Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes

Background: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S. pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. Results: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. Conclusions: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.General Secretariat of Research and Technology (GSRT) {[}09-SYN-23-879]; grant SEE-ERA. NET PLUS {[}ERA 64/01]; grant KRIPIS {[}MIS 448840

Τροποποίηση του σακχαρομύκητα για την παραγωγή φυτικών τερπενίων και τον χαρακτηρισμό των βιοσυνθετικών του ενζύμων απο είδη Salvia: Φυτά Arabidopsis που υπερεκφράζουν επιλεγμένα αντιοξειδωτικά γονίδια παρουσιάζουν αντοχή στην οξειδωτική καταπόνηση και την βακτηριακή μόλυνση

Plants produce an enormous variety of low molecular weight compounds called secondary metabolites, through various biosynthetic pathways. Terpenoids and isoprenoids contribute more than 50,000 compounds to this chemical diversity which include numerous commercial flavors, fragrances and medicines. Artemisinin and taxol are such terpene-based drug compounds. In general, most useful terpenoids are produced in small quantities in plants, which has slowed considerably their commercial utilization. Research has recently targeted the development of microbial fermentative processes as an alternative approach. Saccharomyces cerevisiae is an amenable organism for metabolic engineering and the diversion of the metabolic machinery towards the overproduction of terpenoids compounds. Terpene synthases, the enzymes synthesizing terpenes utilize geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl-geranyl pyrophosphate (GGPP) as substrates. In yeast they are synthesized by the sterol biosynthetic pathway. Although, the whole genetic pathway is present in yeast, it is tightly regulated and the precursors are present in limited quantities. To overcome this problem, a yeast strain producing high sterol levels was identified and tested for its capacity to produce a monoterpene cineole. This was achieved by stable transformation of a plasmid carrying the cineole synthase gene, a monoterpene synthase from Salvia fruticosa, under the control of an inducible promoter, and facile detection of the terpene products as volatiles by Head Space - Solid Phase Microextraction (HS-SPME) coupled with Gas Chromatography/ Mass Spectrometry (GC/MS) analysis. The selected strain was targeted for further modifications. A mutant stabilized version (K6R) of HMG2 under the control of the inducible Galactose promoter was stably integrated into the HO locus to generate strain AM63. The modified strain produces on average 1.5 fold more cineole than the parental strain and exhibited reduced background volatile metabolites when transformed with Sf-CinSyn (RC). Further molecular engineering of AM63 yeast strain aimed to maximize terpene productivity without sacrificing cell viability which could hamper the biofermentation process. Two different modifications targeted the upregulation of FPP synthase encoded by ERG20 gene by GAL promoter integration into the yeast chromosome of AM68 strain, and deletion of one allele of erg9 gene encoding for a squalene synthase in the diploid AM70 strain resulting in further enhancement in terpene production. AM68 strains produces 3 fold more cineole, while AM70 cells produced 3.5 fold more sesquiterpenes than the parental strain. Additional modifications targeted the HMG1 gene by truncation of the N-terminus and expression of the one allele under a stable constitutive promoter. The increased yield of terpenes in yeast enabled the identification of several novel terpene synthases isolated from Salvia fruticosa (Greek sage) and Salvia pomifera. Four of them failed to yield any products when tested as bacterially expressed proteins. One clone was a monoterpene synthase producing mostly cineole, distinct from the previously identified canonical Salvia cineole synthase, while another one was a naturally truncated form which failed to yield any products. The two other genes encoded for sesquiterpene synthases, the first producing beta-farnesene and nerolidol and the second was a multiproduct enzyme synthesizing alpha-cubebene, alpha-copaene, trans-caryophyllene and delta-cadinene. Focusing on the terpene synthase molecule we tested whether additional modifications in the N-terminus of CS in the chloroplastic targeting sequence could further enhance product yield. The truncated SfCinS1(RR) and SfCinS1(RC) catalyzed the formation of multiple monoterpenes using the endogenous GPP pool as substrate with a significant peak of 1,8-cineole. However, SfCinS1(RC) was found to be more stable, efficient, with high activity during long incubation times. Additionally, using the two-hybrid system we screened a Salvia fruticosa glandular trichome library to identify interacting proteins to the Cineole monoterpene synthase. One of the interactors an, HSP90 when co-expressed with cineole synthase (CS) reproducibly increased product yield by 20%. Parallel work in the context of the PENED funded project requirements focused on the in vivo characterization of two recently isolated enzymes involved in secondary metabolism and plant defense by expressing them in Arabidopsis transgenic plants. When exposed to acrolein-induced oxidative stress Arabidopsis plants overexpressing a thioredoxin-peroxidase transgene, LeTpx1, exhibited less sensitivity than wild type plants. Correspondingly, the BI-GST, a plant GST-like protein inhibiting Bax lethality in yeast cells, and the LeTpx1 transgenes significantly increased plant resistance to the microbial pathogen, Pseudomonas syringe pv.tomato DC3000.Μια σημαντική κατηγορία φυσικών προϊόντων, τα τερπενοϊδή και ισοπρενοϊδή συνισφέρουν περισσότερες απο 50,000 ουσίες στην χημική ποικιλότητα των φυτών. Πολλά τερπένια έχουν προσελκύσει εμπορικό ενδιαφέρον για τις βιομηχανικές και ιατρικές τους χρήσεις. Ένα σημαντικό εμπόδιο στην επέκταση της χρήσης τους, είναι οι περιορισμένες ποσότητες που παράγουν τα φυτά. Ο Saccharomyces cerevisiae είναι οργανισμός που επιτρέπει την μεταβολική μηχανική με στόχο την παραγωγή φυτικών τερπενών. Οι συνθάσες τερπενίων είναι τα ένζυμα που συνθέτουν τερπένια αξιοποιώντας το διφωσφοπικό γερανύλιο (GPP), το διφωσφορικό φαρνεσύλιο (FPP) ή το διφωσφορικό γερανυλ-γερανύλιο (GGPP) ως υποστρώματα. Στη ζύμη συνθέτονται από το μονοπάτι βιοσύνθεσης στερολών. Ένα στέλεχος ζύμης που βρέθηκε να παράγει υψηλές στερόλες χρησιμοποιήθηκε για περαιτέρω τροποποιήσεις. Ένα μετάλλαγμα του γονιδίου HMG2 (K6R), με αυξημένη σταθερότητα, ενσωματώθηκε στο ΗΟ τόπο του γονιδιώματος κάτω από τον έλεγχο του επαγώμενου εκκινητή γαλακτόζης (στέλεχος ΑΜ63). Μια σειρά χαρακτηρισμένων και νέων συνθασών τερπενίων που απομονώθηκαν απο τή Salvia fruticosa (φασκόμηλο) εκφράστηκαν στο στέλεχος ΑΜ63 και το πατρικό του. Το τροποποιημένο στέλεχος παράγει κατά μέσο όρο 1,5 φορές περισσότερο από το πατρικό του και εκλύει μειωμένους πτητικούς μη-ειδικούς μεταβολίτες. Αυτό επιτρέπει τον χαρακτηρισμό τριών νέων συνθασών τερπενίων που στο παρελθόν δεν είχαν καταλυτική δράση όταν εκφράστηκαν ως βακτηριακές πρωτεΐνες. Μία απο αυτές ήταν μια συνθάση μονοτερπενίων που παράγει κύρια σινεόλη, αλλά διαφέρει απο την ορθόλογη συνθάση σινεόλης. Δύο άλλες κωδικοποιούν για σεσκιτερπενικές συνθάσες, η πρώτη παράγει β-φαρνεσίνη και νερολιδόλη ενώ ή δεύτερη δ-καδινένιο, α-κουμπεμπένιο, α-κοπαένιο και trans-καρυοφυλένιο. Περαιτέρω μοριακή μηχανική του στελέχους ΑΜ63 στόχευσε στη μεγιστοποίηση της παραγωγικότητας σε τερπένια χωρίς να θυσιαστεί η βιωσιμότητα του οργανισμού κατά την διαδικασία της βιοζύμωσης. Δύο διαφορετικές τροποποιήσεις στόχευσαν στην υπερέκφραση της FPP συνθάσης που κωδικοποιείται από το γονίδιο ERG20 με ενσωμάτωση του GAL εκκινητή στο χρωμόσωμα του ΑΜ68 στελέχους και την αδρανοποίηση ενός αλληλόμορφου του erg9 που κωδικοποιεί την συνθάση σκουαλενίου (στέλεχος ΑΜ70) με αποτέλεσμα την περαιτέρω αυξημένη παραγωγή τερπενίων. Το ΑΜ68 παράγει 3 φορές περισσότερη σινεόλη, ενώ το ΑΜ70 παράγει 3,5 φορές περισσότερα σεσκιτερπένια από το πατρικό τους. Περαιτέρω τροποποιήσεις στόχευσαν το HMG1 γονίδιο αποκόπτωντας το Ν-terminus και υπερεκφράζοντας το αλληλόμορφο με ισχυρό σταθερό εκκινητή. Εστιάζοντας στην μονοτερπενική συνθάση ελέγξαμε κατά πόσο οι περαιτέρω τροποποιήσεις στο Ν-τελικό άκρο της ΣΣ στις αλληλουχίες χλωροπλαστικής στόχευσης μπορούν να επιρέασουν περαιτέρω την παραγωγή. Επιπρόσθετα χρησιμοποιώντας μια βιβλιοθήκη δύο υβριδίων απομονώσαμε πρωτεΐνες που αλληλεπιδρούν με τη συνθάση σινεόλης. Μια από αυτές η HSP90 οταν εκφράζεται μαζί με την συνθάση σινεόλης οδηγεί σε αύξηση της παραγωγής κατα 30%. Παράλληλη δουλειά στα πλαίσια των απαιτήσεων του προγράμματος ΠΕΝΕΔ εστιάστηκε στον in-vivo χαρακτηρισμό δύο πρόσφατα απομονωθέντων ενζύμων που συμμετέχουν στο δευτερογενή μεταβολισμό και την άμυνα, σε φυτά Arabidopsis. Έκθεση στην ακρολεΐνη επάγει οξειδωτικό στρές. Τα φυτά Arabidopsis που υπερεκφράζουν την υπεροξειδάση θειορεδοξίνης LeTPX1 παρουσίασαν μικρότερη ευαισθησία απο τα αγρίου τύπου φυτά. Αντίστοιχα η BI-GST μια τρανσφεράση της γλουταθειόνης και η LeTPX1 αύξησαν σημαντικά την φυτική αντοχή στον παθογόνο μικροοργανισμό Pseudomonas syringae pv.tomato DC3000

Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids

Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

Abstract Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.</p

Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric substrate

Synthetic biology efforts for the production of valuable chemicals are frequently hindered by the structure and regulation of the native metabolic pathways of the chassis. This is particularly evident in the case of monoterpenoid production in Saccharomyces cerevisiae, where the canonical terpene precursor geranyl diphosphate is tightly coupled to the biosynthesis of isoprenoid compounds essential for yeast viability. Here, we establish a synthetic orthogonal monoterpenoid pathway based on an alternative precursor, neryl diphosphate. We identify structural determinants of isomeric substrate selectivity in monoterpene synthases and engineer five different enzymes to accept the alternative substrate with improved efficiency and specificity. We combine the engineered enzymes with dynamic regulation of metabolic flux to harness the potential of the orthogonal substrate and improve the production of industrially-relevant monoterpenes by several-fold compared to the canonical pathway. This approach highlights the introduction of synthetic metabolism as an effective strategy for high-value compound production