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Oryzalexin S biosynthesis: a cross-stitched disappearing pathway

Oryzalexin S biosynthesis: a cross-stitched disappearing pathway aBIOTECH https://doi.org/10.1007/s42994-022-00092-3 aBIOTECH BRIEF COMMUNICATION Oryzalexin S biosynthesis: a cross-stitched disappearing pathway 1 1 1 1 1& Le Zhao , Richard Oyagbenro , Yiling Feng , Meimei Xu , Reuben J. Peters Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA Received: 14 October 2022 / Accepted: 25 December 2022 Abstract Rice produces many diterpenoid phytoalexins and, reflecting the importance of these natural products in this important cereal crop plant, its genome contains three biosynthetic gene clusters (BGCs) for such metabolism. The chromosome 4 BGC (c4BGC) is largely associated with momilactone production, in part due to the presence of the initiating syn-copalyl diphosphate (CPP) synthase gene (OsCPS4). Oryzalexin S is also derived from syn-CPP. However, the relevant subsequently acting syn-stemarene synthase gene (OsKSL8) is not located in the c4BGC. Production of oryzalexin S further requires hydroxylation at carbons 2 and 19 (C2 and C19), presumably catalyzed by cytochrome P450 (CYP) monooxygenases. Here it is reported the closely related CYP99A2 and CYP99A3, whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation, while the closely related CYP71Z21 and CYP71Z22, whose genes are found in the recently reported chromosome 7 BGC (c7BGC), catalyze subsequent hydroxy- lation at C2a. Thus, oryzalexin S biosynthesis utilizes two distinct BGCs, in a pathway cross-stitched together by OsKSL8. Notably, in contrast to the widely conserved c4BGC, the c7BGC is subspecies (ssp.) specific, being prevalent in ssp. japonica and only rarely found in the other major ssp. indica. Moreover, while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8, it has now been reported to be a ssp. indica derived allele at the same genetic loci. Intriguingly, more detailed analysis indicates that OsKSL8(j) is being replaced by OsKSL11 (OsKSL8i), suggesting introgression from ssp. indica to (sub)tropical japonica, with concurrent disappearance of oryzalexin S production. Keywords Biosynthetic gene clusters, Phytoalexins, Diterpenoids, Rice evolution INTRODUCTION (BGCs) associated with such metabolism, which were among the first to be discovered in plants (Nutzmann Rice (Oryza sativa) is well known for producing many et al. 2016). In particular, functional identification of the diterpenoid phytoalexins and has served as model sys- consecutively acting pairs of cyclases led to realization tem for investigation of these natural products, which of the proximity of the relevant genes (Prisic et al. 2004; are prevalent in cereal crops more generally (Murphy Wilderman et al. 2004). Moreover, the two identified and Zerbe 2020). One of the early findings from these regions each also contained a number of genes for studies was discovery of biosynthetic gene clusters cytochrome P450 (CYP) monooxygenases, which were later demonstrated to act on the diterpenes generated by the co-clustered cyclases (Swaminathan et al. 2009; Le Zhao and Richard Oyagbenro contributed equally to this work. Wang et al. 2011, 2012;Wuetal. 2011, 2013; Kitaoka et al. 2015). & Correspondence: rjpeters@iastate.edu (R. J. Peters) The Author(s) 2023 aBIOTECH The use of two consecutively acting cyclases is CYP71Z2 and CYP71Z21, an independent study found characteristic of the labdane-related nature of almost all the c7BGC also contains genes encoding CYP71Z22, rice diterpenoids. Their production is initiated by bicy- which is closely related and functionally redundant with clization of the general diterpenoid precursor (E,E,E)- CYP71Z21, as well as CYP71Z30, albeit this latter is geranylgeranyl diphosphate (GGPP) catalyzed by class II found as a pseudo-gene even in some cultivars (cv.) diterpene cyclases, which prototypically produce the from ssp. japonica (Liang et al. 2021). eponymous labdadienyl/copalyl diphosphate (CPP) as Oryzalexin S is a phytoalexin against the devastating distinct stereoisomers (Peters 2010). For example, all fungal blast pathogen Magnaporthe oryzae, and is land plants must produce ent-CPP and, hence, contain derived from syn-stemar-13-ene via hydroxylation at such a CPP synthase (CPS) for phytohormone biosyn- C2a and C19 (Tamogani et al. 1993). The relevant thesis. For this purpose, ent-CPP must then be further OsKSL8, acting on the syn-CPP produced by OsCPS4, has cyclized to the tetracyclic olefin ent-kaurene by a class I been identified, with the encoding gene found on diterpene synthase. In angiosperms the CPS and kau- chromosome 11 (Nemoto et al. 2004). In addition, a rene synthase (KS) required for gibberellin phytohor- closely related (both mechanistically and phylogeneti- mone biosynthesis have often given rise to expanded cally) syn-stemod-13(17)-ene synthase was then iden- gene families involved in more specialized metabolism, tified (Morrone et al. 2006). Although the relevant gene with the latter generally termed KS-like (KSL) (Zi et al. loci was not found in the available genome sequence, 2014). the relatively low sequence identity (\ 93% nucleotide In rice, functional characterization of the syn-CPP sequence identity) suggested that it was not an allele of synthase OsCPS4 and subsequently acting syn-pimara- OsKSL8, as other KSL alleles (even with functionally diene synthase OsKSL4 led to discovery of co-localiza- distinct activity and from different ssp.) shared[ 99% tion their genes on chromosome 4 (Wilderman et al. identity (Xu et al. 2007), and so this was termed 2004; Xu et al. 2004). Notably, the only other genes in OsKSL11. However, it has since been clarified that the region encoded the closely related paralogs OsKSL11 is a distinct allele of OsKSL8 derived from ssp. CYP99A2 and CYP99A3, and pair of short-chain alcohol indica rice, which can then be termed OsKSL8i, while the dehydrogenases, defining a BGC (c4BGC). Consistent other ssp. japonica derived allele is then OsKSL8j (Toy- with RNAi knock-down studies (Shimura et al. 2007), it omasu et al. 2016). was then shown that CYP99A3 (Wang et al. 2011), and Here the CYPs catalyzing hydroxylation at C19 and, latter CYP99A2 (Kitaoka et al. 2015), react with syn- subsequently, C2a of syn-stemarene to generate oryza- pimaradiene, both introducing oxygen at carbon-19 lexin S were identified as the closely related CYP99A2/3 (C19), as required for momilactone biosynthesis. from the c4BGC and CYP71Z21/22 from the c7BGC, Similarly, functional characterization of the ent-cas- respectively. Intriguingly, utilizing the recently reported sadiene synthase OsKSL7 and upstream ent-CPP syn- phylogenetically representative set of pan-rice genomes thase OsCPS2 dedicated to more specialized metabolism (Zhou et al. 2020), it was found that the relevant syn- led to discovery of another BGC located on chromosome stemarene producing OsKSL8j is actually not present in 2 (Cho et al. 2004; Prisic et al. 2004). This c2BGC con- the examined (sub)tropical spp. japonica cultivars, tains multiple members of the CYP71Z and CYP76M having been replaced by the functionally distinct, syn- sub-families, with some of the latter surprisingly stemodene producing OsKSL8i (OsKSL11), the implica- involved in biosynthesis of the momilactones associated tions of which are discussed. with the c4BGC, demonstrating interdependent evolu- tion of these two BGCs (Kitaoka et al. 2021;Lietal. 2022). Not surprisingly, the presence of CYP76M sub- MATERIALS AND METHODS family members at the c2BGC locus is then conserved throughout the Oryza genus (Miyamoto et al. 2016), Unless otherwise stated, all chemicals were obtained with c4BGC orthologs found even more widely (Wu et al. from Fisher Scientific. The genes utilized here were 2022). largely synthetic, optimized for expression in Escher- More recently reported was another diterpenoid BGC ichia coli, as previously reported—i.e., sCYP99A2 and on chromosome 7 (c7BGC), encoding ent-10-oxode- sCYP99A3 (Wang et al. 2011), as well as sCYP71Z6 and pression production, which seems to be almost entirely sCYP71Z7 (Wu et al. 2011), and sCYP71Z22, but the restricted to the japonica subspecies (ssp.) and is only native CYP71Z21 (Liang et al. 2021). These CYPs were rarely found in the other major ssp. (indica) of rice expressed as described (Kitaoka et al. 2015), either with (Zhan et al. 2020). While this report only described the just the CYP reductase (CPR) from Arabidopsis thaliana phylogenetically related but functionally distinct (AtCPR1) for feeding studies, or in a previously The Author(s) 2023 aBIOTECH described modular metabolic engineering system (Cyr feeding experiments demonstrated these also do not et al. 2007), as adapted for CYP expression (Kitaoka react with syn-stemar-13-en-19-ol (data not shown). et al. 2015), with expression of a GGPP synthase Given the recent discovery of roles for the CYP71Z sub- (AgGGPS), OsCPS4 and sOsKSL8j to produce syn-stemar- family members from the c7BGC in diterpenoid phy- 13-ene. As needed, metabolic flux was increased as toalexin biosynthesis, despite their activity with the previously described (Morrone et al. 2010). The macrocyclic ent-casbene, which is not a labdane-related resulting diterpenoids were extracted and analyzed by diterpene as it is derived from direct cyclization of GGPP GC–MS and, as necessary, purified by flash chromatog- by a class I diterpene synthase, these also were inves- raphy and HPLC, using GC–MS analysis to track the tigated. Fortuitously, feeding experiments found that, targeted compounds, which were then structurally while CYP71Z2 was unreactive, the closely related analyzed by NMR and/or utilized for feeding studies, all CYP71Z21 and CYP71Z22 both efficiently converted as has been previously described—e.g., (Swaminathan syn-stemar-13-en-19-ol to oryzalexin S (Fig. 1Bi, ii and et al. 2009; Wang et al. 2011, 2012;Wuetal. Supplemental Fig. S2). This demonstrates CYP71Z21/22 2011, 2013; Kitaoka et al. 2015). Gene presence was catalyze the necessary C2a-hydroxylation. In addition, determined via BLAST searches of the relevant genes co-expression of CYP71Z22 and CYP99A3 (as well as from cv. Nipponbare (ssp. japonica) against the cDNA AtCPR1) in E. coli also engineered to produce syn-ste- databases for the described (sub)species and, where marene led to production of oryzalexin S (3), along with relevant, cultivars at http://oryza.gramene.org (Tello- a substantial amount of an alternative hydroxylated Ruiz et al. 2022). Phylogenetic analysis of KSL8 was derivative (Fig. 1Biii, iv). To verify this product was the carried out using CLC Main WorkBench 22 (Qiagen expected syn-stemar-13-en-2a-ol (4), CYP71Z22 was Aarhus A/S). utilized in the metabolic engineering system (Supple- mental Figure S3), along with increased metabolic flux to isoprenoids and larger culture volume, enabling RESULTS purification of sufficient amounts of this diterpenoid to feed to CYP99A3, which was able to convert this to Based on the previously reported ability of CYP99A3 to oryzalexin S (Fig. 1Bv). The less efficient production of catalyze at least C19-hydroxylation of syn-stemodene oryzalexin S (3) from syn-stemar-13-en-2a-ol (4)by (Wang et al. 2011), the activity of both this and the CYP99A3, relative to syn-stemar-13-en-19-ol (2)by closely related paralog CYP99A2 with the oryzalexin S CYP71Z22, along with accumulation of 4 in the full- precursor syn-stemar-13-ene (1) was examined. pathway containing E. coli, suggests preferential Specifically, these CYPs were each co-expressed with the hydroxylation, with initial C19-hydroxylation of syn- requisite CPR in E. coli also engineered to produce this stemarene by CYP99A2/3 and subsequent C2a-hydrox- putative substrate. In both cases this led to observation ylation by CYP71Z21/22 in oryzalexin S biosynthesis of what appeared to be hydroxylated derivatives of 1, (Fig. 1Ci). along with the accompanying syn-stemod-12-ene also Given the previously reported ssp. specific occur- made by OsKSL8j in approximately the same ratio rel- rence of both OsKSL8j and the c7BGC, especially the ative to 1 as seen here for these hydroxylated products variation observed for the associated CYP71Z sub-fam- (Xu et al. 2007), as indicated by the increase in molec- ily members, it seemed prudent to investigate distri- ular weight (MW) from 272 to 288 Da (Fig. 1Ai, ii and bution of the genes associated with oryzalexin S Supplemental Fig. S1). For structural analysis, metabolic biosynthesis. For this purpose, the recently reported flux to isoprenoids was increased and the culture vol- phylogenetically representative set of Asian rice genome ume increased with CYP99A3 (as this seems to be more sequences (Zhou et al. 2020), along with a broader set efficient than CYP99A2, see Supplemental Fig. S1), of domesticated and wild rice relatives across the Oryza enabling purification of sufficient amounts of this genus, up to and including Oryza punctata, which served diterpenoid for NMR analysis (Supplemental Table S1), as an outgroup (Stein et al. 2018), were examined here which revealed the major product to be the expected (Fig. 1Cii). As previously reported, the c4BGC is widely C19-hydroxylated derivative syn-stemar-13-en-19-ol conserved (Miyamoto et al. 2016), with OsCPS4 and (2). CYP99A2/3 present in all these genomes, and the c7BGC Previous work indicated that CYP71Z6 and CYP71Z7 is found in all ssp. japonica, although at least CYP71Z21 from the c2BGC act on C2 of specific (ent-CPP derived) is more widely distributed, including other species labdane-related diterpenes (Wu et al. 2011), producing (Zhan et al. 2020). While the O. rufipogon landrace C2a-hydroxy derivatives (Kitaoka et al. 2015). Accord- examined here (W1943) does not contain CYP71Z21/22, ingly, neither was found to modify syn-stemarene. Here, which clarifies the basis for the previously reported The Author(s) 2023 aBIOTECH The Author(s) 2023 aBIOTECH bFig. 1 A CYP99A2/3 catalyze 19-hydroxylation of syn-stemar-13- (Supplemental Figure S4), allowing tentative assign- ene (1). i) Extracted ion count (EIC) chromatogram from GC–MS ment of allele in almost all the AA-genome rice, although analysis of extracts from E. coli co-expressing CYP99A3 and the not the more distant (BB-genome) O. punctata, in which requisite AtCPR1 as well as engineered to produce 1 via co- the most closely related cDNA shared 85–88% ORF expression of a GGPP synthase and OsKSL8j. Peak marked with * is presumed to be 19-hydroxy derivative of syn-stemod-12-ene, sequence identity with the other full-length KSL8 which is produced in approximately the same ratio relative to 1 by examined here. Strikingly, the only other exception is OsKSL8j as seen here for these hydroxylated products (Xu et al. found in the O. sativa ssp. basmati representative, where 2007). ii) Mass spectra of syn-stemar-13-en-19-ol (2). See the most closely related cDNA shared\ 82% ORF Supplemental Fig. S1 for verification of CYP99A2 activity and additional GC–MS data supporting assignment of * as syn-stemod- sequence identity with all other KSL8 examined here 12-en-19-ol. B CYP71Z21/22 catalyze 2a-hydroxylation. i) EIC (including that from O. punctata). chromatogram from GC–MS analysis of extracts from E. coli co- expressing CYP71Z22 and the requisite AtCPR1, and then fed syn- stemar-13-en-19-ol (2). See Supplemental Fig. S2 for verification of CYP71Z21 activity. ii) Mass spectra of oryzalexin S (3). iii) EIC DISCUSSION chromatogram from GC–MS analysis of extracts from E. coli co- expressing CYP99A3, CYP71Z21 and the requisite AtCPR1 as well Here the CYPs catalyzing hydroxylation of syn-stemar- as engineered to produce - 1. iv) Mass spectra of syn-stemar-13- ene at C19 and, as shown here, the subsequent en-2a-ol (4). v) EIC chromatogram from GC–MS analysis of extracts from E. coli co-expressing CYP99A3 and the requisite hydroxylation at C2a required for oryzalexin S biosyn- AtCPR1, and then fed syn-stemar-13-en-2a-ol (4) purified from thesis were identified as the closely related paralogous E. coli co-expressing CYP71Z22 and the requisite AtCPR1 as well pairs CYP99A2/3 and CYP71Z21/22 (respectively). The as engineered to produce 1 (see Supplemental Fig. S3 for EIC activity of CYP71Z21/22 is somewhat surprising given chromatogram from GC–MS analysis of extract). C Conservation of Oryzalexin S. i) Biosynthetic pathway, with previously identified the differences between the macrocyclic ent-casbene diterpene cyclases on top and CYPs identified here below (also that these react upon for ent-10-oxodepressin produc- indicated by bold text). ii) Conservation of relevant genes (bold tion versus the labdane-related (multicyclic) syn-ste- italic text indicates those explicitly discussed here) mar-13-en-19-ol these react with for oryzalexin S biosynthesis. Regardless, the encoding pair of genes are found in the previously identified c4BGC and c7BGC (respectively), with the former also containing that for absence of oryzalexin S therein (Toyomasu et al. 2016), the syn-CPP synthase OsCPS4, which is required for it was previously reported that 12/13 O. rufipogon production of oryzalexin S (Zhang et al. 2021). By con- landraces contain at least CYP71Z21 (Zhan et al. 2020), trast, the gene for the relevant syn-stemarene synthase hence the ‘‘±’’ indication at this point. More interest- OsKSL8(j) is located elsewhere (chromosome 11), and ingly, substantial variation is observed with KSL8. Note then cross-stitches together these two BGCs to form the that while the originally identified OsKSL8j was cloned oryzalexin S biosynthetic pathway. from cv. Nipponbare, only a partial cDNA was found in Given both OsKSL8j and the c7BGC are specifically the database examined here. Thus, while apparently associated with ssp. japonica (Toyomasu et al. 2016; partial cDNA are found for a few other O. sativa cultivars Zhan et al. 2020), and, especially, the discovery that in this database (as indicated by appended ‘‘p’’), these OsKSL11 is a functionally distinct ssp. indica derived OsKSL8 may be functional and were included in the allele (more accurately termed OsKSL8i), a wider phylogenetic analysis, enabling assignment of relevant examination of the genes relevant to oryzalexin S allele. Despite association of the syn-stemarene pro- biosynthesis was carried out here. The results were ducing allele with ssp. japonica, the corresponding consistent with the previous suggestion that such OsKSL8j is only found in the temperate representative metabolism was present in the progenitor of ssp. cv. Nipponbare, as well as the wild-rice relative O. rufi- japonica and O. rufipogon (Toyomasu et al. 2016), with pogon. The other (tropical and subtropical) represen- possibly more ancient origins suggested by the presence tatives contain alleles phylogenetically closer to OsKSL8i of the relevant genes more widely in the Oryza genus (OsKSL11), with full-length open-reading frame (ORF) (although this latter hypothesis needs further investi- sequence identities [ 95.5% versus \ 92.5%, respec- gation). Strikingly, in O. sativa ssp. japonica it appears tively. Consistent with retention in only the temperate the OsKSL8j allele has only been retained in temperate cultivars of ssp. japonica, OsKSL8j was found in the cultivars, with those from (sub)tropical climates con- temperate cv. KitaakeX (Jain et al. 2019), while OsKSL8i taining OsKSL8i (OsKSL11) instead. While gene flow via is found in the tropical cv. Carolina Gold (Vaughn et al. hybridization and introgression from ssp. japonica into 2021). OsKSL8 from the more distant species of rice ssp. indica is well known (Wing et al. 2018), these were included in this phylogenetic analysis results indicate introgression of this functionally The Author(s) 2023 aBIOTECH Shibuya N, Nojiri H, Omori T, Nishiyama M, Yamane H (2004) distinct allele in the opposite direction (i.e., from ssp. Molecular cloning and characterization of a cDNA encoding indica into ssp. japonica), with the accompanying dis- ent-cassa-12,15-diene synthase, a putative diterpenoid phy- appearance of oryzalexin S. Indeed, an even more toalexin biosynthetic enzyme, from suspension-cultured rice extreme case is found in ssp. basmati, where a highly cells treated with a chitin elicitor. Plant J 37:1–8 Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular divergent allele appears. Thus, it will be of interest to approach for facile biosynthesis of labdane-related diter- determine the product of this OsKSL8 variant. In addi- penes. J Am Chem Soc 129:6684–6685 tion, although CYP99A3 from the widespread c4BGC has Jain R, Jenkins J, Shu S, Chern M, Martin JA, Copetti D, Duong PQ, been reported to catalyze oxygenation at C19 of the syn- Pham NT, Kudrna DA, Talag J, Schackwitz WS, Lipzen AM, Dilworth D, Bauer D, Grimwood J, Nelson CR, Xing F, Xie W, stemod-13(17)-ene product of OsKSL8i (OsKSL11) Barry KW, Wing RA, Schmutz J, Li G, Ronald PC (2019) (Wang et al. 2011), this is not sufficient for diterpenoid Genome sequence of the model rice variety KitaakeX. BMC solubility and bioactivity, which invariably requires at Genomics 20:905 least two spatially separated oxy groups (Wu et al. Kitaoka N, Wu Y, Xu M, Peters RJ (2015) Optimization of recombinant expression enables discovery of novel cyto- 2013). However, syn-stemod-13(17)-en-19-ol is not chrome P450 activity in rice diterpenoid biosynthesis. Appl further transformed by CYP71Z21/22 (data not shown). Microbiol Biotechnol 99:7549–7558 Accordingly, no syn-stemod-13(17)-ene derived phy- Kitaoka N, Zhang J, Oyagbenro RK, Brown B, Wu Y, Yang B, Li Z, toalexin has yet been identified, so it will be of interest Peters RJ (2021) Interdependent evolution of biosynthetic gene clusters for momilactone production in rice. Plant Cell to investigate any additional oxygenation of this to 33:290–305 examine bioactivity and determine the basis for intro- Li R, Zhang J, Li Z, Peters RJ, Yang B (2022) Dissecting the labdane- gression of OsKSL8i into (sub)tropical ssp. japonica, as related diterpenoid biosynthetic gene clusters in rice reveals well as the more divergent allele found in ssp. basmati. directional cross-cluster phytotoxicity. New Phytol 233:878–889 Supplementary InformationThe online version contains Liang J, Shen Q, Wang L, Liu J, Fu J, Zhao L, Xu M, Peters RJ, Wang Q supplementary material available at https://doi.org/10.1007/ (2021) Rice contains a biosynthetic gene cluster associated s42994-022-00092-3. with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin. New Phytol 231:85–93 Acknowledgements The authors thank Prof. Robert Coates Miyamoto K, Fujita M, Shenton MR, Akashi S, Sugawara C, Sakai A, (Univ. Illinois, ret.) for an authentic standard of oryzalexin S. This Horie K, Hasegawa M, Kawaide H, Mitsuhashi W, Nojiri H, work was supported by Grants from the NIH (GM131885) and Yamane H, Kurata N, Okada K, Toyomasu T (2016) Evolu- USDA (2020-67013-32557) to R.J.P. tionary trajectory of phytoalexin biosynthetic gene clusters in rice. Plant J 87:293–304 Morrone D, Jin Y, Xu M, Choi SY, Coates RM, Peters RJ (2006) An Data availability All data generated or analyzed during this unexpected diterpene cyclase from rice: functional identifi- study are available from the corresponding author upon reason- cation of a stemodene synthase. Arch Biochem Biophys able request. 448:133–140 Morrone D, Lowry L, Determan MK, Hershey DM, Xu M, Peters RJ Declarations (2010) Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP Conflict of interest On behalf of all authors, the corresponding isoprenoid precursor pathway engineering. Appl Microbiol author states that there is no conflict of interest. Biotechnol 85:1893–1906 Murphy KM, Zerbe P (2020) Specialized diterpenoid metabolism Open Access This article is licensed under a Creative Commons in monocot crops: biosynthesis and chemical diversity. Attribution 4.0 International License, which permits use, sharing, Phytochemistry 172:112289 adaptation, distribution and reproduction in any medium or for- Nemoto T, Cho E-M, Okada A, Okada K, Otomo K, Kanno Y, mat, as long as you give appropriate credit to the original Toyomasu T, Mitsuhashi W, Sassa T, Minami E, Shibuya N, author(s) and the source, provide a link to the Creative Commons Nishiyama M, Nojiri H, Yamane H (2004) Stemar-13-ene licence, and indicate if changes were made. The images or other synthase, a diterpene cyclase involved in the biosynthesis of third party material in this article are included in the article’s the phytoalexin oryzalexin S in rice. FEBS Lett 571:182–186 Creative Commons licence, unless indicated otherwise in a credit Nutzmann HW, Huang A, Osbourn A (2016) Plant metabolic line to the material. If material is not included in the article’s clusters—from genetics to genomics. 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Plant J 65:87–95 Mauleon R, Lorieux M, Zuccolo A, McNally K, Zhang J, Wing Wang Q, Hillwig ML, Okada K, Yamazaki K, Wu Y, Swaminathan S, RA (2020) A platinum standard pan-genome resource that Yamane H, Peters RJ (2012) Characterization of CYP76M5-8 represents the population structure of Asian rice. Sci Data indicates metabolic plasticity within a plant biosynthetic gene 7:113 cluster. J Biol Chem 287:6159–6168 Zi J, Mafu S, Peters RJ (2014) To gibberellins and beyond! Wilderman PR, Xu M, Jin Y, Coates RM, Peters RJ (2004) Surveying the evolution of (di)terpenoid metabolism. Annu Identification of syn-pimara-7,15-diene synthase reveals Rev Plant Biol 65:259–286 functional clustering of terpene synthases involved in rice The Author(s) 2023 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png aBIOTECH Springer Journals

Oryzalexin S biosynthesis: a cross-stitched disappearing pathway

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aBIOTECH https://doi.org/10.1007/s42994-022-00092-3 aBIOTECH BRIEF COMMUNICATION Oryzalexin S biosynthesis: a cross-stitched disappearing pathway 1 1 1 1 1& Le Zhao , Richard Oyagbenro , Yiling Feng , Meimei Xu , Reuben J. Peters Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA Received: 14 October 2022 / Accepted: 25 December 2022 Abstract Rice produces many diterpenoid phytoalexins and, reflecting the importance of these natural products in this important cereal crop plant, its genome contains three biosynthetic gene clusters (BGCs) for such metabolism. The chromosome 4 BGC (c4BGC) is largely associated with momilactone production, in part due to the presence of the initiating syn-copalyl diphosphate (CPP) synthase gene (OsCPS4). Oryzalexin S is also derived from syn-CPP. However, the relevant subsequently acting syn-stemarene synthase gene (OsKSL8) is not located in the c4BGC. Production of oryzalexin S further requires hydroxylation at carbons 2 and 19 (C2 and C19), presumably catalyzed by cytochrome P450 (CYP) monooxygenases. Here it is reported the closely related CYP99A2 and CYP99A3, whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation, while the closely related CYP71Z21 and CYP71Z22, whose genes are found in the recently reported chromosome 7 BGC (c7BGC), catalyze subsequent hydroxy- lation at C2a. Thus, oryzalexin S biosynthesis utilizes two distinct BGCs, in a pathway cross-stitched together by OsKSL8. Notably, in contrast to the widely conserved c4BGC, the c7BGC is subspecies (ssp.) specific, being prevalent in ssp. japonica and only rarely found in the other major ssp. indica. Moreover, while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8, it has now been reported to be a ssp. indica derived allele at the same genetic loci. Intriguingly, more detailed analysis indicates that OsKSL8(j) is being replaced by OsKSL11 (OsKSL8i), suggesting introgression from ssp. indica to (sub)tropical japonica, with concurrent disappearance of oryzalexin S production. Keywords Biosynthetic gene clusters, Phytoalexins, Diterpenoids, Rice evolution INTRODUCTION (BGCs) associated with such metabolism, which were among the first to be discovered in plants (Nutzmann Rice (Oryza sativa) is well known for producing many et al. 2016). In particular, functional identification of the diterpenoid phytoalexins and has served as model sys- consecutively acting pairs of cyclases led to realization tem for investigation of these natural products, which of the proximity of the relevant genes (Prisic et al. 2004; are prevalent in cereal crops more generally (Murphy Wilderman et al. 2004). Moreover, the two identified and Zerbe 2020). One of the early findings from these regions each also contained a number of genes for studies was discovery of biosynthetic gene clusters cytochrome P450 (CYP) monooxygenases, which were later demonstrated to act on the diterpenes generated by the co-clustered cyclases (Swaminathan et al. 2009; Le Zhao and Richard Oyagbenro contributed equally to this work. Wang et al. 2011, 2012;Wuetal. 2011, 2013; Kitaoka et al. 2015). & Correspondence: rjpeters@iastate.edu (R. J. Peters) The Author(s) 2023 aBIOTECH The use of two consecutively acting cyclases is CYP71Z2 and CYP71Z21, an independent study found characteristic of the labdane-related nature of almost all the c7BGC also contains genes encoding CYP71Z22, rice diterpenoids. Their production is initiated by bicy- which is closely related and functionally redundant with clization of the general diterpenoid precursor (E,E,E)- CYP71Z21, as well as CYP71Z30, albeit this latter is geranylgeranyl diphosphate (GGPP) catalyzed by class II found as a pseudo-gene even in some cultivars (cv.) diterpene cyclases, which prototypically produce the from ssp. japonica (Liang et al. 2021). eponymous labdadienyl/copalyl diphosphate (CPP) as Oryzalexin S is a phytoalexin against the devastating distinct stereoisomers (Peters 2010). For example, all fungal blast pathogen Magnaporthe oryzae, and is land plants must produce ent-CPP and, hence, contain derived from syn-stemar-13-ene via hydroxylation at such a CPP synthase (CPS) for phytohormone biosyn- C2a and C19 (Tamogani et al. 1993). The relevant thesis. For this purpose, ent-CPP must then be further OsKSL8, acting on the syn-CPP produced by OsCPS4, has cyclized to the tetracyclic olefin ent-kaurene by a class I been identified, with the encoding gene found on diterpene synthase. In angiosperms the CPS and kau- chromosome 11 (Nemoto et al. 2004). In addition, a rene synthase (KS) required for gibberellin phytohor- closely related (both mechanistically and phylogeneti- mone biosynthesis have often given rise to expanded cally) syn-stemod-13(17)-ene synthase was then iden- gene families involved in more specialized metabolism, tified (Morrone et al. 2006). Although the relevant gene with the latter generally termed KS-like (KSL) (Zi et al. loci was not found in the available genome sequence, 2014). the relatively low sequence identity (\ 93% nucleotide In rice, functional characterization of the syn-CPP sequence identity) suggested that it was not an allele of synthase OsCPS4 and subsequently acting syn-pimara- OsKSL8, as other KSL alleles (even with functionally diene synthase OsKSL4 led to discovery of co-localiza- distinct activity and from different ssp.) shared[ 99% tion their genes on chromosome 4 (Wilderman et al. identity (Xu et al. 2007), and so this was termed 2004; Xu et al. 2004). Notably, the only other genes in OsKSL11. However, it has since been clarified that the region encoded the closely related paralogs OsKSL11 is a distinct allele of OsKSL8 derived from ssp. CYP99A2 and CYP99A3, and pair of short-chain alcohol indica rice, which can then be termed OsKSL8i, while the dehydrogenases, defining a BGC (c4BGC). Consistent other ssp. japonica derived allele is then OsKSL8j (Toy- with RNAi knock-down studies (Shimura et al. 2007), it omasu et al. 2016). was then shown that CYP99A3 (Wang et al. 2011), and Here the CYPs catalyzing hydroxylation at C19 and, latter CYP99A2 (Kitaoka et al. 2015), react with syn- subsequently, C2a of syn-stemarene to generate oryza- pimaradiene, both introducing oxygen at carbon-19 lexin S were identified as the closely related CYP99A2/3 (C19), as required for momilactone biosynthesis. from the c4BGC and CYP71Z21/22 from the c7BGC, Similarly, functional characterization of the ent-cas- respectively. Intriguingly, utilizing the recently reported sadiene synthase OsKSL7 and upstream ent-CPP syn- phylogenetically representative set of pan-rice genomes thase OsCPS2 dedicated to more specialized metabolism (Zhou et al. 2020), it was found that the relevant syn- led to discovery of another BGC located on chromosome stemarene producing OsKSL8j is actually not present in 2 (Cho et al. 2004; Prisic et al. 2004). This c2BGC con- the examined (sub)tropical spp. japonica cultivars, tains multiple members of the CYP71Z and CYP76M having been replaced by the functionally distinct, syn- sub-families, with some of the latter surprisingly stemodene producing OsKSL8i (OsKSL11), the implica- involved in biosynthesis of the momilactones associated tions of which are discussed. with the c4BGC, demonstrating interdependent evolu- tion of these two BGCs (Kitaoka et al. 2021;Lietal. 2022). Not surprisingly, the presence of CYP76M sub- MATERIALS AND METHODS family members at the c2BGC locus is then conserved throughout the Oryza genus (Miyamoto et al. 2016), Unless otherwise stated, all chemicals were obtained with c4BGC orthologs found even more widely (Wu et al. from Fisher Scientific. The genes utilized here were 2022). largely synthetic, optimized for expression in Escher- More recently reported was another diterpenoid BGC ichia coli, as previously reported—i.e., sCYP99A2 and on chromosome 7 (c7BGC), encoding ent-10-oxode- sCYP99A3 (Wang et al. 2011), as well as sCYP71Z6 and pression production, which seems to be almost entirely sCYP71Z7 (Wu et al. 2011), and sCYP71Z22, but the restricted to the japonica subspecies (ssp.) and is only native CYP71Z21 (Liang et al. 2021). These CYPs were rarely found in the other major ssp. (indica) of rice expressed as described (Kitaoka et al. 2015), either with (Zhan et al. 2020). While this report only described the just the CYP reductase (CPR) from Arabidopsis thaliana phylogenetically related but functionally distinct (AtCPR1) for feeding studies, or in a previously The Author(s) 2023 aBIOTECH described modular metabolic engineering system (Cyr feeding experiments demonstrated these also do not et al. 2007), as adapted for CYP expression (Kitaoka react with syn-stemar-13-en-19-ol (data not shown). et al. 2015), with expression of a GGPP synthase Given the recent discovery of roles for the CYP71Z sub- (AgGGPS), OsCPS4 and sOsKSL8j to produce syn-stemar- family members from the c7BGC in diterpenoid phy- 13-ene. As needed, metabolic flux was increased as toalexin biosynthesis, despite their activity with the previously described (Morrone et al. 2010). The macrocyclic ent-casbene, which is not a labdane-related resulting diterpenoids were extracted and analyzed by diterpene as it is derived from direct cyclization of GGPP GC–MS and, as necessary, purified by flash chromatog- by a class I diterpene synthase, these also were inves- raphy and HPLC, using GC–MS analysis to track the tigated. Fortuitously, feeding experiments found that, targeted compounds, which were then structurally while CYP71Z2 was unreactive, the closely related analyzed by NMR and/or utilized for feeding studies, all CYP71Z21 and CYP71Z22 both efficiently converted as has been previously described—e.g., (Swaminathan syn-stemar-13-en-19-ol to oryzalexin S (Fig. 1Bi, ii and et al. 2009; Wang et al. 2011, 2012;Wuetal. Supplemental Fig. S2). This demonstrates CYP71Z21/22 2011, 2013; Kitaoka et al. 2015). Gene presence was catalyze the necessary C2a-hydroxylation. In addition, determined via BLAST searches of the relevant genes co-expression of CYP71Z22 and CYP99A3 (as well as from cv. Nipponbare (ssp. japonica) against the cDNA AtCPR1) in E. coli also engineered to produce syn-ste- databases for the described (sub)species and, where marene led to production of oryzalexin S (3), along with relevant, cultivars at http://oryza.gramene.org (Tello- a substantial amount of an alternative hydroxylated Ruiz et al. 2022). Phylogenetic analysis of KSL8 was derivative (Fig. 1Biii, iv). To verify this product was the carried out using CLC Main WorkBench 22 (Qiagen expected syn-stemar-13-en-2a-ol (4), CYP71Z22 was Aarhus A/S). utilized in the metabolic engineering system (Supple- mental Figure S3), along with increased metabolic flux to isoprenoids and larger culture volume, enabling RESULTS purification of sufficient amounts of this diterpenoid to feed to CYP99A3, which was able to convert this to Based on the previously reported ability of CYP99A3 to oryzalexin S (Fig. 1Bv). The less efficient production of catalyze at least C19-hydroxylation of syn-stemodene oryzalexin S (3) from syn-stemar-13-en-2a-ol (4)by (Wang et al. 2011), the activity of both this and the CYP99A3, relative to syn-stemar-13-en-19-ol (2)by closely related paralog CYP99A2 with the oryzalexin S CYP71Z22, along with accumulation of 4 in the full- precursor syn-stemar-13-ene (1) was examined. pathway containing E. coli, suggests preferential Specifically, these CYPs were each co-expressed with the hydroxylation, with initial C19-hydroxylation of syn- requisite CPR in E. coli also engineered to produce this stemarene by CYP99A2/3 and subsequent C2a-hydrox- putative substrate. In both cases this led to observation ylation by CYP71Z21/22 in oryzalexin S biosynthesis of what appeared to be hydroxylated derivatives of 1, (Fig. 1Ci). along with the accompanying syn-stemod-12-ene also Given the previously reported ssp. specific occur- made by OsKSL8j in approximately the same ratio rel- rence of both OsKSL8j and the c7BGC, especially the ative to 1 as seen here for these hydroxylated products variation observed for the associated CYP71Z sub-fam- (Xu et al. 2007), as indicated by the increase in molec- ily members, it seemed prudent to investigate distri- ular weight (MW) from 272 to 288 Da (Fig. 1Ai, ii and bution of the genes associated with oryzalexin S Supplemental Fig. S1). For structural analysis, metabolic biosynthesis. For this purpose, the recently reported flux to isoprenoids was increased and the culture vol- phylogenetically representative set of Asian rice genome ume increased with CYP99A3 (as this seems to be more sequences (Zhou et al. 2020), along with a broader set efficient than CYP99A2, see Supplemental Fig. S1), of domesticated and wild rice relatives across the Oryza enabling purification of sufficient amounts of this genus, up to and including Oryza punctata, which served diterpenoid for NMR analysis (Supplemental Table S1), as an outgroup (Stein et al. 2018), were examined here which revealed the major product to be the expected (Fig. 1Cii). As previously reported, the c4BGC is widely C19-hydroxylated derivative syn-stemar-13-en-19-ol conserved (Miyamoto et al. 2016), with OsCPS4 and (2). CYP99A2/3 present in all these genomes, and the c7BGC Previous work indicated that CYP71Z6 and CYP71Z7 is found in all ssp. japonica, although at least CYP71Z21 from the c2BGC act on C2 of specific (ent-CPP derived) is more widely distributed, including other species labdane-related diterpenes (Wu et al. 2011), producing (Zhan et al. 2020). While the O. rufipogon landrace C2a-hydroxy derivatives (Kitaoka et al. 2015). Accord- examined here (W1943) does not contain CYP71Z21/22, ingly, neither was found to modify syn-stemarene. Here, which clarifies the basis for the previously reported The Author(s) 2023 aBIOTECH The Author(s) 2023 aBIOTECH bFig. 1 A CYP99A2/3 catalyze 19-hydroxylation of syn-stemar-13- (Supplemental Figure S4), allowing tentative assign- ene (1). i) Extracted ion count (EIC) chromatogram from GC–MS ment of allele in almost all the AA-genome rice, although analysis of extracts from E. coli co-expressing CYP99A3 and the not the more distant (BB-genome) O. punctata, in which requisite AtCPR1 as well as engineered to produce 1 via co- the most closely related cDNA shared 85–88% ORF expression of a GGPP synthase and OsKSL8j. Peak marked with * is presumed to be 19-hydroxy derivative of syn-stemod-12-ene, sequence identity with the other full-length KSL8 which is produced in approximately the same ratio relative to 1 by examined here. Strikingly, the only other exception is OsKSL8j as seen here for these hydroxylated products (Xu et al. found in the O. sativa ssp. basmati representative, where 2007). ii) Mass spectra of syn-stemar-13-en-19-ol (2). See the most closely related cDNA shared\ 82% ORF Supplemental Fig. S1 for verification of CYP99A2 activity and additional GC–MS data supporting assignment of * as syn-stemod- sequence identity with all other KSL8 examined here 12-en-19-ol. B CYP71Z21/22 catalyze 2a-hydroxylation. i) EIC (including that from O. punctata). chromatogram from GC–MS analysis of extracts from E. coli co- expressing CYP71Z22 and the requisite AtCPR1, and then fed syn- stemar-13-en-19-ol (2). See Supplemental Fig. S2 for verification of CYP71Z21 activity. ii) Mass spectra of oryzalexin S (3). iii) EIC DISCUSSION chromatogram from GC–MS analysis of extracts from E. coli co- expressing CYP99A3, CYP71Z21 and the requisite AtCPR1 as well Here the CYPs catalyzing hydroxylation of syn-stemar- as engineered to produce - 1. iv) Mass spectra of syn-stemar-13- ene at C19 and, as shown here, the subsequent en-2a-ol (4). v) EIC chromatogram from GC–MS analysis of extracts from E. coli co-expressing CYP99A3 and the requisite hydroxylation at C2a required for oryzalexin S biosyn- AtCPR1, and then fed syn-stemar-13-en-2a-ol (4) purified from thesis were identified as the closely related paralogous E. coli co-expressing CYP71Z22 and the requisite AtCPR1 as well pairs CYP99A2/3 and CYP71Z21/22 (respectively). The as engineered to produce 1 (see Supplemental Fig. S3 for EIC activity of CYP71Z21/22 is somewhat surprising given chromatogram from GC–MS analysis of extract). C Conservation of Oryzalexin S. i) Biosynthetic pathway, with previously identified the differences between the macrocyclic ent-casbene diterpene cyclases on top and CYPs identified here below (also that these react upon for ent-10-oxodepressin produc- indicated by bold text). ii) Conservation of relevant genes (bold tion versus the labdane-related (multicyclic) syn-ste- italic text indicates those explicitly discussed here) mar-13-en-19-ol these react with for oryzalexin S biosynthesis. Regardless, the encoding pair of genes are found in the previously identified c4BGC and c7BGC (respectively), with the former also containing that for absence of oryzalexin S therein (Toyomasu et al. 2016), the syn-CPP synthase OsCPS4, which is required for it was previously reported that 12/13 O. rufipogon production of oryzalexin S (Zhang et al. 2021). By con- landraces contain at least CYP71Z21 (Zhan et al. 2020), trast, the gene for the relevant syn-stemarene synthase hence the ‘‘±’’ indication at this point. More interest- OsKSL8(j) is located elsewhere (chromosome 11), and ingly, substantial variation is observed with KSL8. Note then cross-stitches together these two BGCs to form the that while the originally identified OsKSL8j was cloned oryzalexin S biosynthetic pathway. from cv. Nipponbare, only a partial cDNA was found in Given both OsKSL8j and the c7BGC are specifically the database examined here. Thus, while apparently associated with ssp. japonica (Toyomasu et al. 2016; partial cDNA are found for a few other O. sativa cultivars Zhan et al. 2020), and, especially, the discovery that in this database (as indicated by appended ‘‘p’’), these OsKSL11 is a functionally distinct ssp. indica derived OsKSL8 may be functional and were included in the allele (more accurately termed OsKSL8i), a wider phylogenetic analysis, enabling assignment of relevant examination of the genes relevant to oryzalexin S allele. Despite association of the syn-stemarene pro- biosynthesis was carried out here. The results were ducing allele with ssp. japonica, the corresponding consistent with the previous suggestion that such OsKSL8j is only found in the temperate representative metabolism was present in the progenitor of ssp. cv. Nipponbare, as well as the wild-rice relative O. rufi- japonica and O. rufipogon (Toyomasu et al. 2016), with pogon. The other (tropical and subtropical) represen- possibly more ancient origins suggested by the presence tatives contain alleles phylogenetically closer to OsKSL8i of the relevant genes more widely in the Oryza genus (OsKSL11), with full-length open-reading frame (ORF) (although this latter hypothesis needs further investi- sequence identities [ 95.5% versus \ 92.5%, respec- gation). Strikingly, in O. sativa ssp. japonica it appears tively. Consistent with retention in only the temperate the OsKSL8j allele has only been retained in temperate cultivars of ssp. japonica, OsKSL8j was found in the cultivars, with those from (sub)tropical climates con- temperate cv. KitaakeX (Jain et al. 2019), while OsKSL8i taining OsKSL8i (OsKSL11) instead. While gene flow via is found in the tropical cv. Carolina Gold (Vaughn et al. hybridization and introgression from ssp. japonica into 2021). OsKSL8 from the more distant species of rice ssp. indica is well known (Wing et al. 2018), these were included in this phylogenetic analysis results indicate introgression of this functionally The Author(s) 2023 aBIOTECH Shibuya N, Nojiri H, Omori T, Nishiyama M, Yamane H (2004) distinct allele in the opposite direction (i.e., from ssp. Molecular cloning and characterization of a cDNA encoding indica into ssp. japonica), with the accompanying dis- ent-cassa-12,15-diene synthase, a putative diterpenoid phy- appearance of oryzalexin S. Indeed, an even more toalexin biosynthetic enzyme, from suspension-cultured rice extreme case is found in ssp. basmati, where a highly cells treated with a chitin elicitor. Plant J 37:1–8 Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular divergent allele appears. Thus, it will be of interest to approach for facile biosynthesis of labdane-related diter- determine the product of this OsKSL8 variant. In addi- penes. J Am Chem Soc 129:6684–6685 tion, although CYP99A3 from the widespread c4BGC has Jain R, Jenkins J, Shu S, Chern M, Martin JA, Copetti D, Duong PQ, been reported to catalyze oxygenation at C19 of the syn- Pham NT, Kudrna DA, Talag J, Schackwitz WS, Lipzen AM, Dilworth D, Bauer D, Grimwood J, Nelson CR, Xing F, Xie W, stemod-13(17)-ene product of OsKSL8i (OsKSL11) Barry KW, Wing RA, Schmutz J, Li G, Ronald PC (2019) (Wang et al. 2011), this is not sufficient for diterpenoid Genome sequence of the model rice variety KitaakeX. BMC solubility and bioactivity, which invariably requires at Genomics 20:905 least two spatially separated oxy groups (Wu et al. Kitaoka N, Wu Y, Xu M, Peters RJ (2015) Optimization of recombinant expression enables discovery of novel cyto- 2013). However, syn-stemod-13(17)-en-19-ol is not chrome P450 activity in rice diterpenoid biosynthesis. Appl further transformed by CYP71Z21/22 (data not shown). Microbiol Biotechnol 99:7549–7558 Accordingly, no syn-stemod-13(17)-ene derived phy- Kitaoka N, Zhang J, Oyagbenro RK, Brown B, Wu Y, Yang B, Li Z, toalexin has yet been identified, so it will be of interest Peters RJ (2021) Interdependent evolution of biosynthetic gene clusters for momilactone production in rice. Plant Cell to investigate any additional oxygenation of this to 33:290–305 examine bioactivity and determine the basis for intro- Li R, Zhang J, Li Z, Peters RJ, Yang B (2022) Dissecting the labdane- gression of OsKSL8i into (sub)tropical ssp. japonica, as related diterpenoid biosynthetic gene clusters in rice reveals well as the more divergent allele found in ssp. basmati. directional cross-cluster phytotoxicity. New Phytol 233:878–889 Supplementary InformationThe online version contains Liang J, Shen Q, Wang L, Liu J, Fu J, Zhao L, Xu M, Peters RJ, Wang Q supplementary material available at https://doi.org/10.1007/ (2021) Rice contains a biosynthetic gene cluster associated s42994-022-00092-3. with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin. New Phytol 231:85–93 Acknowledgements The authors thank Prof. Robert Coates Miyamoto K, Fujita M, Shenton MR, Akashi S, Sugawara C, Sakai A, (Univ. Illinois, ret.) for an authentic standard of oryzalexin S. This Horie K, Hasegawa M, Kawaide H, Mitsuhashi W, Nojiri H, work was supported by Grants from the NIH (GM131885) and Yamane H, Kurata N, Okada K, Toyomasu T (2016) Evolu- USDA (2020-67013-32557) to R.J.P. tionary trajectory of phytoalexin biosynthetic gene clusters in rice. Plant J 87:293–304 Morrone D, Jin Y, Xu M, Choi SY, Coates RM, Peters RJ (2006) An Data availability All data generated or analyzed during this unexpected diterpene cyclase from rice: functional identifi- study are available from the corresponding author upon reason- cation of a stemodene synthase. Arch Biochem Biophys able request. 448:133–140 Morrone D, Lowry L, Determan MK, Hershey DM, Xu M, Peters RJ Declarations (2010) Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP Conflict of interest On behalf of all authors, the corresponding isoprenoid precursor pathway engineering. Appl Microbiol author states that there is no conflict of interest. Biotechnol 85:1893–1906 Murphy KM, Zerbe P (2020) Specialized diterpenoid metabolism Open Access This article is licensed under a Creative Commons in monocot crops: biosynthesis and chemical diversity. Attribution 4.0 International License, which permits use, sharing, Phytochemistry 172:112289 adaptation, distribution and reproduction in any medium or for- Nemoto T, Cho E-M, Okada A, Okada K, Otomo K, Kanno Y, mat, as long as you give appropriate credit to the original Toyomasu T, Mitsuhashi W, Sassa T, Minami E, Shibuya N, author(s) and the source, provide a link to the Creative Commons Nishiyama M, Nojiri H, Yamane H (2004) Stemar-13-ene licence, and indicate if changes were made. The images or other synthase, a diterpene cyclase involved in the biosynthesis of third party material in this article are included in the article’s the phytoalexin oryzalexin S in rice. FEBS Lett 571:182–186 Creative Commons licence, unless indicated otherwise in a credit Nutzmann HW, Huang A, Osbourn A (2016) Plant metabolic line to the material. If material is not included in the article’s clusters—from genetics to genomics. 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Journal

aBIOTECH – Springer Journals

Published: Mar 1, 2023

Keywords: Biosynthetic gene clusters; Phytoalexins; Diterpenoids; Rice evolution

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