Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles

Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles Seiji Takahashi, Miki Suenaga-Hiromori, Tomoki Ishii, Nadia Nur Shazana Binti Abu Talib Khan, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3615345/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Natural rubber (NR) is a sustainable biopolymer consisting mainly of cis -1,4-polyisoprene. Modifying an NR biosynthetic enzyme is a promising strategy to bioproduce novel polymers. Here, we have elucidated the NR biosynthetic mechanism and successfully developed novel enzymes that synthesise NR-sized polyisoprenes with unnatural substrates. NR is synthesised by a cis -prenyltransferase (cPT) on rubber particles (RPs), NR-harbouring lipid monolayer membrane organelles. However, the key to NR biosynthesis is not specialised cPTs, but the proper arrangement of cPTs on RPs since cPTs from various non-NR-producing organisms, such as humans, synthesise NR when introduced into the RPs. A tomato cPT, which condenses only one isoprene unit, was engineered to synthesise novel NR-sized polyisoprenes with artificial substrates by modifying residues for product size determination. Furthermore, the introduction of a modified trans-prenyltransferase into RPs led to the synthesis of NR-sized trans -1,4-polyisoprenes. This RP system could be used as a versatile platform for enzymatic polyisoprenoid synthesis. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Biotechnology/Molecular engineering/Protein design Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography Biological sciences/Biological techniques/Molecular engineering/Protein design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Methodsandinformation.pdf Supplementaryfiles.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3615345","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":253877016,"identity":"9c0838e4-993d-4f77-81a2-421218f76505","order_by":0,"name":"Seiji Takahashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYFACNgZmIJkApA4wMDYwGEBEeXBr4EFoYUtsIE0LkGmIpAUPsGc/lvi5oKYuj4//zPcHH3fYGBscYH74gUHmDm5beNIOS884driYTSJ3Y+PMM2lmBgfYjCUYeJ7hcVh6gzQP24HENgnejc28bYdtDA4wmAHFD+PWwv+8+TfPv7rENv4zD6Fa2L/h1yKRdkyat405sY0hhxGkBegwHgK23HiWZs3bdxjosDTDmTPb0owlD/MUSyTg8Qt7f5rxbZ5vdYnz+w8/+PCxzcaw73j7xg8fe3CHGBYAiqbEngOkaAGDH6RrGQWjYBSMgmELAJ2iVGogUMKRAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2288-4340","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Seiji","middleName":"","lastName":"Takahashi","suffix":""},{"id":253877017,"identity":"eeda3d3e-be92-4414-8162-7fd773d38ebc","order_by":1,"name":"Miki Suenaga-Hiromori","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Miki","middleName":"","lastName":"Suenaga-Hiromori","suffix":""},{"id":253877019,"identity":"59b38d85-ae9e-439e-bec4-566439b8dbd6","order_by":2,"name":"Tomoki Ishii","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Tomoki","middleName":"","lastName":"Ishii","suffix":""},{"id":253877020,"identity":"7c5fa117-41ab-4767-9bff-92997d5b5329","order_by":3,"name":"Nadia Nur Shazana Binti Abu Talib Khan","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"Nur Shazana Binti Abu Talib","lastName":"Khan","suffix":""},{"id":253877021,"identity":"59ffc0cb-6203-425d-90fa-a8f4069eb63f","order_by":4,"name":"Tomoyo Mikami","email":"","orcid":"","institution":"Tohoku 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University","correspondingAuthor":false,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Yanbe","suffix":""},{"id":253877025,"identity":"551e1079-a1a1-4359-995b-da6b716b594e","order_by":8,"name":"Toshiyuki Waki","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Toshiyuki","middleName":"","lastName":"Waki","suffix":""},{"id":253877026,"identity":"81704c5c-799a-4ef9-abbf-a6293c0867e9","order_by":9,"name":"Toru Nakayama","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Nakayama","suffix":""},{"id":253877027,"identity":"d916229d-7958-4733-bc2a-22ea7304d49d","order_by":10,"name":"Riki Imaizumi","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Riki","middleName":"","lastName":"Imaizumi","suffix":""},{"id":253877028,"identity":"861e405f-65a6-43e9-a6f3-3ec4042832fe","order_by":11,"name":"Taro Yanai","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Taro","middleName":"","lastName":"Yanai","suffix":""},{"id":253877029,"identity":"79307a05-f450-4352-87dd-f1804fd6c89a","order_by":12,"name":"Kunishige Kataoka","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Kunishige","middleName":"","lastName":"Kataoka","suffix":""},{"id":253877030,"identity":"b8ea5a22-b95d-4d3d-83e2-4b98f3050438","order_by":13,"name":"Satoshi Yamashita","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Yamashita","suffix":""},{"id":253877031,"identity":"85ea1952-43aa-4f08-95a2-f5c69adf48af","order_by":14,"name":"Kohei Takeshita","email":"","orcid":"","institution":"RIKEN SPring-8 Center","correspondingAuthor":false,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Takeshita","suffix":""},{"id":253877032,"identity":"3c9d3d0a-a4ad-4328-b692-d99c8fd5daf6","order_by":15,"name":"Hiroaki Matsuura","email":"","orcid":"","institution":"RIKEN SPring-8 Center","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Matsuura","suffix":""},{"id":253877033,"identity":"98d88adc-b137-409e-8fb1-924e0185ccd3","order_by":16,"name":"Naoki Sakai","email":"","orcid":"","institution":"RIKEN SPring-8 Center","correspondingAuthor":false,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Sakai","suffix":""},{"id":253877034,"identity":"3e6d6bb8-4d33-4fee-8f4f-e8fd7ec45868","order_by":17,"name":"Masaki Yamamoto","email":"","orcid":"","institution":"RIKEN SPring-8 Center","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Yamamoto","suffix":""},{"id":253877035,"identity":"85186fda-88a9-46d1-a996-70bf3a1cb872","order_by":18,"name":"Haruhiko Yamaguchi","email":"","orcid":"https://orcid.org/0009-0004-6544-7265","institution":"Sumitomo Rubber Industries, Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Haruhiko","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":253877036,"identity":"0fd617e2-1285-4c85-9eb6-969196dab4a1","order_by":19,"name":"Yukino Miyagi-Inoue","email":"","orcid":"","institution":"Sumitomo Rubber Industries, Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Yukino","middleName":"","lastName":"Miyagi-Inoue","suffix":""},{"id":253877037,"identity":"e71de67d-3000-417e-9dc9-53209eb29906","order_by":20,"name":"Kazuhisa Fushihara","email":"","orcid":"","institution":"Sumitomo Rubber Industries, Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kazuhisa","middleName":"","lastName":"Fushihara","suffix":""},{"id":253877038,"identity":"af4b4041-3dde-49b7-9ab3-d56f31fb965d","order_by":21,"name":"Yuzuru Tozawa","email":"","orcid":"https://orcid.org/0000-0002-3885-1105","institution":"Saitama University","correspondingAuthor":false,"prefix":"","firstName":"Yuzuru","middleName":"","lastName":"Tozawa","suffix":""}],"badges":[],"createdAt":"2023-11-15 13:21:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3615345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3615345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59540813,"identity":"137efbe4-8f86-4037-a8a3-d90e2c159316","added_by":"auto","created_at":"2024-07-03 03:49:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":904353,"visible":true,"origin":"","legend":"\u003cp\u003ePolyisoprenoid biosynthesis by the WRP system. a, Reactions catalysed by PTs. The upper scheme represents the reactions catalysed by the conventional tPTs and cPTs. The lower scheme represents the reactions catalysed by modified cPTs that can use DMAPP and artificial prenyl diphosphates as a primer. b, Schematic in vitro protein expression workflow on the detergent-washed rubber particles with the wheat germ cell-free system.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/9711224dc41bb2f357013bd0.jpg"},{"id":59541403,"identity":"a0049583-9bcc-4b3c-9b23-d3da2b16d210","added_by":"auto","created_at":"2024-07-03 03:57:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1866460,"visible":true,"origin":"","legend":"\u003cp\u003eNR-sized polymer synthesis on WRPs by L- and M-cPTs from non-NR producing eukaryotes. a, Analysis of the proteins derived from the WRP system. After the in vitro translation, the reaction mixtures (Total) were separated into RP-containing (WRP) and aqueous fractions (Soluble) by ultracentrifugation, and proteins in each fraction were analysed on SDS-PAGE and stained with Coomassie brilliant blue (CBB). 1, vector control; 2, HRT1; 3, AtcPT8; 4, AtcPT5; 5, HRT1+HRBP; 6, AtcPT8-Lew1; 7, AtcPT5+Lew1. b, f, PT activities of WRPs with the recombinant protein(s) in the reaction with FPP and 14C-IPP as substrates. The activity is expressed as net IPP incorporation into the BuOH and T/H extracts, calculated by subtracting the values for the vector control WRPs from those with the WRPs carrying protein(s) as indicated. Results are the mean of three independent determinations ± S.D. *, lower value than the vector control. c., g. Radio-GPC analysis of 14C-labelled reaction products in the T/H extracts from PT assays with WRPs carrying protein(s) indicated. Elution peaks of commercially available polyisoprene standards (molecular weights 103, 104, and 106) are indicated at the top of the graph. d. Autoradiogram of reversedphase TLC developing enzymatic reaction products. Prenyl alcohols obtained by acid phosphatase treatment of the BuOH extracts were developed on reversed-phase TLC using acetone/water (39:1). Arrowheads indicate the positions of authentic standards: C20, all-(E)-geranylgeraniol; C35, all-(E)- heptaprenol; C55, tri-trans,hepta-cis-undecaprenol; C60, tri-trans,octa-cis-dodecaprenol. Ori., origin; S.F., solvent front. e. SDS-PAGE analysis of the proteins derived from the WRP system as Fig. 1a. 1, vector control; 2, HRT1; 3, TbCPT1; 4, HDS; 5, SRT1. h. Physical interaction analyses of cPT and cPTL through a SU-Y2H assay. Yeast strains harbouring proteins fused with N-terminal or C-terminal halves of split ubiquitin fragments were grown on SD(-WL) and SD(-WLH). A five-fold dilution series of cultures was spotted in each panel, with cell densities adjusted to be equivalent for each strain. P.C., positive control plasmid harbouring Ost1-Nub; V.C., empty vector as negative control; N, pPR3-N; SUC, pPR3-SUC.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/147a5ed74a053ed8879df7dd.jpg"},{"id":59540818,"identity":"8ed4a9f0-8a55-46c3-98d6-cde3e124b64a","added_by":"auto","created_at":"2024-07-03 03:49:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1681104,"visible":true,"origin":"","legend":"\u003cp\u003eNR-sized polymer synthesis on WRPs by engineered enzymes of NDPS1 and MlUPS. a, d, h, In vitro PT activities of WRPs with the recombinant protein(s) in the reaction with 14C-IPP and an oligoprenyl diphosphate indicated (a, d) or (E,E)-FPP (h) as a primer. Results are the mean of three independent determinations ± S.D. C5, DMAPP; C10, GPP; C15, (E,E)-FPP. *, lower value than the vector control. b, Comparisons of the hydrophobic pockets of NDPS1 and its mutants, NAt-GW and NHb-GW. The NDPS1 crystal structure (PDB ID: 7VPC) and structural models of the NDPS1 mutants constructed by AlphaFold2_advanced53 are shown by a ribbon diagram with a computationally predicted interior cavity of the hydrophobic cleft/pocket visualized as transparent surface representations. Key residues are virtually shown in ball and stick models. Yellow, helix-3 domain (H3) of NDPS1; orange, swapped residues derived from AtcPT4; green, swapped residues derived from HRT1; medium purple, helix-2 domain (H2). c. SDS PAGE analysis of the proteins derived from the WRP system as Fig. 1a. 1, vector control; 2, trNDPS1; 3, NAt; 4, NAt-GW; 5, NHb; 6, NHb-GW. e, i, Autoradiogram of reversed-phase TLC developing prenyl alcohols obtained by acid phosphatase treatment of the reaction products. Solvent system: acetone/water (19:1). Arrowheads: C20, all-(E)-geranylgeraniol; C35, all-(E)-heptaprenol; C55, tri-trans,hepta-cis undecaprenol; C60, tri-trans,octa-cis-dodecaprenol. Ori., origin; S.F., solvent front. f, j, Radio-GPC analysis of 14C-labelled reaction products in the T/H extracts from PT assays with WRPs carrying protein(s) indicated. Elution peaks of commercially available polyisoprene standards (molecular weights 103, 104, and 106) are indicated at the top of the graph. g, SDS-PAGE analysis of the proteins derived from the WRP system. Total, total reaction mixture; WRP, WRP-containing fraction; Soluble, aqueous fraction. 1, vector control; 2, HRT1; 3, MlUPS; 4, UHb.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/1e5bc841480adbe97ece0371.jpg"},{"id":59540815,"identity":"c97576bf-7b71-4195-a2c0-66b37634377a","added_by":"auto","created_at":"2024-07-03 03:49:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1901211,"visible":true,"origin":"","legend":"\u003cp\u003eStructural insights into the prenyl chain elongation in NAt. a, Close-up views of the structures of trNDPS1 (left) and NAt (right) shown by a ribbon diagram with a computationally predicted interior cavity of the hydrophobic cleft/pocket visualized as transparent surface representations. Key residues are virtually shown in ball and stick models. Yellow, helix-3 domain of NDPS1; orange, swapped residues derived from AtcPT4; medium purple, helix-2 domains. b, Comparison of structures of monomer subunits of the substrate binding form consisting of the NAt homodimer. The introduced helix-3 from AtcPT4 is highlighted in orange. The substrates (GSPP and IPP) are shown in ball and stick models. Magnesium ions are shown in sphere models. Interior cavities calculated by CASTp 3.0 server are shown as transparent surface representations. c, Heterogeneity of the introduced helix-3 in the substrate binding form of NAt. Top panels: Differences in the helix-3 of trNDPS1, NAt (apo), and its substrate-binding form [NAt (IPP+GSPP)]. Helix-3 in each monomer in the three dimer structures is highlighted and the remainder of the monomer structures are shown in transparent. Bottom panels: color-coded main-chain trace of each monomer according to the B-factor generated using the PyMOL program. Helix-3 moieties are highlighted as in the top panels.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/d1780afdf0e394955c009606.jpg"},{"id":59540817,"identity":"004c69dc-4005-4c2f-bd85-0e2d52d2834c","added_by":"auto","created_at":"2024-07-03 03:49:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1327065,"visible":true,"origin":"","legend":"\u003cp\u003eNR-sized polymer synthesis on WRPs by engineered enzymes of NDPS1 with artificial primer substrates. a, Schematic models of prenyl chain elongation in NDPS1 and its mutants on WRPs. The main structural domains comprising the hydrophobic pocket of NDPS1, helix-2 (purple), helix-3 (yellow), and b-strands (grey) are represented schematically. In the mutants, the swapped residues derived from AtcPT4 and HRT1 are indicated in orange and green, respectively. Dotted red arrows indicate a putative prenyl chain elongation pathway. An allylic prenyl diphosphate [GPP or (E,E)-FPP] and IPP are shown as substrates bound to the S1 and S2 sites, respectively. In NAt and NHb, the entire prenyl moiety of (E,E)- FPP cannot be accommodated in the hydrophobic pocket due to the bulky residue of Ile-106 (blue), and the nascent prenyl chain elongates outside the hydrophobic pocket. In NAt-GW, the nascent prenyl chain can elongate in the hydrophobic cleft due to the I106G substitution until the w-terminus of the prenyl chain reaches a putative bulky residue derived from AtcPT4 (black), whereas in NHb-GW the prenyl chain can elongate through the hydrophobic cleft due to the absence of a bottom formed by a bulky residue. b, In vitro PT activities of WRPs with the recombinant protein in the reaction with 14C-IPP and a primer substrate indicated. Results are the mean of three independent determinations ± S.D. P values were obtained using the two-tailed Student’s t-test. c. Weight average molecular weight, number average molecular weight, and polydispersity index of the reaction products calculated by the results of radio-GPC analysis of the T/H extracts in the assay with WRP introduced enzymes (Supplementary Fig. 8).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/40e8147a23d8efa7149a8ce2.jpg"},{"id":59541404,"identity":"96854ac3-9f3e-4b8a-b729-6d30f0f82516","added_by":"auto","created_at":"2024-07-03 03:57:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":527201,"visible":true,"origin":"","legend":"\u003cp\u003eTPI synthesis by HbSDSD introduced on WRPs. a, A structural model of the HbSDSD homodimer constructed by AlphaFold2_advanced shown by a ribbon diagram. Substrate binding motifs, the first Asp-rich motif (FARM) and the second Asp-rich motif (SARM), in each subunit are shown in magenta and cyan, respectively. b, SDS-PAGE analysis of the proteins derived from the WRP system. Total, total reaction mixture; WRP, WRP-containing fraction; Soluble, aqueous fraction. 1, vector control; 2, HRT1; 3, HbSDSD. c, In vitro PT activities of WRPs with the recombinant protein indicated in the reaction with 14C-IPP and an oligoprenyl diphosphate indicated as a primer. C15, (E,E)-FPP; C20, all-(E)- GGPP. Results are the mean of three independent determinations ± S.D. *, lower value than the vector control. d, Radio-GPC analysis of reaction products in the T/H extracts from PT assays with WRPs carrying protein indicated. Elution peaks of commercially available polyisoprene standards (molecular weights 103, 104, and 106) are indicated at the top of the graph.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/3a44bd395ac8cc38c5416a86.jpg"},{"id":59541823,"identity":"454d39e0-570a-4011-b5ef-cc72819c822a","added_by":"auto","created_at":"2024-07-03 04:05:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8688857,"visible":true,"origin":"","legend":"Article File","description":"","filename":"SuenagaHiromorietal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1_covered_862e96d2-cbfe-4fba-aead-5dc77328c8f2.pdf"},{"id":59540812,"identity":"8357da45-5a33-4560-9997-560f49cf2f6e","added_by":"auto","created_at":"2024-07-03 03:49:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":246227,"visible":true,"origin":"","legend":"","description":"","filename":"Methodsandinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/bf33582c3fd62097e68e9510.pdf"},{"id":59540819,"identity":"f124b717-7894-4fa8-9d2a-6e8e12083391","added_by":"auto","created_at":"2024-07-03 03:49:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7160008,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3615345/v1/687b3b0d5c556573f1fc13ff.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3615345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3615345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Natural rubber (NR) is a sustainable biopolymer consisting mainly of \u003ci\u003ecis\u003c/i\u003e-1,4-polyisoprene. Modifying an NR biosynthetic enzyme is a promising strategy to bioproduce novel polymers. Here, we have elucidated the NR biosynthetic mechanism and successfully developed novel enzymes that synthesise NR-sized polyisoprenes with unnatural substrates. NR is synthesised by a \u003ci\u003ecis\u003c/i\u003e-prenyltransferase (cPT) on rubber particles (RPs), NR-harbouring lipid monolayer membrane organelles. However, the key to NR biosynthesis is not specialised cPTs, but the proper arrangement of cPTs on RPs since cPTs from various non-NR-producing organisms, such as humans, synthesise NR when introduced into the RPs. A tomato cPT, which condenses only one isoprene unit, was engineered to synthesise novel NR-sized polyisoprenes with artificial substrates by modifying residues for product size determination. Furthermore, the introduction of a modified trans-prenyltransferase into RPs led to the synthesis of NR-sized \u003ci\u003etrans\u003c/i\u003e-1,4-polyisoprenes. This RP system could be used as a versatile platform for enzymatic polyisoprenoid synthesis.","manuscriptTitle":"Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 03:49:42","doi":"10.21203/rs.3.rs-3615345/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9af1781b-215f-48c8-9651-ab55bf80013d","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":26801337,"name":"Biological sciences/Biochemistry/Enzyme mechanisms"},{"id":26801338,"name":"Biological sciences/Biotechnology/Molecular engineering/Protein design"},{"id":26801339,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":26801340,"name":"Biological sciences/Structural biology/X-ray crystallography/Nanocrystallography"},{"id":26801341,"name":"Biological sciences/Biological techniques/Molecular engineering/Protein design"}],"tags":[],"updatedAt":"2025-02-20T14:57:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 03:49:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3615345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3615345","identity":"rs-3615345","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}