list of replies and improvements in the following table. The page and line number mentioned below correspond to the pdf file with track change.
Reviewer 1 1. The case of the solanopyrones could be mentioned as being related -there is an interesting redox cycle here from alcohol to activated aldehyde and then back to alcohol after the DA step.
i) We mentioned solanopyrone as a related case to the oxidative diene activation of sordaricin biosynthesis in the first submission. In the revised manuscript, we expanded the corresponding sentence (as showing below) and added an additional citation to further elaborate.
Page18, line 345-348: "Such redox activation of diene/dienophile is analogous to the role played by the flavin-dependent enzyme Sol5 catalyzing a decalin-forming IMDA in solanapyrone biosynthesis where Sol5 is required to activate the dienophile by oxidizing an electron donating hydroxymethyl group conjugated to the dienophile to an electron withdrawing aldehyde 45,46 ".

Position numbers are normally C-8, O-15 H-4/C-5 etc
ii) A hyphen was inserted in between atom labels and atom numbers throughout the revised manuscript.
iii) This was corrected throughout the revised manuscript.
4. The calls to the 'extended' data are annoying. Its 2022 -why can't this be included in the main paper? Its not like the journal has to save on ink and paper.... iv) We moved Extended Data Figs 3, 4, and 5 to the main text where they are now Figs 3, 5, and 6. Extended Data Figs 1 and 2 were moved to the Supplementary Information as Supplementary Fig. 1  and 3.

Reviewer 2
Authors are advised to include enlargements of the two-dimensional spectra, making it easier for readers to use them.
We attempted to enlarge the spectra but the figure width is already at the limit of the page width. However, the pdf file we resubmitted, when zoomed in, provides reasonable resolution for the 2D spectra. We hope this will satisfy the readers.
i) This has been corrected throughout the revised manuscript.
2. Line 136 -Extended data fig. 3a does not demonstrate oxidative cleavage of 2 to 4, as only 3 is observed. Instead, it is a combination of that experiment and the one shown in extended data fig. 3d, where feeding of 4 is shown result in efficient conversion to 3. Thus, both should be cited here.
ii) We added a trace showing efficient conversion of 4 to 3 by A. nidulans to Extended data fig. 3a (now Fig. 3a, relabeled following the suggestion of another reviewer). We also revised the corresponding paragraph as follows to clarify this point.
Page 7, line 132-151: "We proposed that 3 is likely formed via oxidative cleavage of the C-8,C-9 diol in 2 by SdnB (Fig. 2b), which was confirmed through direct feeding of 2 to A. nidulans expressing only SdnB (Fig. 3a). Compound 2 is stable under the same feeding conditions when an empty plasmid control was used. Since the conversion of 2 to 3 is a net redox-neutral process, the product of SdnB oxidation is likely dialdehyde 4 instead of 3. In the absence of downstream enzymes, the C-7 acrolein moiety in 4 can be reduced by endogenous reductases in A. nidulans to afford alcohol 3 as a cellular detoxification mechanism to remove the unsaturated aldehyde 33,34 . We chemically synthesized 4 by selectively oxidizing the allylic alcohol in 3 to the corresponding unsaturated aldehyde via activated MnO2 35 (Supplementary Notes,Supplementary Table 7,Supplementary Figs. 2,(23)(24)(25)(26)(27). Consistent with our hypothesis, 4 was ready converted to 3 when fed to A. nidulans expressing only empty plasmids (Fig.  3a). Overall, our results suggest that SdnB both acts as a canonical monooxygenase and also a "thwarted oxygenase", an oxygenase that consumes oxygen to generate strong enzymatic oxidant but does not result in formal incorporation of oxygen atom into the product 32 (Supplementary Fig. 3). The diol cleavage activity of SdnB enables rotation of the C-6-C-7 bond and thereby "freed" the C-7-C-17 double bond which is the proposed dienophile for the IMDA reaction (Fig. 2b)." 3. Line 146 & Fig. 2b -Please discuss how 6 is generated in the absence of SdnF.
iii) We believe in the absence of SdnF, 6 can be generated from 5 either by auto-oxidation or host oxidases. As shown in Extended Data Fig. 4b (now Fig. 5b, relabeled following the suggestion of another reviewer), feeding of 5 to A. nidulans expressing only empty plasmid resulted in formation of 6. We modified the corresponding sentence as follows to incorporate this discussion.
Page 10, line 167-170: "Similar to the formation of 3 from 4, 5 is likely a redox shunt product derived from cyclopentadienedialdehyde 7, which should be the product of sequential actions of SdnB and SdnH starting from cycloaraneosene (and 2). Auto-oxidation or host oxidases may subsequently convert 5 to 6." Page 13, line 235-240: "To assay the oxidation activities of SdnF separately from cyclization of 10, 5 was supplemented to the SdnF expression strain and a control strain expressing only empty plasmids. While the control strain is able to convert ~30% of 5 to the carboxylate 6 likely via auto-oxidation or host oxidases, the SdnF expression strain led to near complete conversion of 5 to 6 (Fig. 5b), demonstrating that SdnF is able to selectively oxidize the C-8 aldehyde to carboxylate."