Carotenoid cleavage dioxygenase genes negatively regulate 2-phenylethanol biosynthesis in yeast

Carotenoid cleavage dioxygenase genes negatively regulate 2-phenylethanol biosynthesis in yeast | 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 Research Article Carotenoid cleavage dioxygenase genes negatively regulate 2-phenylethanol biosynthesis in yeast Xiaowei GONG, Fan Li, Guanghui Ma, Xiulin Han, Mengliang Wen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9333171/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 This study reveals for the first time that overexpression of the plant-derived CCD genes NtCCD1-2 , NtCCD10 , ZmCCD1 , and CaCCD4C significantly inhibits 2-phenylethanol biosynthesis in engineered β -ionone-producing Saccharomyces cerevisiae strains ( p <0.001), whereas PhCCD1 and NtCCD1-3 exhibit no inhibitory effect. Using comparative transcriptomics and phenotypic analysis, we identified a novel regulatory mechanism by which these inhibitory CCD genes suppress 2-phenylethanol production through impairing yeast growth and downregulating its de novo biosynthetic pathway. Mechanistically, inhibitory CCD genes trigger biotic stress, downregulate the ribosome and multiple metabolic pathways, and upregulate the MAPK signaling pathway, thereby reducing cell biomass. In the inhibitory group, the high cell density-induced transcription factor Aro80 is downregulated, leading to the repression of aro10 and a series of genes involved in 2-phenylethanol synthesis, together with lpd1 and multiple thiamine regulon genes. These combined effects result in decreased 2-phenylethanol yield. This work deepens our understanding of the crosstalk between 2-phenylethanol and β -ionone biosynthesis in yeast, and offers a novel strategy for coordinating yeast growth and terpenoid metabolism. Carotenoid cleavage dioxygenase Saccharomyces cerevisiae 2-Phenylethanol Inhibition Molecular mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Key Points • Certain plant-derived CCD genes inhibit 2-PE production and impair yeast cell growth. • Inhibitory CCD genes induce biotic stress and decrease cell biomass in yeast. • The de novo 2-PE synthesis pathway is downregulated by inhibitory CCD genes. Introduction Carotenoids are a class of lipophilic isoprenoid compounds, typically consisting of a 40-carbon backbone (Hirschberg 2001 ; Maresca et al. 2008 ). Carotenoids are synthesized in all photosynthetic organisms (such as cyanobacteria, algae, and plants) and some non-photosynthetic organisms (such as fungi and bacteria) (Nisar et al. 2015 ). In plants, carotenoids are involved in various biological processes, including photosynthesis, photomorphogenesis, photoprotection, and development (Nisar et al. 2015 ). Apocarotenoids are oxidation cleavage products of carotenoids, formed through either enzymatic or non-enzymatic processes, that play roles in regulating plant development, participating in stress responses, and attracting pollinators and seed-dispersing organisms in plants (Hou et al. 2016 ). The enzymatic cleavage of carotenoids is mediated by carotenoid cleavage dioxygenases (CCDs). In plants, the CCD family includes 11 members: CCD1, CCD2, CCD4, CCD7, CCD8, CCD10, NCED2, NCED3, NCED5, NCED6, and NCED9. The latter five members belong to the NCED (nine-cis-epoxy carotenoid dioxygenase) subfamily (Auldridge et al. 2006 ; Frusciante et al. 2014 ; Zhong et al. 2020 ; Li et al. 2022 ). Studies on CCD1 from various species have shown that it has a broad substrate specificity. CCD1 can cleave a variety of linear, monocyclic, and bicyclic carotenoids at different positions on the carbon backbone, generating apocarotenoids such as β -ionone, pseudoionone, 6-methyl-5-hepten-2-one, geranylacetone, 3-hydroxy- β -ionone, geranial, and β -cyclocitral (Vogel et al. 2008 ; Ilg et al. 2014 ; Meng et al. 2019 ). It has been reported that CCD2 from Crocus sativus and Crocus ancyrensis is involved in crocin synthesis and has activity to cleave the C7-C8 (C7’-C8’) double bond of zeaxanthine (Frusciante et al. 2014 ; Ahrazem et al. 2016 ). CCD4 typically cleaves carotenoids at the C9-C10 (C9’-C10’) double bond to generate β -ionone, but can also asymmetrically cleave the C7-C8 double bond of β -cryptoxanthin, zeaxanthine, or β -carotene to produce β -citraurin, apo-8’- β -carotenal, β -cyclocitral, and others (Huang et al. 2009 ; Ma et al. 2013 ; Rodrigo et al. 2013 ; Zhang et al. 2016 ). CCD7 and CCD8 are involved in the synthesis of the plant hormone strigolactones (Alder et al. 2012 ). CCD10 catalyzes the cleavage of carotenoids at the C9-C10 (C9’-C10’) and C5-C6 (C5’-C6’) double bonds, and in maize, it enhances plant tolerance to low-phosphorus stress (Zhong et al. 2020 ; Li et al. 2022 ). NCEDs are involved in the synthesis of abscisic acid (ABA) (Tan et al. 2003 ). CCDs participate in plant growth, development, and stress responses through their cleavage products, apocarotenoids (Moreno et al. 2021 ). β -Ionone is involved in a plant herbivore interaction (Wei et al. 2011 ). In planta, β -cyclocitral participates in 1 O2 signaling, enhances high light and drought tolerance, and serves as a root growth regulator (D’Alessandro et al. 2018; D’Alessandro et al. 2019; Dickinson et al. 2019 ). Strigolactones, a class of apocarotenoids, act as germination stimulants for parasitic plants, root-derived symbiotic signals for arbuscular mycorrhizal fungi, and inhibitors of shoot branching (Cook et al. 1966 ; Akiyama et al. 2005 ; Gomez-Roldan et al. 2008 ). ABA as plant hormone, performs many important functions in plant, such as root and shoot development, hypocotyl elongation, fruit development and ripening, responses to high salinity, drought and nutrient depletion (Gόmez-Cadenas et al. 2000; Hasegawa et al. 2000 ; Iuchi et al., 2001 ; Galpaz et al. 2008 ; Felemban et al. 2019 ). 2-Phenylethanol is a quorum-sensing molecule in yeast that facilitates the transition from a unicellular to a filamentous morphology (Chen and Fink 2006 ). The main biosynthetic pathways for 2-phenylethanol in yeast are the Ehrlich pathway and the de novo pathway. The Ehrlich pathway is the primary route for 2-phenylethanol biosynthesis in yeast and involves three steps: transamination, decarboxylation, and reduction. In the first step, the transaminases Aro8 and Aro9 catalyze the reaction; in the second step, the aromatic decarboxylase Aro10 and the pyruvate decarboxylases Pdc1, Pdc5, and Pdc6 catalyze the reaction; and in the third step, the reduction is mainly catalyzed by the reductases Adh1, Adh2, Adh3, Adh4, Adh5, and Sfa1 (Iraqui et al. 1998 ; Dickinson et al. 2003 ). In Saccharomyces cerevisiae , the biosynthesis of 2-phenylethanol is also regulated by other enzymes and regulatory factors. Dickinson et al. ( 2003 ) found that 2-phenylethanol production was reduced in an lpd1 gene knockout strain, suggesting that Lpd1 is involved in 2-phenylethanol synthesis. Lpd1 is involved in the citrate cycle, and the carbon flux in the fermentative pathway can be increased by fine-tuning the expression of the lpd1 gene (Chen et al. 2025 ). The decarboxylation step in the Ehrlich pathway for 2-phenylethanol synthesis depends on thiamine pyrophosphate (Dai et al. 2021 ), so the thiamine-regulating gene this is indirectly involved in 2-phenylethanol synthesis. In our previous study, we found that the 2-phenylethanol production increased when thi4 gene was overexpressed (Gong et al. 2022 ). It has been reported that the transcriptional activator Aro80 plays a role in activating the transcription of aro9 and aro10 genes during 2-phenylethanol synthesis (Iraqui et al. 1999 ). The transcription of aro9 and aro10 is suppressed by nitrogen catabolite repression (NCR), while Gln3 and Gat1 can activate the expression of these genes through the intracellular amino acid sensing system (Target of Rapamycin pathway, TOR pathway) (Cooper 2002 ; Dai et al. 2021 ). Overexpression of gln3 and gat1 in S. cerevisiae resulted in a significant increase in 2-phenylethanol production (Chen et al. 2017 ), whereas knockout of gln3 led to significant decrease (Xia et al. 2022 ). It has been reported that knocking out the mig1 gene or overexpressing the cat8 gene increases the expression levels of aro9 and aro10 , leading to higher 2-phenylethanol production in S. cerevisiae (Wang et al. 2017 ), indicating that Mig1 negatively regulates 2-phenylethanol synthesis, while Cat8 positively regulates it. Currently, there have been no reports indicating that expression of CCDs genes in S. cerevisiae inhibits 2-phenylethanol synthesis. In this study, an unexpected phenomenon was found for the first time that genes NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C from plants inhibited 2-phenylethanol production when they were expressed in S. cerevisiae , but PhCCD1 and NtCCD1-3 cannot. The biomass of yeast was consistent with the production of 2-phenylethanol. Transcriptome analysis was carried out to elucidate the molecular mechanism. This study offers valuable insights into the biosynthesis of 2-phenylethanol and β -ionone using yeast as a host organism. Additionally, it presents a novel strategy for regulating yeast growth. Materials and methods Strains, plasmids, and reagents The key strains and plasmids utilized in this work are summarized in Table 1 . The PhCCD1 , ZmCCD1 , and CaCCD4C genes originate from Petunia hybrida , Zea mays , and Crocus ancyrensis , respectively. In contrast, the NtCCD1-3 , NtCCD1-2 , and NtCCD10 genes are all derived from Nicotiana tabacum . Table 1 Strains and plasmids used in this study Strains/plasmids Description Source Strians BY4741 MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Yuchun Biology DH5 α F − φ80 lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk − , mk + ) phoA, supE44 thi-1 gyrA96 relA1 λ − Vazyme A10 BY4741 ura3 ::P GAP -crtE-T CYC1 ,P GAP -crtYB-T CYC1 ,P GAP -crtI-T CYC1 Gong et al., 2022 A10- δ -PhCCD1 (Ph1) A10 δ ::P ADH1 -PhCCD1-T CYC1 , P Leu2 -Leu2-T Leu2 This study A10- δ -NtCCD1-3 (N3) A10 δ ::P ADH1 -NtCCD1-3-T CYC1 , P Leu2 -Leu2-T Leu2 This study A10- δ -NtCCD1-2 (N1) A10 δ ::P ADH1 -NtCCD1-2-T CYC1 , P Leu2 -Leu2-T Leu2 This study A10- δ -NtCCD10 (N10) A10 δ ::P ADH1 -NtCCD10-T CYC1 , P Leu2 -Leu2-T Leu2 This study A10- δ -ZmCCD1 (Zm1) A10 δ ::P ADH1 -ZmCCD1-T CYC1 , P Leu2 -Leu2-T Leu2 This study A10- δ -CaCCD4C (Ca4) A10 δ ::P ADH1 -CaCCD4C-T CYC1 , P Leu2 -Leu2-T Leu2 This study Plasmids tpLADH1 ColE1 origin, F1 origin, P ADH1 , P LEU -LEU2-T LEU , Amp R Wang et al., 2021 tpLADH1-PhCCD1 tpLADH1 carrying P ADH1 -PhCCD1-T CYC1 , P LEU -LEU2-T LEU This study tpLADH1-NtCCD1-3 tpLADH1 carrying P ADH1 -NtCCD1-3-T CYC1 , P LEU -LEU2-T LEU This study tpLADH1-NtCCD1-2 tpLADH1 carrying P ADH1 -NtCCD1-2-T CYC1 , P LEU -LEU2-T LEU This study tpLADH1-NtCCD10 tpLADH1 carrying P ADH1 -NtCCD10-T CYC1 , P LEU -LEU2-T LEU This study tpLADH1-ZmCCD1 tpLADH1 carrying P ADH1 -ZmCCD1-T CYC1 , P LEU -LEU2-T LEU This study tpLADH1-CaCCD4C tpLADH1 carrying P ADH1 -CaCCD4C-T CYC1 , P LEU -LEU2-T LEU This study pColdTF ColE1 origin, His-Tag, lac I, Amp R TaKaRa pColdTF- δ -PhCCD1-LEU2 Derived from pColdTF, contains PhCCD1 and Leu cassette, delta homologous This study pColdTF- δ -NtCCD1-3-LEU2 Derived from pColdTF, contains NtCCD1-3 and Leu cassette, delta homologous This study pColdTF- δ -NtCCD1-2-LEU2 Derived from pColdTF, contains NtCCD1-2 and Leu cassette, delta homologous This study pColdTF- δ -NtCCD10-LEU2 Derived from pColdTF, contains NtCCD10 and Leu cassette, delta homologous This study pColdTF- δ -ZmCCD1-LEU2 Derived from pColdTF, contains ZmCCD1 and Leu cassette, delta homologous This study pColdTF- δ -CaCCD4C-LEU2 Derived from pColdTF, contains CaCCD4C and Leu cassette, delta homologous This study β -Ionone (> 97%) was sourced from Sigma-Aldrich. pEASY ® -Basic Seamless Cloning and Assembly Kit and PrimeScript™ RT reagent Kit were obtained from TaKaRa. TIANgel Midi Purification Kit was obtained from TianGen Biotech Co., Ltd. (Beijing, China). E.Z.N.A. ® Plant RNA Kit and 2×TSINGKE ® Master qPCR Mix (SYBR Green I) Kit were obtained from Omega Bio-Tek and Tsingke Biotechnology Co., Ltd. (Beijing, China), respectively. Dodecane (analytical standard) and other reagents were purchased from aladdin (Shanghai, China). Construction of β -ionone producing yeast strains The β -carotene producing strain A10 was used as the start strain (Gong et al., 2022 ). PhCCD1 , NtCCD1-3 , NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes which were codon-optimized for S. cerevisiae expression, were selected, synthesized by GenScript (Nanjing, China), and then cloned into the plasmid tpLADH1 to obtain tpLADH1-PhCCD1, tpLADH1-NtCCD1-3, tpLADH1-NtCCD1-2, tpLADH1-NtCCD10, tpLADH1-ZmCCD1 and tpLADH1-CaCCD4C, respectively. Strain A10- δ -PhCCD1 was used as the case for elaborating the construction process of the β -ionone producing yeast strains. The delta locus was selected for PhCCD1 gene integration. The PhCCD1 cassette containing promoter ADH1 , PhCCD1 gene, and terminator CYC1 , was amplified from plasmid tpLADH1-PhCCD1 by using primers CCDs-F and CCDs-R. Then, selection marker LEU2 cassette was amplified from the tpLADH1 plasmid with primers LEU- δ -F and LEU- δ -R, and the δ -up and δ -down fragments were amplified from S. cerevisiae BY4741 genome using primers δ- up-F/ δ- up-R and δ- down-F/ δ- down-R, respectively. The PhCCD1 cassette, the selection marker LEU2 cassette, the δ -up and δ -down fragments, as well as the Eco R I linearized pColdTF plasmid were infused with pEASY ® -Basic Seamless Cloning and Assembly Kit, and the transformants were screened on LB solid medium containing 50 µg/mL ampicillin. The recombinant plasmid pColdTF- δ -PhCCD1-LEU2 was verified by Eco R I digestion and sequencing. The pColdTF- δ -PhCCD1-LEU2 plasmid was digested by Pst I and Bam H I to obtain the δ -up-P ADH1 -PhCCD1-T CYC1 -P LEU -LEU2-T LEU - δ -down fragment, which was purified and transformed into strain A10 by chemical transformation (Chen et al. 1992 ). Finally, the transformants were screened by incubating on leucine-deficient synthetic agar plate at 30°C, and verified by sequencing. Several correct transformants named A10- δ -PhCCD1 8#, A10- δ -PhCCD1 14# and A10- δ -PhCCD1 21# respectively, were used for the subsequent experiments. Strains A10- δ -NtCCD1-3 (transformants 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8# and 9#), A10- δ -NtCCD1-2 (transformants 2# and 3#), A10- δ -NtCCD10 (transformants 2# and 3#), A10- δ -ZmCCD1 (transformants 8#, 9#, 10#, 11# and 12#) and A10- δ -CaCCD4C (transformants 1#, 7#, 8#, 10# and 11#) were constructed with the same method as A10- δ -PhCCD1. All primers used in this study are shown in Table S1 , and all strains and plasmids used in this study are shown in Table 1 . Fermentation and volatile extraction To analyze the volatile compounds via headspace solid-phase micro-extraction gas chromatography-mass spectrometry (SPME-GC-MS), various transformants of strains A10- δ -PhCCD1, A10- δ -NtCCD1-3, A10- δ -NtCCD1-2, A10- δ -NtCCD10, A10- δ -ZmCCD1, and A10- δ -CaCCD4C were cultured on YPD solid medium at 28°C for 5 days. A 0.15 g cell pellet was collected from each strain for SPME-GC-MS analysis. The control strain, A10, was treated in the same manner. Strain A10 was inoculated into 20 mL uracil-deficient medium and incubated at 28°C with shaking at 200 rpm for 48 h. Strains A10- δ -PhCCD1 (8# and 14#), A10- δ -NtCCD1-3 (1# and 9#), A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#), and A10- δ -CaCCD4C (1# and 11#) were inoculated into 20 mL leucine-deficient medium and incubated under the same conditions as A10. Afterward, 4 mL of each culture and 5 mL of dodecane were transferred to 100 mL fresh YPD medium and incubated for 2 days at 28°C with shaking at 200 rpm. After fermentation, the organic phase was separated by centrifugation at 21,734 × g for 10 min, then dehydrated with anhydrous sodium sulfate before gas chromatography-mass spectrometry (GC-MS) analysis for β -ionone quantification. The aqueous phase was collected for 2-phenylethanol quantification using high-performance liquid chromatography (HPLC). The cells were harvested and lyophilized for dry weight measurement to determine the biomass. Three biological replicates were performed for each sample. SPME-GC-MS analysis of volatile compounds The volatile compounds produced by A10- δ -PhCCD1, A10- δ -NtCCD1-3, A10- δ -NtCCD1-2, A10- δ -NtCCD10, A10- δ -ZmCCD1, A10- δ -CaCCD4C, and the control strain A10 were analyzed using SPME-GC-MS. A 65 µm polydimethylsiloxane / divinylbenzene SPME fiber (Supelco, USA) was placed into a headspace vial containing 0.15 g of cell pellet. The vial was stirred at 80°C and 250 rpm for 20 min. The volatile compounds on the fiber were analyzed by GC-MS (Agilent 5977B, USA) equipped with a DB-5MS column (Agilent, USA; 30 m × 0.25 mm ID, 0.25 µm film thickness), using helium as the carrier gas at a flow rate of 2 mL/min. The temperature program was as follows: 40°C for 1 min, ramped to 250°C at 2°C/min, and held at 250°C for 10 min. Electron ionization was performed at 70 eV, with a mass range of m/z 30–500 and an ion source temperature of 200°C. Volatile compounds were identified using the NIST 2.0 spectral library, and β -ionone was confirmed by comparing the mass spectrum and chromatographic behavior with that of an authentic standard. β -ionone and 2-phenylethanol quantification β -ionone production by different strains was quantified using GC-MS (Agilent 5977B, USA) equipped with a DB-5MS column (Agilent, USA; 30 m × 0.25 mm ID, 0.25 µm film thickness), with helium as the carrier gas at a flow rate of 2 mL/min. A 1 µL sample was injected. The temperature program started at 100°C, held for 2 min, then ramped to 210°C at 10°C/min, followed by an increase to 290°C at 20°C/min, and held at 290°C for 5 min. Electron ionization was performed at 70 eV, with a mass range of m/z 40–400 and an ion source temperature of 200°C. β -ionone quantification was carried out using the external standard method. A standard curve was prepared by dissolving β -ionone in dodecane and recording the peak areas at different concentrations. β -ionone in the dodecane extraction liquid from the samples was detected by GC-MS, and the concentration of β -ionone in each sample was calculated using the standard curve. Finally, the titers and yields of β -ionone were determined based on the volume of dodecane used for extraction and the dry cell weight (DCW). 2-Phenylethanol production by different strains was quantified using an HPLC system (Agilent 1260, USA) equipped with an Acclaim Explosives E2 column (4.6 mm × 250 mm, 5 µm, 120 Å; Thermo Scientific). A 10 µL sample was injected, and Isogradient elution was performed using a methanol / H 2 O solution (70:30, v/v) for 20 min at 25°C and a flow rate of 0.5 mL/min. 2-Phenylethanol was monitored at 210 nm, and its concentration was determined by integrating the calibration curves from the standards. Each sample was measured in triplicate. Real-time quantitative PCR analysis of CCD genes Strains A10- δ -PhCCD1 (8# and 14#), A10- δ -NtCCD1-3 (1# and 9#), A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#) and A10- δ -CaCCD4C (1# and 11#) cells pellets within logarithmic growth phase were collected and used for real-time quantitative PCR (qPCR) assay. Total RNA extraction, cDNA synthesis, and qPCR were carried out according to E.Z.N.A. ® Plant RNA Kit, PrimeScript™ RT reagent Kit, 2×TSINGKE ® Master qPCR Mix (SYBR Green I) Kit, respectively. Primers PhCCD1-qPCR-F / PhCCD1-qPCR-R, NtCCD1-3-qPCR-F / NtCCD1-3-qPCR-R, NtCCD1-2-qPCR-F / NtCCD1-2-qPCR-R, NtCCD10-qPCR-F / NtCCD10-qPCR-R, ZmCCD1-qPCR-F / ZmCCD1-qPCR-R, as well as CaCCD4C-qPCR-F / CaCCD4C-qPCR-R were used for PhCCD1 , NtCCD1-3 , NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes amplified respectively, and Act1-qPCR-F and Act1-qPCR-R for the actin gene ( act1 ) amplified. Differences in the expression of the CCD genes and act1 gene were calculated according to the 2 -ΔΔCT method (Livak and Schmittgen, 2001 ) using the act1 gene as the reference. Phylogenetic analysis, sequence alignment The amino acid sequences of PhCCD1 (AAT68189), NtCCD1-3 (NP_001312918), NtCCD1-2 (AHH25650), NtCCD10 (XP_016482179), ZmCCD1 (ABF85668), CaCCD4C (AKN09911), AtCCD1 (CAA06712), OfCCD1 (BAJ05401), VvCCD1 (AAX48772), RdCCD1 (ABY47994), CsCCD2 (AIG94929), CangCCD2 (ALM23546), CsCCD4 (ACD62476), AtCCD4 (AAM97019), CmCCD4 (ABY60885), AtNCED3 (NP_188062), AtNCED9 (NP_177960), AtNCED5 (NP_174302), VP14 (O24592), AtNCED6 (NP_189064), ZmCCD7 (NP_001183928), AtCCD7 (AEC10494), MmRPE65 (AAL01119), CsCCD8a (AIF27229), AtCCD8 (NP_195007), ZmCCD10a (XP_020396187), and OsZAS (EAZ36971) were retrieved from the NCBI protein database for phylogenetic tree construction. Multiple protein sequence alignments were performed using CLUSTAL X (Thompson et al. 1997 ). A neighbor-joining phylogenetic tree was generated with 1000 bootstrap replications using MEGA X, based on the aforementioned multiple sequence alignments (Kumar et al. 2018 ). Sequence alignment was performed using the amino acid sequences of NtCCD1-2, NtCCD10, ZmCCD1, CaCCD4C, PhCCD1, NtCCD1-3, and 9-cis-epoxycarotenoid dioxygenase viviparous 14 (VP14) with Espript 3.0 (Robert and Gouet 2014 ). The VP14 model (PDB 3NPE) was selected to depict its secondary structure. Product inhibition test β -cyclocitral and dihydroactinidiolide, which may be the metabolites preventing yeast from producing 2-phenylethanol, were screened through the SPME-GC-MS analysis. Strains A10, A10- δ -NtCCD1-3 9# (N3) as well as A10- δ -NtCCD1-2 2# (N1) were selected for verification test. Strains A10 was inoculated into 20 mL uracil-deficient medium, and incubated at 28°C with shaking at 200 rpm for 48 h, while N3 and N1 were inoculated into 20 mL leucine-deficient medium, which were incubated as A10. After that, 4 mL culture above were transferred to 100 mL fresh YPD medium with different concentration of β -cyclocitral or dihydroactinidiolide respectively, and finally incubated for 2 days at 28°C with shaking at 200 rpm. Four gradients of 0, 1, 5 and 25 mg/L were set for the final concentrations of the two candidate metabolites. After fermentation, the aqueous phase for 2-phenylethanol quantification by HPLC analysis was collected by centrifuging at 21,734 × g for 10 min. Three biological replicates were carried out for each sample. Sample preparation for transcriptomic sequencing According to the quantitative measurement results of β -ionone and 2-phenylethanol produced by different transformants, combined with the results of CCDs gene expression level, transformants with the similar 2-phenylethanol yields were selected in the inhibition group to explore the molecular mechanism of CCDs inhibiting 2-phenylethanol synthesis in S. cerevisiae , including A10- δ -NtCCD1-2 2# (N1), A10- δ -NtCCD10 3# (N10), A10- δ -ZmCCD1 8# (Zm1) and A10- δ -CaCCD4C 1# (Ca4). The 2-phenylethanol produced by different transformants were similar to each other in the non-inhibited group, so the transformants with high β -ionone yield and high CCD genes expression level were selected, including A10- δ -PhCCD1 14# (Ph1) and A10- δ -NtCCD1-3 9# (N3). Strain A10 was set as the control. To determine the incubated time of samples for transcriptome analysis, A10, N3 and N1 strains were selected to carry out the experiments. To determine the yeast samples’ fermentation time for transcriptome analysis, five fermentation time points 0, 2, 4, 6, and 7 d were set, and the production of β -ionone and 2-phenylethanol producing by each strain was determined. According to the β -ionone and 2-phenylethanol results, cell samples of A10, Ph1, N3, N1, N10, Zm1 and Ca4 incubated for 2 d were selected and collected for transcriptome analysis and biomass determination. The preparation of transcriptome analysis samples was as follows. Strains A10 was inoculated into 20 mL uracil-deficient medium, and incubated at 28°C with shaking at 200 rpm for 48 h, and Ph1, N3, N1, N10, Zm1 and Ca4 were inoculated into 20 mL leucine-deficient medium, which were incubated as A10. After that, 6 mL culture above were transferred to 150 mL fresh YPD medium respectively, and finally incubated for 2 days at 28°C with shaking at 200 rpm. After fermentation, cell pellets for transcriptome analysis and biomass determination were collected by centrifuging at 8,000 rpm and 4°C for 10 min, and the cell pellets were frozen in liquid nitrogen for 20 min and used for transcriptome analysis, while the aqueous phase was collected for 2-phenylethanol quantification. Three biological replicates were carried out for each sample. Transcriptome analysis Total RNA extracted from A10, Ph1, N3, N1, N10, Zm1 and Ca4 was used for sequencing with an Illumina HiSeq 4000 platform. The clean reads were mapped to the genome of S. cerevisiae S288C (GCF_000146045.2) using HISAT2 (Kim et al. 2015 ) with the default settings. Differential expression analysis were carried out using DESeq2 (Love et al. 2014 ). Differentially expressed genes (DEGs) between two samples were identified according to the conditions of |log 2 (fold change) | ≥ 1 and a false discovery rate (FDR) < 0.05. Log 2 (fold change) ≥ 1 was the up-regulated genes' standard, and values ≤ − 1 was the down-regulated genes'. Heat map analysis of gene expression levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the online data analysis platform OmicShare ( www.omicshare.com/tools ). In the heat map analysis, the value of the individual sample was shown by Z-scores, and the Z-scores were normalized values by scaling the row values of the gene expression levels. Results NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes inhibits 2-phenylethanol production in engineered S. cerevisiae Previously, we found that the delta integrating site for NtCCD1-3 gene in S. cerevisiae was suitable for promoting the yield of β -ionone (Gong et al. 2022 ). Therefore, the delta integrating site was selected for PhCCD1 , NtCCD1-3 , NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes integration in S. cerevisiae to construct β -ionone producing strains A10- δ -PhCCD1, A10- δ -NtCCD1-3, A10- δ -NtCCD1-2, A10- δ -NtCCD10, A10- δ -ZmCCD1 and A10- δ -CaCCD4C, respectively. The yield of β -ionone produced by different transformants of strains was determined by SPME-GC-MS, and the transformants producing higher yield of β -ionone were screened preliminarily (Fig. S1 , S 2, S3, S4, S5 and S6). The transformants A10- δ -PhCCD1 14#, A10- δ -NtCCD1-3 9# as well as A10- δ -NtCCD1-2 3# produced higher yield of β -ionone. Meanwhile, all the transformants could generate β -ionone (peak 3), indicating that the four CCDs above all could cleave 9,10 (9’, 10’) double bonds. Besides A10- δ -NtCCD10, other transformants all could produce geranylacetone (peak 2), and the transformants of strains A10- δ -PhCCD1, A10- δ -NtCCD1-3 and A10- δ -ZmCCD1 could synthesize 6-methyl-5-heptene-2-one (MHO) (peak 4). Different from other strains, A10- δ -PhCCD1 transformants all could produce pseudoionone (peak 5). In addition, all the transformants could give birth to 2-phenylethanol (peak 1). Confusingly, the yield of 2-phenylethanol in transformants of A10- δ -PhCCD1 and A10- δ -NtCCD1-3 was similar to that of the control strain A10, while the yield of 2-phenylethanol in transformants of A10- δ -NtCCD1-2, A10- δ -NtCCD10, A10- δ -ZmCCD1 and A10- δ -CaCCD4C was much lower than that of A10. The results suggested that the integrated expression of NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes in S. cerevisiae could inhibit the synthesis of 2-phenylethanol, while PhCCD1 and NtCCD1-3 genes had no such effect on 2-phenylethanol synthesis. To verify the conclusion above, the β -ionone and 2-phenylethanol generated by A10- δ -PhCCD1 (8# and 14#), A10- δ -NtCCD1-3 (1# and 9#), A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#) and A10- δ -CaCCD4C (1# and 11#) was quantified by GC-MS. The titer and yield of β -ionone in A10- δ -PhCCD1 14# and A10- δ -NtCCD1-3 9# were significantly higher than those of A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#) and A10- δ -CaCCD4C (1# and 11#) (Fig. 1 ), indicating that PhCCD1 and NtCCD1-3 may have higher activity of cleaving β -carotene to produce β -ionone than NtCCD1-2, NtCCD10, ZmCCD1 as well as CaCCD4C. The titer and yield of β -ionone in A10- δ -PhCCD1 (8# and 14#) exhibited a positive correlation with the expression levels of the PhCCD1 gene, a trend that was similarly observed in A10- δ -NtCCD1-3 (1# and 9#), A10- δ -NtCCD1-2 (2# and 3#), as well as A10- δ -CaCCD4C (1# and 11#) (Fig. 1 and Fig. 2 ). The reason may be that CCD enzymes catalyze the cleavage of β -carotene to produce β -ionone; thus, a higher expression level of the CCD gene leads to increased synthesis of CCD proteins, which in turn results in higher β -ionone production. The titer and yield of 2-phenylethanol in A10- δ -PhCCD1 (8# and 14#) were similar to that of the control strain A10, and those of A10- δ -NtCCD1-3 (1# and 9#) were even higher than those of A10 (Fig. 1 ). While, the titer and yield of 2-phenylethanol in A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#) and A10- δ -CaCCD4C (1# and 11#) were all significantly lower ( p < 0.05) than those of A10 (Fig. 1 ). Specifically, the titer and yield of 2-phenylethanol in A10 were 14.50 mg/L and 2.69 mg/g DCW, respectively. For the strains A10- δ -NtCCD1-2 (2# and 3#), A10- δ -NtCCD10 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#), and A10- δ -CaCCD4C (1# and 11#), the titer and yield of 2-phenylethanol ranged from 0.87 to 6.02 mg/L and from 0.28 to 1.48 mg/g DCW, respectively. Meanwhile, the production of 2-phenylethanol in transformants A10- δ -PhCCD1 8# and 14# was comparable, despite significant differences in the expression levels of the PhCCD1 gene between the two strains (Fig. 2 ). Similarly, 2-phenylethanol yields in A10- δ -NtCCD1-3 1# and 9# were equivalent, even though the expression levels of the NtCCD1-3 gene differed markedly between these two transformants (Fig. 2 ). Differently, the production of 2-phenylethanol in A10- δ -NtCCD1-2 (2# and 3#), A10- δ -ZmCCD1 (8# and 9#) and A10- δ -CaCCD4C (1# and 11#) was inversely proportional to the expression level of NtCCD1-2 , ZmCCD1 or CaCCD4C , respectively (Fig. 1 and Fig. 2 ). Those results further confirmed that the integrated expression of NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C genes in S. cerevisiae could inhibit the production of 2-phenylethanol, while PhCCD1 and NtCCD1-3 genes had no such effect on 2-phenylethanol producing. Inhibition of 2-phenylethanol synthesis by CCDs in S. cerevisiae may more common To understand the distribution of inhibitory CCDs (NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C) and non-inhibitory CCDs (PhCCD1 and NtCCD1-3) in CCD protein family, a neighbor joining tree was constructed (Fig. 3 ). Inhibitory CCDs distributed in three subclades (CCD1, CCD10, and CCD4), while non-inhibitory CCDs distributed in one subclade (CCD1). Meanwhile, the non-inhibitory CCDs were closely clustered together. The results above suggested that CCDs inhibition of 2-phenylethanol generation may be more common in the CCDs family. PhCCD1 shares the highest identity (92.9%) with NtCCD1-3, and both of them showed non-inhibition effect on 2-phenylethanol producing. Interestingly, NtCCD1-2 and ZmCCD1, both also belonging to CCD1, showed inhibition effect on 2-phenylethanol generating, with NtCCD1-2 sharing 83.9% and 82.85% identity with PhCCD1 and NtCCD1-3, respectively. It is speculated that the possible reason is that most members of the CCD protein family had the ability to inhibit the synthesis of 2-phenylethanol in S. cerevisiae . Over time, some members in CCD1 subclade undergone mutation event, generating CCDs that did not inhibit the synthesis of 2-phenylethanol (such as PhCCD1 and NtCCD1-3). According to the results of phylogenetic analysis, it is presumed that most members of CCD protein family can inhibit the synthesis of 2-phenylethanol in S. cerevisiae . Due to the mutation of some key sites of PhCCD1 and NtCCD1-3 genes, the corresponding amino acid sites were changed, which made them lose the ability to inhibit the synthesis of 2-phenylethanol in S. cerevisiae . It has been reported that a single site mutation in CCDs significantly reduced or increased the catalytic activity of the enzyme (Messing et al. 2010 ; Werner et al. 2019 ; Gong et al. 2022 ), indicating that the mutation of a few key amino acids could greatly affect the function of the enzyme. Therefore, the inhibition or non-inhibition of CCDs on the synthesis of 2-phenylethanol in S. cerevicae might be caused by different sites of a few key amino acids. So, the amino acid sites, which are the same in PhCCD1 and NtCCD1-3, while different from NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C, may be the amino acid sites related to the loss of inhibition of 2-phenylethanol synthesis function of PhCCD1 and NtCCD1-3. Therefore, the amino acid sequences of PhCCD1, NtCCD1-3, NtCCD1-2, NtCCD10, ZmCCD1, CaCCD4C and VP14 were aligned (Fig. 4 ), and a total of 20 amino acid sites with the above characteristics were selected. In PhCCD1 and NtCCD1-3, they are Asp-7, Lys-25, Ile-41, Tyr-53, Leu-67, Asp-69, Leu-200, Ile-259, Ile-272, Cys-292, Gln-303, Phe-306, Ser-325, Ala-327, Met-366, Val-371, Ser-379, Thr-425, Val-473 and Ala-503, respectively (Shown in green boxes in Fig. 4 ). Meanwhile, both inhibited and non-inhibited CCDs contain four histidine residues (Marked with red star in Fig. 4 ) that bind to Fe 2+ active centers and three Glu/Asp/Gly residues (Marked with blue diamond in Fig. 4 ), which is consistent with the results of VP14 and ZmCCD10a (Messing et al. 2010 ; Zhong et al. 2020 ). Neither β -Cyclocitral nor dihydroactinidiolide can inhibit yeast from producing 2-phenylethanol The metabolites producing by A10 and different A10- δ -CCDs transformants were detected by SPME-GC-MS (Table S2). β -Cyclocitral was detected in all the inhibitory group strains, but not in the control A10 and non-inhibitory group strains. Dihydroactinidiolide was also detected in all the inhibitory group strains, but not in the non-inhibitory group strains. Although dihydroactinidiolide was detected in the control A10, but the area of dihydroactinidiolide in A10 sample was far low compared with those of inhibitory group strains. Therefore, β -cyclocitral and dihydroactinidiolide might be the metabolites that inhibit the synthesis of 2-phenylethanol in yeast. Product inhibition test was performed to confirm whether β -cyclocitral and dihydroactinidiolide could inhibit the synthesis of 2-phenylethanol in yeast. The results showed that β -cyclocitral had no significant effect on the 2-phenylethanol production of strains A10, N3 and N1 (Fig. 5 a), indicating that β -cyclocitral can not inhibit the synthesis of 2-phenylethanol in S. cerevisiae . Meanwhile, dihydroactinidiolide also had no significant effect on the 2-phenylethanol production of strains A10 and N3. Although dihydroactinidiolide had a certain impact on the 2-phenylethanol production of N1, considering that the p -value was between 0.03 and 0.05 and the 2-phenylethanol production of N1 was relatively low (Fig. 5 b), suggesting that dihydroactinidiolide did not have the ability to prevent S. cerevisiae from producing 2-phenylethanol. Both β -cyclocitral and dihydroactinidiolide are derivatives of carotenoid (Havaux 2020 ; Klok et al. 1984 ; Sung et al. 2010 ), and carotenoid share the glycolytic pathway with 2-phenylethanol. Therefore, the accumulation of β -cyclocitral and dihydroactinidiolide in the strains of the inhibitory group may be one of the reasons for the inhibition of 2-phenylethanol production. Cells incubated for 2 d were suitable for transcriptome analysis Production curves of 2-phenylethanol and β -ionone in strains A10, N3 and N1 indicated that the 2-phenylethanol production of all the three strains reached a plateau after 2 days cultivation, and after reaching the plateau, the 2-phenylethanol production of strains A10 and N3 was comparable and much higher than that of strain N1 (Fig. 6 a). Meanwhile, the synthesis of β -ionone by A10, N3 and N1 showed different trends: (1) A10 can not produce β -ionone; (2) the yield of β -ionone producing by N1 reached the plateau stage at 2 days cultivation, but the yield was low (below 1.0 mg/L); and (3) the yield of β -ionone generating by N3 increased linearly within 0–7 days and reached 10 mg/L after 7 days cultivation (Fig. 6 b). The results suggested that there was no significant correlation between the synthesis of 2-phenylethanol and β -ionone by S. cerevisiae . Considering that the yield of 2-phenylethanol of the three strains reached the plateau stage after 2 days cultavition, so the cell culture time for transcriptome assay was determined to be 2 days. Inhibitory CCDs severely impair cell growth, correlating with decreased 2-phenylethanol yields The 2-phenylethanol production and biomass (Dry cell weight) of A10, Ph1, N3, N1, N10, Zm1 and Ca4, which were fermented for 2 days, were determined. The 2-phenylethanol production of the non-inhibited group (Ph1 and N3) was similar to that of A10, while 2-phenylethanol production of the inhibited group (N1, N10, Zm1 and Ca4) were significantly lower ( p < 0.001) than that of A10 (Fig. 7 a). In detail, the 2-phenylethanol titer of strain A10 was 14.50 mg/L, while that of strains N1, N10, Zm1, and Ca4 ranged from 1.59 to 3.34 mg/L. The variation trend of biomass of different samples was basically consistent with that of 2-phenylethanol yield (Fig. 7 b), which was consistent with the results reported by Chen and Fink ( 2006 ), indicating that the biomass of strains affected the synthesis of 2-phenylethanol, and the high concentration of cells promoted the synthesis of 2-phenylethanol. According to the results above, it is speculated that inhibitory CCDs genes may reduce the synthesis of 2-phenylethanol through inhibiting cell growth. Inhibitory CCDs trigger global transcriptional reprogramming and activate stress response pathways Transcriptome analysis of yeast cell samples showed that gene expression profiles of Ph1 and N3 were similar to that of A10, while gene expression profiles of N1, N10, Zm1 and Ca4 were different from that of A10 (Fig. 8 a, 8 b, 8 c; Fig. S7). There were 63, 108 and 8 DEGs identified between A10 and Ph1, A10 and N3, as well as N3 and Ph1, respectively. Meanwhile, there were 9, 112, 9, 6, 27 and 5 DEGs identified between N1 and N10, N1 and Zm1, Ca4 and N1, Ca4 and N10, Ca4 and Zm1, as well as N10 and Zm1, respectively. While there were more than 2000 DEGs identified between control and inhibitory group strains, and non-inhibitory group and inhibitory group strains, respectively (Fig. 8 d). The results above suggest that gene expression profiles of A10, Ph1 and N3 were similar to each other, and those of N1, N10, Zm1 and Ca4 were also similar to each other, while those of N1, N10, Zm1 and Ca4 were different from those of A10, Ph1 and N3. The analysis revealed that several stress response-related pathways, including the ribosome pathway, MAPK signaling pathway - yeast, longevity regulating pathway - multiple species, and ABC transporters pathway, were enriched. Notably, the ribosome pathway was downregulated in N1, N10, Zm1, and Ca4 compared to A10, suggesting a shift in cellular priorities under stress. In contrast, the MAPK signaling pathway, longevity-regulating pathway, and ABC transporters pathway were all upregulated, highlighting the activation of key adaptive mechanisms that support stress resilience, cellular maintenance, and detoxification processes. Meanwhile, many metabolic pathways (carbon metabolism, biosynthesis of amino acids, glycolysis / gluconeogenesis, methane metabolism, oxidative phosphorylation, citrate cycle, pyruvate metabolism, thiamine metabolism and pentose phosphate pathway) were also enriched and all downregulated in N1, N10, Zm1 and Ca4 compared to A10 (Fig. 8 e; Fig. S8). Downregulation of the 2-phenylethanol de novo synthesis pathway reduces its production in the inhibitory group The genes related to 2-phenylethanol de novo synthesis, 2-phenylethanol synthesis pathway regulation, MAPK signaling pathway and ergosterol synthesis were selected for heat map cluster analysis. Compare to A10 and non-inhibitory group, many 2-phenylethanol de novo synthesis genes aro1 , aro2 , aro3 , aro4 , aro8 , bat1 , aro10 , pdc1 , pdc6 , adh1 , adh2 and adh7 were downregulated in the inhibitory group (Fig. 9 a). This is consistent with the trend of extracellular 2-phenylethanol production, suggesting that these gene downregulation may be the direct cause of the decline in 2-phenylethanol production. The aro80 whose expression product can positive regulate aro10 transcription, lpd1 and plenty of thiamine regulon genes ( thi2 , thi4 , thi5 , thi7 , thi11 , thi12 , thi13 , thi20 and thi73 ) were downregulated in the inhibitory group compared to A10 and non-inhibitory group (Fig. 9 b), which reduced the synthesis of 2-phenylethanol. In the MAPK signaling pathway, there were 6 genes ( ste2 , sst2 , ste4 , rag1 , far1 , and ptp2 ), 6 genes ( pkh1 , mkk1 , mkk2 , ptp2 , paf1 and fks3 ), 4 genes ( hog1 , ptp2 , smp1 and hsl7 ) as well as 2 genes ( ptp2 and tec1 ) related to pheromone, cell wall, high osmolarity and starvation stress upregulated respectively in the inhibitory group compared to A10 and non-inhibitory group (Fig. 9 c). This suggests that the inhibitory CCDs genes expression in the yeast leads to some kind of stress, and the stress inhibits yeast growth. And a lot of genes ( hmg1 , erg3 , erg10 , erg25 , erg5 , erg1 , erg6 , erg2 , erg11 , erg13 and erg20 ) of ergosterol synthesis expressed downregulation for the inhibitory group compared to A10 and non-inhibitory group (Fig. 9 d), and this may give rise to yeast growth slowdown. Discussion Carotenoid cleavage dioxygenases are involved in plant growth, development, and stress response thought their catalytic products apocarotenoids (Moreno et al. 2021 ). 2-Phenylethanol is a kind of quorum sensing compound in S. cerevisiae , which is controlled by cell density (Chen and Fink 2006 ). In this study, an interesting phenomenon was found that some CCDs ( NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C ) from plants inhibited 2-phenylethanol production when they were expressed in S. cerevisiae , but others ( PhCCD1 and NtCCD1-3 ) cannot (Fig. 1 ). And the biomass of yeast was consistent with the production of 2-phenylethanol (Fig. 7 ). Thus, we propose that the expression of inhibitory group genes ( NtCCD1-2 , NtCCD10 , ZmCCD1 and CaCCD4C ) in yeast leads to biotic stress, which inhibits host growth and ultimately reduces the production of 2-phenylethanol, whereas the non-inhibitory group genes ( PhCCD1 and NtCCD1-3 ) do not have such an effect. This discovery has not been previously reported in the literature. The non-inhibitory CCDs (PhCCD1 and NtCCD1-3) are closely clustered in the CCD1 subclade, while the inhibitory CCDs (NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C) distributed in three subclades (CCD1, CCD10, and CCD4) (Fig. 3 ). Accordingly, CCDs inhibition of 2-phenylethanol generation is more common in the CCDs family. We propose that the CCDs originally possessed the ability to inhibit the synthesis of 2-phenylethanol in S. cerevisiae . However, some members of the CCD1 subclade underwent mutations, resulting in CCDs that no longer inhibited the synthesis of 2-phenylethanol, such as PhCCD1 and NtCCD1-3. Hence, 20 amino acid sites that may be the mutations were screened based on amino acid sequence alignment (Fig. 4 ). Apocarotenoids such as β -cyclocitral, dihydroactinidiolide (Fig. 5 ), β -ionone (Fig. 1 ), and geranylacetone (data not shown) were not responsible for inhibiting yeast production of 2-phenylethanol. Therefore, the biotic stress that inhibits yeast growth may be not triggered by the apocarotenoids produced through the cleavage of carotenoids by the inhibitory CCDs . This contrasts with the CCDs in plants, which exert their effects through the catalytic production of apocarotenoids (Moreno et al. 2021 ). The transcriptome profiles of N1, N10, Zm1, and Ca4 differ from those of A10, whereas the profiles of Ph1 and N3 are similar to those of A10. The result is consistent with both the biomass and 2-phenylethanol production data, suggesting that the transcriptional changes contributed to the biomass and production of 2-phenylethanol. Just as a paper remarked, the transcriptome can affect cellular phenotypes (Pelechano 2017 ). Ribosomes act as key stress sensors, triggering signaling pathways that influence cell fate (Iordanov et al. 1997 ; Iordanov et al. 1998 ). MAPK signaling pathway play a crucial role in fungal signal transduction, mediating responses to various stresses and regulating developmental processes (González-Rubio et al. 2019 ). In the study, the ribosome pathway was significantly enriched in the first place and downregulated in N1, N10, Zm1, and Ca4 compared to A10, and many metabolic pathways were also enriched and downregulated, while MAPK signaling pathway was upregulated. These results indicate that the expression of the inhibitory CCDs may trigger biotic stress, which lead to downregulation of ribosome pathway and some metabolic pathways, as well as upregulation of MAPK signaling pathway. Therefore, the ergosterol synthesis is downregulated and the growth of yeast slow in the inhibitory group, resulting in cell biomass reduction which means low cell density. Transcription factor Aro80 inducing by high cell density (Chen and Fink 2006 ) is downregulated in the inhibitory group. Aro80 positively regulates the expression of aro9 and aro10 (Iraqui et al. 1999 ). Therefore, the gene aro10 was downregulated in the inhibitory group with the downregulation of aro80 . In the inhibitory group, the expression levels of key enzymes involved in the biosynthesis of phenylpyruvate (PPA) from glucose, such as Aro1, Aro2, Aro3, and Aro4, as well as the enzymes catalyzing the decarboxylation of phenylpyruvate to phenylacetaldehyde (Pdc1 and Pdc6), and those catalyzing the reduction of phenylpyruvate to 2-phenylethanol (Adh1, Adh2, and Adh7) were downregulated. The downregulation of the expression of these enzymes greatly weakened the de novo biosynthetic pathway of 2-phenylethanol, resulting in its suppression in the inhibitory group. At the same time, the expression levels of a series of thiamine regulon genes (i.e., thi2 , thi4 , thi5 , thi7 , thi11 , thi12 , thi13 , thi20 , and thi73 ) were also downregulated in the inhibitory group. These genes are involved in the synthesis of thiamine pyrophosphate (TPP), a cofactor of enzymes that catalyze the decarboxylation of phenylpyruvate (Kneen et al. 2011 ). In our previous research on the molecular mechanisms regulating yeast tryptophol synthesis, we also found that 13 of these thiamine regulon genes were upregulated in high-yield 2-phenylethanol-producing cells (Gong et al. 2022 ). Since tryptophol and 2-phenylethanol share the Ehrlich pathway, this result is consistent with our current findings, suggesting that thiamine regulon genes positively regulate 2-phenylethanol synthesis in yeast. Furthermore, studies have shown that mutations in the lipoamide dehydrogenase gene ( lpd1 ) result in a decreased 2-phenylethanol yield, suggesting that 2-phenylethanol synthesis requires the involvement of Lpd1 (Dickinson et al. 2003 ). In this study, the expression of the lpd1 gene was downregulated in the inhibitory group. Therefore, the downregulation of both the thiamine regulon genes and the lpd1 gene in the inhibitory group further weakened the yeast Ehrlich pathway, leading to a further suppression of 2-phenylethanol synthesis. Based on the above research results, we have preliminarily revealed the potential regulatory mechanism by which the inhibitory CCDs suppress 2-phenylethanol synthesis in yeast (Fig. 10 ). Specifically, the inhibitory CCDs genes inhibit 2-phenylethanol production by suppressing yeast cell growth and downregulating the de novo biosynthetic pathway of 2-phenylethanol. This study provides important insights into the biosynthesis of 2-phenylethanol and β -ionone using yeast as a chassis. It also offers a new approach to regulating yeast growth. However, further research is needed, such as identifying the key sites at which inhibitory CCDs genes suppress yeast growth and 2-phenylethanol synthesis, as well as further exploration of the genes through which inhibitory CCDs regulate 2-phenylethanol synthesis in yeast. Declarations Supplementary Information The online version contains supplementary material available at Author contribution XH, MW and XG conceived and designed the study. FL and XG conducted the experiments. XG and GM analysed the data and drafted the manuscript. XH and MW revised the manuscript. All authors read and approved the manuscript. Funding This work was supported by the National Natural Science Foundation of China (32560036), the Science and Technology Talent and Platform Project of Yunnan Provincial (202405AD350017), the Caiyun Postdoctoral Innovation Project of Yunnan Province (C615300504053), and the Key Scientific Research Project of China Tobacco Yunnan Industrial Co., Ltd. (2025XY01). 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N.D\u003cem\u003e.\u003c/em\u003e, not detected. \u003cem\u003ep\u003c/em\u003e values were determined by one-way ANOVA (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/ae8b6780dd552ac89d06cd5b.png"},{"id":108491775,"identity":"10df0dc0-83b0-45e0-81f5-f909d0dba9c1","added_by":"auto","created_at":"2026-05-05 09:55:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84872,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003ePhCCD1\u003c/em\u003e (a), \u003cem\u003eNtCCD1-3\u003c/em\u003e (b), \u003cem\u003eNtCCD1-2\u003c/em\u003e (c), \u003cem\u003eZmCCD1\u003c/em\u003e (d) and \u003cem\u003eCaCCD4C\u003c/em\u003e (e) genes in different transformants. The expression level of \u003cem\u003ePhCCD1\u003c/em\u003e gene in A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 8# (PhCCD1 8#) transformant, that of \u003cem\u003eNtCCD1-3 \u003c/em\u003egene in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 1# (NtCCD1-3 1#) transformant, that of \u003cem\u003eNtCCD1-2\u003c/em\u003e gene in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 2# (NtCCD1-2 2#) transformant, that of \u003cem\u003eZmCCD1 \u003c/em\u003egene in A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 8# (ZmCCD1 8#) transformant, and that of \u003cem\u003eCaCCD4C \u003c/em\u003egene in A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C 1# (CaCCD4C 1#) transformant were set as 1, respectively. \u003cem\u003ep\u003c/em\u003e values were determined by one-way ANOVA (**\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/25072bff3d5604d1611b7eed.png"},{"id":108491780,"identity":"345a53f0-1dbb-40ce-b9be-0d754c5e2cbb","added_by":"auto","created_at":"2026-05-05 09:55:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8096476,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of CCD protein family. The inhibitory CCDs containing NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C are in in red and bold, and the non-inhibitory CCDs PhCCD1 as well as NtCCD1-3 are in green and bold. The CCD protein family is composed by CCD1, CCD2, CCD4, CCD7, CCD8, CCD10 and NCED clades. The neighbor joining tree was constructed by using MEGA X, based on 1000 replicates. Bootstrap values above 50 are shown on the branches. Rd, \u003cem\u003eRosa damascena\u003c/em\u003e; Vv, \u003cem\u003eVitis vinifera\u003c/em\u003e; Of, \u003cem\u003eOsmanthus fragrans\u003c/em\u003e; At, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; Nt, \u003cem\u003eNicotiana tabacum\u003c/em\u003e; Ph, \u003cem\u003ePetunia hybrida\u003c/em\u003e; Zm, \u003cem\u003eZea mays\u003c/em\u003e; Cs, \u003cem\u003eCrocus sativus\u003c/em\u003e; Cang, \u003cem\u003eCrocus Crocus angustifolius\u003c/em\u003e; Cm, \u003cem\u003eCucumis melo\u003c/em\u003e; Ca, \u003cem\u003eCrocus ancyrensis\u003c/em\u003e; VP14, Viviparous 14; Os, \u003cem\u003eOryza sativa\u003c/em\u003e; Mm, \u003cem\u003eMus musculus\u003c/em\u003e; RPE65, Retinal pigment epithelial protein 65kDa.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/959ef47903c406bbed8c1dcb.png"},{"id":108233150,"identity":"a2c7848b-052c-4e14-91d6-10eafae2788a","added_by":"auto","created_at":"2026-04-30 18:06:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18100154,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid sequence alignment of NtCCD1-2, NtCCD10, ZmCCD1, CaCCD4C, PhCCD1, NtCCD1-3 and VP14. The conserved iron-binding His (catalytic active center) are marked with red stars, and the conserved Glu/Asp/Gly are marked with blue diamond. Amino acids, which may be associated with the loss of inhibition of PhCCD1 and NtCCD1-3 for 2-phenylethanol synthesis in yeast, are shown in the green boxes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/b4bb0bc6c9a5a05be54f5d6e.png"},{"id":108492034,"identity":"983612b9-fc88-4b6b-991b-d19bce69072b","added_by":"auto","created_at":"2026-05-05 09:56:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":93108,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eβ\u003c/em\u003e-cyclocitral(a) and dihydroactinidiolide (b) at different concentrations on the yield of 2-phenylethanol (2-PE) producing by strains A10, N3 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 9#) and N1 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 2#), respectively. Significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) are indicated by different lowercase letters above the bars (a to c).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/60837e910d1b99b585fb1033.png"},{"id":108492181,"identity":"c5959de3-12fb-4801-8ffa-9f31568c065a","added_by":"auto","created_at":"2026-05-05 09:57:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115277,"visible":true,"origin":"","legend":"\u003cp\u003eProduction curve of 2-phenylethanol (a) and \u003cem\u003eβ\u003c/em\u003e-ionone (b) in strains A10, N3 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 9#) and N1 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 2#).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/fa15204c60fb662eba8a656d.png"},{"id":108491250,"identity":"2e56bd99-e0c3-44c3-82a9-8405bedf82d3","added_by":"auto","created_at":"2026-05-05 09:53:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":66423,"visible":true,"origin":"","legend":"\u003cp\u003eProduction of 2-phenylethanol (a) in strains A10, Ph1 (A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 14#), N3 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 9#), N1 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 2#), N10 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10 3#), Zm1 (A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 8#) and Ca4 (A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C 1#), as well as biomass (b) of the strains above. Significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) are indicated by different lowercase letters above the bars (a to f).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/00912df2d75169a7e692ebb4.png"},{"id":108233153,"identity":"9a193381-fc4b-45e9-9d8e-98c2419c3f22","added_by":"auto","created_at":"2026-04-30 18:06:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":147694,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis of different yeast cell samples. (a) Principal component analysis (PCA) of yeast transcriptome samples. (b) and (c) Heat map analysis of the upregulated and downregulated genes in Ph1 (A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 14#) and N1 (A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 2#) compared to A10. (d) Numbers of the upregulated and downregulated genes in the yeast transcriptome samples compared to each other. C, NON and INH represent Control (A10), non-inhibitory group strains and inhibitory group strains, respectively. (e) KEGG enrichment analysis of DEGs between N1 and A10, showing 22 enriched KEGG pathways (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.1; #\u003cem\u003ep\u003c/em\u003e\u0026gt;0.1).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/1a56b63440ae1e206d28142d.png"},{"id":108491117,"identity":"ad8826fa-368e-4559-8335-b74264bfc1a5","added_by":"auto","created_at":"2026-05-05 09:52:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":137126,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map analysis of the expression levels of genes related to 2-phenylethanol de novo synthesis (a), 2-phenylethanol synthesis pathway regulation (b), MAPK signaling pathway (c) and ergosterol synthesis (d).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/74087177b655ed1abff03a17.png"},{"id":108233156,"identity":"fd3929e2-50e3-42ee-8dd5-c522ccbcabb1","added_by":"auto","created_at":"2026-04-30 18:06:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":154083,"visible":true,"origin":"","legend":"\u003cp\u003eHypothetical molecular regulation model of 2-phenylethanol synthesis inhibition by \u003cem\u003eCCDs\u003c/em\u003egene in \u003cem\u003eS. cerevisiae\u003c/em\u003e. Phe phenylalanine, PPA phenylpyruvate, PAA phenylacetaldehyde, 2-PE 2-phenylethanol. The solid arrows represent one-step reactions, and the dashed arrows represent multistep reactions or transmembrane transport of substances. The green lines show repression. Red fonts indicate the up-regulated pathway, and the green fonts indicate the down-regulated proteins or compounds of decreased abundance.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/e9d54aca7aa703f4157d6ea9.png"},{"id":108807641,"identity":"49d63ab0-3b5c-4e76-9624-adc48b934ccf","added_by":"auto","created_at":"2026-05-08 15:31:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24670841,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/caa8de1a-1426-4e1d-b663-195fe732a780.pdf"},{"id":108233147,"identity":"42d98866-e6ca-43e6-9ff0-ee5f5c35c0c0","added_by":"auto","created_at":"2026-04-30 18:06:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8549147,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9333171/v1/fc5cc45705613e9b2d1db0fa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carotenoid cleavage dioxygenase genes negatively regulate 2-phenylethanol biosynthesis in yeast","fulltext":[{"header":"Key Points","content":"\u003cp\u003e\u003cstrong\u003e• Certain plant-derived \u003cem\u003eCCD\u003c/em\u003e genes inhibit 2-PE production and impair yeast cell growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e•\u0026nbsp;\u003c/strong\u003eInhibitory \u003cem\u003eCCD\u003c/em\u003e genes induce biotic stress and decrease cell biomass in yeast.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e• The de novo 2-PE synthesis pathway is\u0026nbsp;\u003c/strong\u003edownregulated by inhibitory \u003cem\u003eCCD\u003c/em\u003e genes.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCarotenoids are a class of lipophilic isoprenoid compounds, typically consisting of a 40-carbon backbone (Hirschberg \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Maresca et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Carotenoids are synthesized in all photosynthetic organisms (such as cyanobacteria, algae, and plants) and some non-photosynthetic organisms (such as fungi and bacteria) (Nisar et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In plants, carotenoids are involved in various biological processes, including photosynthesis, photomorphogenesis, photoprotection, and development (Nisar et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Apocarotenoids are oxidation cleavage products of carotenoids, formed through either enzymatic or non-enzymatic processes, that play roles in regulating plant development, participating in stress responses, and attracting pollinators and seed-dispersing organisms in plants (Hou et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe enzymatic cleavage of carotenoids is mediated by carotenoid cleavage dioxygenases (CCDs). In plants, the CCD family includes 11 members: CCD1, CCD2, CCD4, CCD7, CCD8, CCD10, NCED2, NCED3, NCED5, NCED6, and NCED9. The latter five members belong to the NCED (nine-cis-epoxy carotenoid dioxygenase) subfamily (Auldridge et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Frusciante et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies on CCD1 from various species have shown that it has a broad substrate specificity. CCD1 can cleave a variety of linear, monocyclic, and bicyclic carotenoids at different positions on the carbon backbone, generating apocarotenoids such as \u003cem\u003eβ\u003c/em\u003e-ionone, pseudoionone, 6-methyl-5-hepten-2-one, geranylacetone, 3-hydroxy-\u003cem\u003eβ\u003c/em\u003e-ionone, geranial, and \u003cem\u003eβ\u003c/em\u003e-cyclocitral (Vogel et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ilg et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that CCD2 from \u003cem\u003eCrocus sativus\u003c/em\u003e and \u003cem\u003eCrocus ancyrensis\u003c/em\u003e is involved in crocin synthesis and has activity to cleave the C7-C8 (C7\u0026rsquo;-C8\u0026rsquo;) double bond of zeaxanthine (Frusciante et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ahrazem et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). CCD4 typically cleaves carotenoids at the C9-C10 (C9\u0026rsquo;-C10\u0026rsquo;) double bond to generate \u003cem\u003eβ\u003c/em\u003e-ionone, but can also asymmetrically cleave the C7-C8 double bond of \u003cem\u003eβ\u003c/em\u003e-cryptoxanthin, zeaxanthine, or \u003cem\u003eβ\u003c/em\u003e-carotene to produce \u003cem\u003eβ\u003c/em\u003e-citraurin, apo-8\u0026rsquo;-\u003cem\u003eβ\u003c/em\u003e-carotenal, \u003cem\u003eβ\u003c/em\u003e-cyclocitral, and others (Huang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rodrigo et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). CCD7 and CCD8 are involved in the synthesis of the plant hormone strigolactones (Alder et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). CCD10 catalyzes the cleavage of carotenoids at the C9-C10 (C9\u0026rsquo;-C10\u0026rsquo;) and C5-C6 (C5\u0026rsquo;-C6\u0026rsquo;) double bonds, and in maize, it enhances plant tolerance to low-phosphorus stress (Zhong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NCEDs are involved in the synthesis of abscisic acid (ABA) (Tan et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCCDs participate in plant growth, development, and stress responses through their cleavage products, apocarotenoids (Moreno et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eβ\u003c/em\u003e-Ionone is involved in a plant herbivore interaction (Wei et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In planta, \u003cem\u003eβ\u003c/em\u003e-cyclocitral participates in \u003csup\u003e1\u003c/sup\u003eO2 signaling, enhances high light and drought tolerance, and serves as a root growth regulator (D\u0026rsquo;Alessandro et al. 2018; D\u0026rsquo;Alessandro et al. 2019; Dickinson et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Strigolactones, a class of apocarotenoids, act as germination stimulants for parasitic plants, root-derived symbiotic signals for arbuscular mycorrhizal fungi, and inhibitors of shoot branching (Cook et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1966\u003c/span\u003e; Akiyama et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gomez-Roldan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). ABA as plant hormone, performs many important functions in plant, such as root and shoot development, hypocotyl elongation, fruit development and ripening, responses to high salinity, drought and nutrient depletion (Gόmez-Cadenas et al. 2000; Hasegawa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Iuchi et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Galpaz et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Felemban et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e2-Phenylethanol is a quorum-sensing molecule in yeast that facilitates the transition from a unicellular to a filamentous morphology (Chen and Fink \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The main biosynthetic pathways for 2-phenylethanol in yeast are the Ehrlich pathway and the de novo pathway. The Ehrlich pathway is the primary route for 2-phenylethanol biosynthesis in yeast and involves three steps: transamination, decarboxylation, and reduction. In the first step, the transaminases Aro8 and Aro9 catalyze the reaction; in the second step, the aromatic decarboxylase Aro10 and the pyruvate decarboxylases Pdc1, Pdc5, and Pdc6 catalyze the reaction; and in the third step, the reduction is mainly catalyzed by the reductases Adh1, Adh2, Adh3, Adh4, Adh5, and Sfa1 (Iraqui et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Dickinson et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the biosynthesis of 2-phenylethanol is also regulated by other enzymes and regulatory factors. Dickinson et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) found that 2-phenylethanol production was reduced in an \u003cem\u003elpd1\u003c/em\u003e gene knockout strain, suggesting that Lpd1 is involved in 2-phenylethanol synthesis. Lpd1 is involved in the citrate cycle, and the carbon flux in the fermentative pathway can be increased by fine-tuning the expression of the \u003cem\u003elpd1\u003c/em\u003e gene (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The decarboxylation step in the Ehrlich pathway for 2-phenylethanol synthesis depends on thiamine pyrophosphate (Dai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), so the thiamine-regulating gene \u003cem\u003ethis\u003c/em\u003e is indirectly involved in 2-phenylethanol synthesis. In our previous study, we found that the 2-phenylethanol production increased when \u003cem\u003ethi4\u003c/em\u003e gene was overexpressed (Gong et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has been reported that the transcriptional activator Aro80 plays a role in activating the transcription of \u003cem\u003earo9\u003c/em\u003e and \u003cem\u003earo10\u003c/em\u003e genes during 2-phenylethanol synthesis (Iraqui et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The transcription of \u003cem\u003earo9\u003c/em\u003e and \u003cem\u003earo10\u003c/em\u003e is suppressed by nitrogen catabolite repression (NCR), while Gln3 and Gat1 can activate the expression of these genes through the intracellular amino acid sensing system (Target of Rapamycin pathway, TOR pathway) (Cooper \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overexpression of \u003cem\u003egln3\u003c/em\u003e and \u003cem\u003egat1\u003c/em\u003e in \u003cem\u003eS. cerevisiae\u003c/em\u003e resulted in a significant increase in 2-phenylethanol production (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), whereas knockout of \u003cem\u003egln3\u003c/em\u003e led to significant decrease (Xia et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has been reported that knocking out the \u003cem\u003emig1\u003c/em\u003e gene or overexpressing the \u003cem\u003ecat8\u003c/em\u003e gene increases the expression levels of \u003cem\u003earo9\u003c/em\u003e and \u003cem\u003earo10\u003c/em\u003e, leading to higher 2-phenylethanol production in \u003cem\u003eS. cerevisiae\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), indicating that Mig1 negatively regulates 2-phenylethanol synthesis, while Cat8 positively regulates it. Currently, there have been no reports indicating that expression of \u003cem\u003eCCDs\u003c/em\u003e genes in \u003cem\u003eS. cerevisiae\u003c/em\u003e inhibits 2-phenylethanol synthesis.\u003c/p\u003e \u003cp\u003eIn this study, an unexpected phenomenon was found for the first time that genes \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e from plants inhibited 2-phenylethanol production when they were expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e, but \u003cem\u003ePhCCD1 and NtCCD1-3\u003c/em\u003e cannot. The biomass of yeast was consistent with the production of 2-phenylethanol. Transcriptome analysis was carried out to elucidate the molecular mechanism. This study offers valuable insights into the biosynthesis of 2-phenylethanol and \u003cem\u003eβ\u003c/em\u003e-ionone using yeast as a host organism. Additionally, it presents a novel strategy for regulating yeast growth.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStrains, plasmids, and reagents\u003c/h2\u003e\n \u003cp\u003eThe key strains and plasmids utilized in this work are summarized in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The \u003cem\u003ePhCCD1\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e, and \u003cem\u003eCaCCD4C\u003c/em\u003e genes originate from \u003cem\u003ePetunia hybrida\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, and \u003cem\u003eCrocus ancyrensis\u003c/em\u003e, respectively. In contrast, the \u003cem\u003eNtCCD1-3\u003c/em\u003e, \u003cem\u003eNtCCD1-2\u003c/em\u003e, and \u003cem\u003eNtCCD10\u003c/em\u003e genes are all derived from \u003cem\u003eNicotiana tabacum\u003c/em\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStrains and plasmids used in this study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eStrains/plasmids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrians\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eBY4741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eMAT\u0026alpha; his3\u0026Delta;1 leu2\u0026Delta;0 met15\u0026Delta;0 ura3\u0026Delta;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eYuchun Biology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eDH5\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026phi;80 lacZ\u0026Delta;M15 \u0026Delta;(lacZYA-argF)U169 recA1 endA1 hsdR17(rk\u003csup\u003e\u0026minus;\u003c/sup\u003e, mk\u003csup\u003e+\u003c/sup\u003e) phoA, supE44 thi-1 gyrA96 relA1 \u0026lambda;\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eVazyme\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eBY4741 \u003cem\u003eura3\u003c/em\u003e::P\u003csub\u003eGAP\u003c/sub\u003e-crtE-T\u003csub\u003eCYC1\u003c/sub\u003e,P\u003csub\u003eGAP\u003c/sub\u003e-crtYB-T\u003csub\u003eCYC1\u003c/sub\u003e,P\u003csub\u003eGAP\u003c/sub\u003e-crtI-T\u003csub\u003eCYC1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eGong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 (Ph1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-PhCCD1-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 (N3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD1-3-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 (N1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD1-2-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10 (N10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD10-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1 (Zm1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-ZmCCD1-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eA10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C (Ca4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eA10 \u003cem\u003e\u0026delta;\u003c/em\u003e::P\u003csub\u003eADH1\u003c/sub\u003e-CaCCD4C-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLeu2\u003c/sub\u003e-Leu2-T\u003csub\u003eLeu2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eColE1 origin, F1 origin, P\u003csub\u003eADH1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e, Amp\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eWang et al., 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-PhCCD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-PhCCD1-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-NtCCD1-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD1-3-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-NtCCD1-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD1-2-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-NtCCD10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-NtCCD10-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-ZmCCD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-ZmCCD1-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003etpLADH1-CaCCD4C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003etpLADH1 carrying P\u003csub\u003eADH1\u003c/sub\u003e-CaCCD4C-T\u003csub\u003eCYC1\u003c/sub\u003e, P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eColE1 origin, His-Tag, \u003cem\u003elac\u003c/em\u003e I, Amp\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eTaKaRa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains PhCCD1 and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains NtCCD1-3 and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains NtCCD1-2 and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains NtCCD10 and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains ZmCCD1 and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C-LEU2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eDerived from pColdTF, contains CaCCD4C and Leu cassette, delta homologous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e-Ionone (\u0026gt;\u0026thinsp;97%) was sourced from Sigma-Aldrich. pEASY\u003csup\u003e\u0026reg;\u003c/sup\u003e-Basic Seamless Cloning and Assembly Kit and PrimeScript\u0026trade; RT reagent Kit were obtained from TaKaRa. TIANgel Midi Purification Kit was obtained from TianGen Biotech Co., Ltd. (Beijing, China). E.Z.N.A.\u003csup\u003e\u0026reg;\u003c/sup\u003ePlant RNA Kit and 2\u0026times;TSINGKE\u003csup\u003e\u0026reg;\u003c/sup\u003e Master qPCR Mix (SYBR Green I) Kit were obtained from Omega Bio-Tek and Tsingke Biotechnology Co., Ltd. (Beijing, China), respectively. Dodecane (analytical standard) and other reagents were purchased from aladdin (Shanghai, China).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eConstruction of\u003c/strong\u003e \u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e-ionone producing yeast strains\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe \u003cem\u003e\u0026beta;\u003c/em\u003e-carotene producing strain A10 was used as the start strain (Gong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePhCCD1\u003c/em\u003e, \u003cem\u003eNtCCD1-3\u003c/em\u003e, \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e genes which were codon-optimized for \u003cem\u003eS. cerevisiae\u003c/em\u003e expression, were selected, synthesized by GenScript (Nanjing, China), and then cloned into the plasmid tpLADH1 to obtain tpLADH1-PhCCD1, tpLADH1-NtCCD1-3, tpLADH1-NtCCD1-2, tpLADH1-NtCCD10, tpLADH1-ZmCCD1 and tpLADH1-CaCCD4C, respectively. Strain A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 was used as the case for elaborating the construction process of the \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone producing yeast strains. The delta locus was selected for \u003cem\u003ePhCCD1\u003c/em\u003e gene integration. The PhCCD1 cassette containing promoter \u003cem\u003eADH1\u003c/em\u003e, \u003cem\u003ePhCCD1\u003c/em\u003e gene, and terminator \u003cem\u003eCYC1\u003c/em\u003e, was amplified from plasmid tpLADH1-PhCCD1 by using primers CCDs-F and CCDs-R. Then, selection marker LEU2 cassette was amplified from the tpLADH1 plasmid with primers LEU-\u003cem\u003e\u0026delta;\u003c/em\u003e-F and LEU-\u003cem\u003e\u0026delta;\u003c/em\u003e-R, and the \u003cem\u003e\u0026delta;\u003c/em\u003e-up and \u003cem\u003e\u0026delta;\u003c/em\u003e-down fragments were amplified from \u003cem\u003eS. cerevisiae\u003c/em\u003e BY4741 genome using primers \u003cem\u003e\u0026delta;-\u003c/em\u003eup-F/\u003cem\u003e\u0026delta;-\u003c/em\u003eup-R and \u003cem\u003e\u0026delta;-\u003c/em\u003edown-F/\u003cem\u003e\u0026delta;-\u003c/em\u003edown-R, respectively. The PhCCD1 cassette, the selection marker LEU2 cassette, the \u003cem\u003e\u0026delta;\u003c/em\u003e-up and \u003cem\u003e\u0026delta;\u003c/em\u003e-down fragments, as well as the \u003cem\u003eEco\u003c/em\u003eR I linearized pColdTF plasmid were infused with pEASY\u003csup\u003e\u0026reg;\u003c/sup\u003e-Basic Seamless Cloning and Assembly Kit, and the transformants were screened on LB solid medium containing 50 \u0026micro;g/mL ampicillin. The recombinant plasmid pColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1-LEU2 was verified by \u003cem\u003eEco\u003c/em\u003eR I digestion and sequencing. The pColdTF-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1-LEU2 plasmid was digested by \u003cem\u003ePst\u003c/em\u003e I and \u003cem\u003eBam\u003c/em\u003eH I to obtain the \u003cem\u003e\u0026delta;\u003c/em\u003e-up-P\u003csub\u003eADH1\u003c/sub\u003e-PhCCD1-T\u003csub\u003eCYC1\u003c/sub\u003e-P\u003csub\u003eLEU\u003c/sub\u003e-LEU2-T\u003csub\u003eLEU\u003c/sub\u003e-\u003cem\u003e\u0026delta;\u003c/em\u003e-down fragment, which was purified and transformed into strain A10 by chemical transformation (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Finally, the transformants were screened by incubating on leucine-deficient synthetic agar plate at 30\u0026deg;C, and verified by sequencing. Several correct transformants named A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 8#, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 14# and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 21# respectively, were used for the subsequent experiments. Strains A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 (transformants 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8# and 9#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 (transformants 2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10 (transformants 2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1 (transformants 8#, 9#, 10#, 11# and 12#) and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C (transformants 1#, 7#, 8#, 10# and 11#) were constructed with the same method as A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1. All primers used in this study are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and all strains and plasmids used in this study are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFermentation and volatile extraction\u003c/h3\u003e\n\u003cp\u003eTo analyze the volatile compounds via headspace solid-phase micro-extraction gas chromatography-mass spectrometry (SPME-GC-MS), various transformants of strains A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1, and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C were cultured on YPD solid medium at 28\u0026deg;C for 5 days. A 0.15 g cell pellet was collected from each strain for SPME-GC-MS analysis. The control strain, A10, was treated in the same manner.\u003c/p\u003e\n\u003cp\u003eStrain A10 was inoculated into 20 mL uracil-deficient medium and incubated at 28\u0026deg;C with shaking at 200 rpm for 48 h. Strains A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 (8# and 14#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 (1# and 9#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1 (8# and 9#), and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C (1# and 11#) were inoculated into 20 mL leucine-deficient medium and incubated under the same conditions as A10. Afterward, 4 mL of each culture and 5 mL of dodecane were transferred to 100 mL fresh YPD medium and incubated for 2 days at 28\u0026deg;C with shaking at 200 rpm. After fermentation, the organic phase was separated by centrifugation at 21,734 \u0026times; g for 10 min, then dehydrated with anhydrous sodium sulfate before gas chromatography-mass spectrometry (GC-MS) analysis for \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone quantification. The aqueous phase was collected for 2-phenylethanol quantification using high-performance liquid chromatography (HPLC). The cells were harvested and lyophilized for dry weight measurement to determine the biomass. Three biological replicates were performed for each sample.\u003c/p\u003e\n\u003ch3\u003eSPME-GC-MS analysis of volatile compounds\u003c/h3\u003e\n\u003cp\u003eThe volatile compounds produced by A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C, and the control strain A10 were analyzed using SPME-GC-MS. A 65 \u0026micro;m polydimethylsiloxane / divinylbenzene SPME fiber (Supelco, USA) was placed into a headspace vial containing 0.15 g of cell pellet. The vial was stirred at 80\u0026deg;C and 250 rpm for 20 min. The volatile compounds on the fiber were analyzed by GC-MS (Agilent 5977B, USA) equipped with a DB-5MS column (Agilent, USA; 30 m \u0026times; 0.25 mm ID, 0.25 \u0026micro;m film thickness), using helium as the carrier gas at a flow rate of 2 mL/min. The temperature program was as follows: 40\u0026deg;C for 1 min, ramped to 250\u0026deg;C at 2\u0026deg;C/min, and held at 250\u0026deg;C for 10 min. Electron ionization was performed at 70 eV, with a mass range of m/z 30\u0026ndash;500 and an ion source temperature of 200\u0026deg;C. Volatile compounds were identified using the NIST 2.0 spectral library, and \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone was confirmed by comparing the mass spectrum and chromatographic behavior with that of an authentic standard.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e \u003cstrong\u003e-ionone and 2-phenylethanol quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e-ionone production by different strains was quantified using GC-MS (Agilent 5977B, USA) equipped with a DB-5MS column (Agilent, USA; 30 m \u0026times; 0.25 mm ID, 0.25 \u0026micro;m film thickness), with helium as the carrier gas at a flow rate of 2 mL/min. A 1 \u0026micro;L sample was injected. The temperature program started at 100\u0026deg;C, held for 2 min, then ramped to 210\u0026deg;C at 10\u0026deg;C/min, followed by an increase to 290\u0026deg;C at 20\u0026deg;C/min, and held at 290\u0026deg;C for 5 min. Electron ionization was performed at 70 eV, with a mass range of m/z 40\u0026ndash;400 and an ion source temperature of 200\u0026deg;C. \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone quantification was carried out using the external standard method. A standard curve was prepared by dissolving \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone in dodecane and recording the peak areas at different concentrations. \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone in the dodecane extraction liquid from the samples was detected by GC-MS, and the concentration of \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone in each sample was calculated using the standard curve. Finally, the titers and yields of \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone were determined based on the volume of dodecane used for extraction and the dry cell weight (DCW).\u003c/p\u003e\n\u003cp\u003e2-Phenylethanol production by different strains was quantified using an HPLC system (Agilent 1260, USA) equipped with an Acclaim Explosives E2 column (4.6 mm \u0026times; 250 mm, 5 \u0026micro;m, 120 \u0026Aring;; Thermo Scientific). A 10 \u0026micro;L sample was injected, and Isogradient elution was performed using a methanol / H\u003csub\u003e2\u003c/sub\u003eO solution (70:30, v/v) for 20 min at 25\u0026deg;C and a flow rate of 0.5 mL/min. 2-Phenylethanol was monitored at 210 nm, and its concentration was determined by integrating the calibration curves from the standards. Each sample was measured in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time quantitative PCR analysis of\u003c/strong\u003e \u003cstrong\u003eCCD\u003c/strong\u003e \u003cstrong\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 (8# and 14#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 (1# and 9#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1 (8# and 9#) and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C (1# and 11#) cells pellets within logarithmic growth phase were collected and used for real-time quantitative PCR (qPCR) assay. Total RNA extraction, cDNA synthesis, and qPCR were carried out according to E.Z.N.A.\u003csup\u003e\u0026reg;\u003c/sup\u003ePlant RNA Kit, PrimeScript\u0026trade; RT reagent Kit, 2\u0026times;TSINGKE\u003csup\u003e\u0026reg;\u003c/sup\u003e Master qPCR Mix (SYBR Green I) Kit, respectively. Primers PhCCD1-qPCR-F / PhCCD1-qPCR-R, NtCCD1-3-qPCR-F / NtCCD1-3-qPCR-R, NtCCD1-2-qPCR-F / NtCCD1-2-qPCR-R, NtCCD10-qPCR-F / NtCCD10-qPCR-R, ZmCCD1-qPCR-F / ZmCCD1-qPCR-R, as well as CaCCD4C-qPCR-F / CaCCD4C-qPCR-R were used for \u003cem\u003ePhCCD1\u003c/em\u003e, \u003cem\u003eNtCCD1-3\u003c/em\u003e, \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e genes amplified respectively, and Act1-qPCR-F and Act1-qPCR-R for the actin gene (\u003cem\u003eact1\u003c/em\u003e) amplified. Differences in the expression of the \u003cem\u003eCCD\u003c/em\u003e genes and \u003cem\u003eact1\u003c/em\u003e gene were calculated according to the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method (Livak and Schmittgen, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) using the \u003cem\u003eact1\u003c/em\u003e gene as the reference.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis, sequence alignment\u003c/h3\u003e\n\u003cp\u003eThe amino acid sequences of PhCCD1 (AAT68189), NtCCD1-3 (NP_001312918), NtCCD1-2 (AHH25650), NtCCD10 (XP_016482179), ZmCCD1 (ABF85668), CaCCD4C (AKN09911), AtCCD1 (CAA06712), OfCCD1 (BAJ05401), VvCCD1 (AAX48772), RdCCD1 (ABY47994), CsCCD2 (AIG94929), CangCCD2 (ALM23546), CsCCD4 (ACD62476), AtCCD4 (AAM97019), CmCCD4 (ABY60885), AtNCED3 (NP_188062), AtNCED9 (NP_177960), AtNCED5 (NP_174302), VP14 (O24592), AtNCED6 (NP_189064), ZmCCD7 (NP_001183928), AtCCD7 (AEC10494), MmRPE65 (AAL01119), CsCCD8a (AIF27229), AtCCD8 (NP_195007), ZmCCD10a (XP_020396187), and OsZAS (EAZ36971) were retrieved from the NCBI protein database for phylogenetic tree construction. Multiple protein sequence alignments were performed using CLUSTAL X (Thompson et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). A neighbor-joining phylogenetic tree was generated with 1000 bootstrap replications using MEGA X, based on the aforementioned multiple sequence alignments (Kumar et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSequence alignment was performed using the amino acid sequences of NtCCD1-2, NtCCD10, ZmCCD1, CaCCD4C, PhCCD1, NtCCD1-3, and 9-cis-epoxycarotenoid dioxygenase viviparous 14 (VP14) with Espript 3.0 (Robert and Gouet \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The VP14 model (PDB 3NPE) was selected to depict its secondary structure.\u003c/p\u003e\n\u003ch3\u003eProduct inhibition test\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e-cyclocitral and dihydroactinidiolide, which may be the metabolites preventing yeast from producing 2-phenylethanol, were screened through the SPME-GC-MS analysis. Strains A10, A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 9# (N3) as well as A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 2# (N1) were selected for verification test. Strains A10 was inoculated into 20 mL uracil-deficient medium, and incubated at 28\u0026deg;C with shaking at 200 rpm for 48 h, while N3 and N1 were inoculated into 20 mL leucine-deficient medium, which were incubated as A10. After that, 4 mL culture above were transferred to 100 mL fresh YPD medium with different concentration of \u003cem\u003e\u0026beta;\u003c/em\u003e-cyclocitral or dihydroactinidiolide respectively, and finally incubated for 2 days at 28\u0026deg;C with shaking at 200 rpm. Four gradients of 0, 1, 5 and 25 mg/L were set for the final concentrations of the two candidate metabolites. After fermentation, the aqueous phase for 2-phenylethanol quantification by HPLC analysis was collected by centrifuging at 21,734 \u0026times; g for 10 min. Three biological replicates were carried out for each sample.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSample preparation for transcriptomic sequencing\u003c/h2\u003e\n \u003cp\u003eAccording to the quantitative measurement results of \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone and 2-phenylethanol produced by different transformants, combined with the results of \u003cem\u003eCCDs\u003c/em\u003e gene expression level, transformants with the similar 2-phenylethanol yields were selected in the inhibition group to explore the molecular mechanism of \u003cem\u003eCCDs\u003c/em\u003e inhibiting 2-phenylethanol synthesis in \u003cem\u003eS. cerevisiae\u003c/em\u003e, including A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-2 2# (N1), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD10 3# (N10), A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-ZmCCD1 8# (Zm1) and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-CaCCD4C 1# (Ca4). The 2-phenylethanol produced by different transformants were similar to each other in the non-inhibited group, so the transformants with high \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone yield and high \u003cem\u003eCCD\u003c/em\u003e genes expression level were selected, including A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-PhCCD1 14# (Ph1) and A10-\u003cem\u003e\u0026delta;\u003c/em\u003e-NtCCD1-3 9# (N3). Strain A10 was set as the control. To determine the incubated time of samples for transcriptome analysis, A10, N3 and N1 strains were selected to carry out the experiments. To determine the yeast samples\u0026rsquo; fermentation time for transcriptome analysis, five fermentation time points 0, 2, 4, 6, and 7 d were set, and the production of \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone and 2-phenylethanol producing by each strain was determined.\u003c/p\u003e\n \u003cp\u003eAccording to the \u003cem\u003e\u0026beta;\u003c/em\u003e-ionone and 2-phenylethanol results, cell samples of A10, Ph1, N3, N1, N10, Zm1 and Ca4 incubated for 2 d were selected and collected for transcriptome analysis and biomass determination. The preparation of transcriptome analysis samples was as follows. Strains A10 was inoculated into 20 mL uracil-deficient medium, and incubated at 28\u0026deg;C with shaking at 200 rpm for 48 h, and Ph1, N3, N1, N10, Zm1 and Ca4 were inoculated into 20 mL leucine-deficient medium, which were incubated as A10. After that, 6 mL culture above were transferred to 150 mL fresh YPD medium respectively, and finally incubated for 2 days at 28\u0026deg;C with shaking at 200 rpm. After fermentation, cell pellets for transcriptome analysis and biomass determination were collected by centrifuging at 8,000 rpm and 4\u0026deg;C for 10 min, and the cell pellets were frozen in liquid nitrogen for 20 min and used for transcriptome analysis, while the aqueous phase was collected for 2-phenylethanol quantification. Three biological replicates were carried out for each sample.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA extracted from A10, Ph1, N3, N1, N10, Zm1 and Ca4 was used for sequencing with an Illumina HiSeq 4000 platform. The clean reads were mapped to the genome of \u003cem\u003eS. cerevisiae\u003c/em\u003e S288C (GCF_000146045.2) using HISAT2 (Kim et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) with the default settings. Differential expression analysis were carried out using DESeq2 (Love et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Differentially expressed genes (DEGs) between two samples were identified according to the conditions of |log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(fold change)\u003c/sup\u003e| \u0026ge; 1 and a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Log\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e(fold change)\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;1 was the up-regulated genes\u0026apos; standard, and values\u0026thinsp;\u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;1 was the down-regulated genes\u0026apos;. Heat map analysis of gene expression levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the online data analysis platform OmicShare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.omicshare.com/tools\u003c/span\u003e\u003c/span\u003e). In the heat map analysis, the value of the individual sample was shown by Z-scores, and the Z-scores were normalized values by scaling the row values of the gene expression levels.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eNtCCD1-2\u003c/b\u003e, \u003cb\u003eNtCCD10\u003c/b\u003e, \u003cb\u003eZmCCD1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCaCCD4C\u003c/b\u003e \u003cb\u003egenes inhibits 2-phenylethanol production in engineered\u003c/b\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePreviously, we found that the delta integrating site for \u003cem\u003eNtCCD1-3\u003c/em\u003e gene in \u003cem\u003eS. cerevisiae\u003c/em\u003e was suitable for promoting the yield of \u003cem\u003eβ\u003c/em\u003e-ionone (Gong et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the delta integrating site was selected for \u003cem\u003ePhCCD1\u003c/em\u003e, \u003cem\u003eNtCCD1-3\u003c/em\u003e, \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e genes integration in \u003cem\u003eS. cerevisiae\u003c/em\u003e to construct \u003cem\u003eβ\u003c/em\u003e-ionone producing strains A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10, A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C, respectively. The yield of \u003cem\u003eβ\u003c/em\u003e-ionone produced by different transformants of strains was determined by SPME-GC-MS, and the transformants producing higher yield of \u003cem\u003eβ\u003c/em\u003e-ionone were screened preliminarily (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S 2, S3, S4, S5 and S6). The transformants A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 14#, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 9# as well as A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 3# produced higher yield of \u003cem\u003eβ\u003c/em\u003e-ionone. Meanwhile, all the transformants could generate \u003cem\u003eβ\u003c/em\u003e-ionone (peak 3), indicating that the four CCDs above all could cleave 9,10 (9\u0026rsquo;, 10\u0026rsquo;) double bonds. Besides A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10, other transformants all could produce geranylacetone (peak 2), and the transformants of strains A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 and A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 could synthesize 6-methyl-5-heptene-2-one (MHO) (peak 4). Different from other strains, A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 transformants all could produce pseudoionone (peak 5). In addition, all the transformants could give birth to 2-phenylethanol (peak 1). Confusingly, the yield of 2-phenylethanol in transformants of A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 and A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 was similar to that of the control strain A10, while the yield of 2-phenylethanol in transformants of A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2, A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10, A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C was much lower than that of A10. The results suggested that the integrated expression of \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e genes in \u003cem\u003eS. cerevisiae\u003c/em\u003e could inhibit the synthesis of 2-phenylethanol, while \u003cem\u003ePhCCD1\u003c/em\u003e and \u003cem\u003eNtCCD1-3\u003c/em\u003e genes had no such effect on 2-phenylethanol synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the conclusion above, the \u003cem\u003eβ\u003c/em\u003e-ionone and 2-phenylethanol generated by A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 (8# and 14#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 (1# and 9#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 (8# and 9#) and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#) was quantified by GC-MS. The titer and yield of \u003cem\u003eβ\u003c/em\u003e-ionone in A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 14# and A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 9# were significantly higher than those of A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 (8# and 9#) and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that PhCCD1 and NtCCD1-3 may have higher activity of cleaving \u003cem\u003eβ\u003c/em\u003e-carotene to produce \u003cem\u003eβ\u003c/em\u003e-ionone than NtCCD1-2, NtCCD10, ZmCCD1 as well as CaCCD4C. The titer and yield of \u003cem\u003eβ\u003c/em\u003e-ionone in A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 (8# and 14#) exhibited a positive correlation with the expression levels of the \u003cem\u003ePhCCD1\u003c/em\u003e gene, a trend that was similarly observed in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 (1# and 9#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), as well as A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The reason may be that CCD enzymes catalyze the cleavage of \u003cem\u003eβ\u003c/em\u003e-carotene to produce \u003cem\u003eβ\u003c/em\u003e-ionone; thus, a higher expression level of the \u003cem\u003eCCD\u003c/em\u003e gene leads to increased synthesis of CCD proteins, which in turn results in higher \u003cem\u003eβ\u003c/em\u003e-ionone production. The titer and yield of 2-phenylethanol in A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 (8# and 14#) were similar to that of the control strain A10, and those of A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 (1# and 9#) were even higher than those of A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While, the titer and yield of 2-phenylethanol in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 (8# and 9#) and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#) were all significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Specifically, the titer and yield of 2-phenylethanol in A10 were 14.50 mg/L and 2.69 mg/g DCW, respectively. For the strains A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD10 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 (8# and 9#), and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#), the titer and yield of 2-phenylethanol ranged from 0.87 to 6.02 mg/L and from 0.28 to 1.48 mg/g DCW, respectively. Meanwhile, the production of 2-phenylethanol in transformants A10-\u003cem\u003eδ\u003c/em\u003e-PhCCD1 8# and 14# was comparable, despite significant differences in the expression levels of the \u003cem\u003ePhCCD1\u003c/em\u003e gene between the two strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, 2-phenylethanol yields in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-3 1# and 9# were equivalent, even though the expression levels of the \u003cem\u003eNtCCD1-3\u003c/em\u003e gene differed markedly between these two transformants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Differently, the production of 2-phenylethanol in A10-\u003cem\u003eδ\u003c/em\u003e-NtCCD1-2 (2# and 3#), A10-\u003cem\u003eδ\u003c/em\u003e-ZmCCD1 (8# and 9#) and A10-\u003cem\u003eδ\u003c/em\u003e-CaCCD4C (1# and 11#) was inversely proportional to the expression level of \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e or \u003cem\u003eCaCCD4C\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Those results further confirmed that the integrated expression of \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e genes in \u003cem\u003eS. cerevisiae\u003c/em\u003e could inhibit the production of 2-phenylethanol, while \u003cem\u003ePhCCD1\u003c/em\u003e and \u003cem\u003eNtCCD1-3\u003c/em\u003e genes had no such effect on 2-phenylethanol producing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of 2-phenylethanol synthesis by CCDs in\u003c/b\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003emay more common\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand the distribution of inhibitory CCDs (NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C) and non-inhibitory CCDs (PhCCD1 and NtCCD1-3) in CCD protein family, a neighbor joining tree was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Inhibitory CCDs distributed in three subclades (CCD1, CCD10, and CCD4), while non-inhibitory CCDs distributed in one subclade (CCD1). Meanwhile, the non-inhibitory CCDs were closely clustered together. The results above suggested that CCDs inhibition of 2-phenylethanol generation may be more common in the CCDs family. PhCCD1 shares the highest identity (92.9%) with NtCCD1-3, and both of them showed non-inhibition effect on 2-phenylethanol producing. Interestingly, NtCCD1-2 and ZmCCD1, both also belonging to CCD1, showed inhibition effect on 2-phenylethanol generating, with NtCCD1-2 sharing 83.9% and 82.85% identity with PhCCD1 and NtCCD1-3, respectively. It is speculated that the possible reason is that most members of the CCD protein family had the ability to inhibit the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevisiae\u003c/em\u003e. Over time, some members in CCD1 subclade undergone mutation event, generating CCDs that did not inhibit the synthesis of 2-phenylethanol (such as PhCCD1 and NtCCD1-3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the results of phylogenetic analysis, it is presumed that most members of CCD protein family can inhibit the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevisiae\u003c/em\u003e. Due to the mutation of some key sites of \u003cem\u003ePhCCD1\u003c/em\u003e and \u003cem\u003eNtCCD1-3\u003c/em\u003e genes, the corresponding amino acid sites were changed, which made them lose the ability to inhibit the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevisiae\u003c/em\u003e. It has been reported that a single site mutation in CCDs significantly reduced or increased the catalytic activity of the enzyme (Messing et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Werner et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gong et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), indicating that the mutation of a few key amino acids could greatly affect the function of the enzyme. Therefore, the inhibition or non-inhibition of CCDs on the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevicae\u003c/em\u003e might be caused by different sites of a few key amino acids. So, the amino acid sites, which are the same in PhCCD1 and NtCCD1-3, while different from NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C, may be the amino acid sites related to the loss of inhibition of 2-phenylethanol synthesis function of PhCCD1 and NtCCD1-3. Therefore, the amino acid sequences of PhCCD1, NtCCD1-3, NtCCD1-2, NtCCD10, ZmCCD1, CaCCD4C and VP14 were aligned (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and a total of 20 amino acid sites with the above characteristics were selected. In PhCCD1 and NtCCD1-3, they are Asp-7, Lys-25, Ile-41, Tyr-53, Leu-67, Asp-69, Leu-200, Ile-259, Ile-272, Cys-292, Gln-303, Phe-306, Ser-325, Ala-327, Met-366, Val-371, Ser-379, Thr-425, Val-473 and Ala-503, respectively (Shown in green boxes in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Meanwhile, both inhibited and non-inhibited CCDs contain four histidine residues (Marked with red star in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) that bind to Fe\u003csup\u003e2+\u003c/sup\u003e active centers and three Glu/Asp/Gly residues (Marked with blue diamond in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is consistent with the results of VP14 and ZmCCD10a (Messing et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNeither\u003c/b\u003e \u003cb\u003eβ\u003c/b\u003e\u003cb\u003e-Cyclocitral nor dihydroactinidiolide can inhibit yeast from producing 2-phenylethanol\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe metabolites producing by A10 and different A10-\u003cem\u003eδ\u003c/em\u003e-CCDs transformants were detected by SPME-GC-MS (Table S2). \u003cem\u003eβ\u003c/em\u003e-Cyclocitral was detected in all the inhibitory group strains, but not in the control A10 and non-inhibitory group strains. Dihydroactinidiolide was also detected in all the inhibitory group strains, but not in the non-inhibitory group strains. Although dihydroactinidiolide was detected in the control A10, but the area of dihydroactinidiolide in A10 sample was far low compared with those of inhibitory group strains. Therefore, \u003cem\u003eβ\u003c/em\u003e-cyclocitral and dihydroactinidiolide might be the metabolites that inhibit the synthesis of 2-phenylethanol in yeast.\u003c/p\u003e \u003cp\u003eProduct inhibition test was performed to confirm whether \u003cem\u003eβ\u003c/em\u003e-cyclocitral and dihydroactinidiolide could inhibit the synthesis of 2-phenylethanol in yeast. The results showed that \u003cem\u003eβ\u003c/em\u003e-cyclocitral had no significant effect on the 2-phenylethanol production of strains A10, N3 and N1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), indicating that \u003cem\u003eβ\u003c/em\u003e-cyclocitral can not inhibit the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevisiae\u003c/em\u003e. Meanwhile, dihydroactinidiolide also had no significant effect on the 2-phenylethanol production of strains A10 and N3. Although dihydroactinidiolide had a certain impact on the 2-phenylethanol production of N1, considering that the \u003cem\u003ep\u003c/em\u003e-value was between 0.03 and 0.05 and the 2-phenylethanol production of N1 was relatively low (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), suggesting that dihydroactinidiolide did not have the ability to prevent \u003cem\u003eS. cerevisiae\u003c/em\u003e from producing 2-phenylethanol. Both \u003cem\u003eβ\u003c/em\u003e-cyclocitral and dihydroactinidiolide are derivatives of carotenoid (Havaux \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Klok et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Sung et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and carotenoid share the glycolytic pathway with 2-phenylethanol. Therefore, the accumulation of \u003cem\u003eβ\u003c/em\u003e-cyclocitral and dihydroactinidiolide in the strains of the inhibitory group may be one of the reasons for the inhibition of 2-phenylethanol production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCells incubated for 2 d were suitable for transcriptome analysis\u003c/h2\u003e \u003cp\u003eProduction curves of 2-phenylethanol and \u003cem\u003eβ\u003c/em\u003e-ionone in strains A10, N3 and N1 indicated that the 2-phenylethanol production of all the three strains reached a plateau after 2 days cultivation, and after reaching the plateau, the 2-phenylethanol production of strains A10 and N3 was comparable and much higher than that of strain N1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Meanwhile, the synthesis of \u003cem\u003eβ\u003c/em\u003e-ionone by A10, N3 and N1 showed different trends: (1) A10 can not produce \u003cem\u003eβ\u003c/em\u003e-ionone; (2) the yield of \u003cem\u003eβ\u003c/em\u003e-ionone producing by N1 reached the plateau stage at 2 days cultivation, but the yield was low (below 1.0 mg/L); and (3) the yield of \u003cem\u003eβ\u003c/em\u003e-ionone generating by N3 increased linearly within 0\u0026ndash;7 days and reached 10 mg/L after 7 days cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The results suggested that there was no significant correlation between the synthesis of 2-phenylethanol and \u003cem\u003eβ\u003c/em\u003e-ionone by \u003cem\u003eS. cerevisiae\u003c/em\u003e. Considering that the yield of 2-phenylethanol of the three strains reached the plateau stage after 2 days cultavition, so the cell culture time for transcriptome assay was determined to be 2 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibitory\u003c/b\u003e \u003cb\u003eCCDs\u003c/b\u003e \u003cb\u003eseverely impair cell growth, correlating with decreased 2-phenylethanol yields\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 2-phenylethanol production and biomass (Dry cell weight) of A10, Ph1, N3, N1, N10, Zm1 and Ca4, which were fermented for 2 days, were determined. The 2-phenylethanol production of the non-inhibited group (Ph1 and N3) was similar to that of A10, while 2-phenylethanol production of the inhibited group (N1, N10, Zm1 and Ca4) were significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than that of A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In detail, the 2-phenylethanol titer of strain A10 was 14.50 mg/L, while that of strains N1, N10, Zm1, and Ca4 ranged from 1.59 to 3.34 mg/L. The variation trend of biomass of different samples was basically consistent with that of 2-phenylethanol yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), which was consistent with the results reported by Chen and Fink (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), indicating that the biomass of strains affected the synthesis of 2-phenylethanol, and the high concentration of cells promoted the synthesis of 2-phenylethanol. According to the results above, it is speculated that inhibitory \u003cem\u003eCCDs\u003c/em\u003e genes may reduce the synthesis of 2-phenylethanol through inhibiting cell growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibitory\u003c/b\u003e \u003cb\u003eCCDs\u003c/b\u003e \u003cb\u003etrigger global transcriptional reprogramming and activate stress response pathways\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTranscriptome analysis of yeast cell samples showed that gene expression profiles of Ph1 and N3 were similar to that of A10, while gene expression profiles of N1, N10, Zm1 and Ca4 were different from that of A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec; Fig. S7). There were 63, 108 and 8 DEGs identified between A10 and Ph1, A10 and N3, as well as N3 and Ph1, respectively. Meanwhile, there were 9, 112, 9, 6, 27 and 5 DEGs identified between N1 and N10, N1 and Zm1, Ca4 and N1, Ca4 and N10, Ca4 and Zm1, as well as N10 and Zm1, respectively. While there were more than 2000 DEGs identified between control and inhibitory group strains, and non-inhibitory group and inhibitory group strains, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). The results above suggest that gene expression profiles of A10, Ph1 and N3 were similar to each other, and those of N1, N10, Zm1 and Ca4 were also similar to each other, while those of N1, N10, Zm1 and Ca4 were different from those of A10, Ph1 and N3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis revealed that several stress response-related pathways, including the ribosome pathway, MAPK signaling pathway - yeast, longevity regulating pathway - multiple species, and ABC transporters pathway, were enriched. Notably, the ribosome pathway was downregulated in N1, N10, Zm1, and Ca4 compared to A10, suggesting a shift in cellular priorities under stress. In contrast, the MAPK signaling pathway, longevity-regulating pathway, and ABC transporters pathway were all upregulated, highlighting the activation of key adaptive mechanisms that support stress resilience, cellular maintenance, and detoxification processes. Meanwhile, many metabolic pathways (carbon metabolism, biosynthesis of amino acids, glycolysis / gluconeogenesis, methane metabolism, oxidative phosphorylation, citrate cycle, pyruvate metabolism, thiamine metabolism and pentose phosphate pathway) were also enriched and all downregulated in N1, N10, Zm1 and Ca4 compared to A10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee; Fig. S8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDownregulation of the 2-phenylethanol de novo synthesis pathway reduces its production in the inhibitory group\u003c/h2\u003e \u003cp\u003eThe genes related to 2-phenylethanol de novo synthesis, 2-phenylethanol synthesis pathway regulation, MAPK signaling pathway and ergosterol synthesis were selected for heat map cluster analysis. Compare to A10 and non-inhibitory group, many 2-phenylethanol de novo synthesis genes \u003cem\u003earo1\u003c/em\u003e, \u003cem\u003earo2\u003c/em\u003e, \u003cem\u003earo3\u003c/em\u003e, \u003cem\u003earo4\u003c/em\u003e, \u003cem\u003earo8\u003c/em\u003e, \u003cem\u003ebat1\u003c/em\u003e, \u003cem\u003earo10\u003c/em\u003e, \u003cem\u003epdc1\u003c/em\u003e, \u003cem\u003epdc6\u003c/em\u003e, \u003cem\u003eadh1\u003c/em\u003e, \u003cem\u003eadh2\u003c/em\u003e and \u003cem\u003eadh7\u003c/em\u003e were downregulated in the inhibitory group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). This is consistent with the trend of extracellular 2-phenylethanol production, suggesting that these gene downregulation may be the direct cause of the decline in 2-phenylethanol production. The \u003cem\u003earo80\u003c/em\u003e whose expression product can positive regulate \u003cem\u003earo10\u003c/em\u003e transcription, \u003cem\u003elpd1\u003c/em\u003e and plenty of thiamine regulon genes (\u003cem\u003ethi2\u003c/em\u003e, \u003cem\u003ethi4\u003c/em\u003e, \u003cem\u003ethi5\u003c/em\u003e, \u003cem\u003ethi7\u003c/em\u003e, \u003cem\u003ethi11\u003c/em\u003e, \u003cem\u003ethi12\u003c/em\u003e, \u003cem\u003ethi13\u003c/em\u003e, \u003cem\u003ethi20\u003c/em\u003e and \u003cem\u003ethi73\u003c/em\u003e) were downregulated in the inhibitory group compared to A10 and non-inhibitory group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), which reduced the synthesis of 2-phenylethanol. In the MAPK signaling pathway, there were 6 genes (\u003cem\u003este2\u003c/em\u003e, \u003cem\u003esst2\u003c/em\u003e, \u003cem\u003este4\u003c/em\u003e, \u003cem\u003erag1\u003c/em\u003e, \u003cem\u003efar1\u003c/em\u003e, and \u003cem\u003eptp2\u003c/em\u003e), 6 genes (\u003cem\u003epkh1\u003c/em\u003e, \u003cem\u003emkk1\u003c/em\u003e, \u003cem\u003emkk2\u003c/em\u003e, \u003cem\u003eptp2\u003c/em\u003e, \u003cem\u003epaf1\u003c/em\u003e and \u003cem\u003efks3\u003c/em\u003e), 4 genes (\u003cem\u003ehog1\u003c/em\u003e, \u003cem\u003eptp2\u003c/em\u003e, \u003cem\u003esmp1\u003c/em\u003e and \u003cem\u003ehsl7\u003c/em\u003e) as well as 2 genes (\u003cem\u003eptp2\u003c/em\u003e and \u003cem\u003etec1\u003c/em\u003e) related to pheromone, cell wall, high osmolarity and starvation stress upregulated respectively in the inhibitory group compared to A10 and non-inhibitory group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). This suggests that the inhibitory \u003cem\u003eCCDs\u003c/em\u003e genes expression in the yeast leads to some kind of stress, and the stress inhibits yeast growth. And a lot of genes (\u003cem\u003ehmg1\u003c/em\u003e, \u003cem\u003eerg3\u003c/em\u003e, \u003cem\u003eerg10\u003c/em\u003e, \u003cem\u003eerg25\u003c/em\u003e, \u003cem\u003eerg5\u003c/em\u003e, \u003cem\u003eerg1\u003c/em\u003e, \u003cem\u003eerg6\u003c/em\u003e, \u003cem\u003eerg2\u003c/em\u003e, \u003cem\u003eerg11\u003c/em\u003e, \u003cem\u003eerg13\u003c/em\u003e and \u003cem\u003eerg20\u003c/em\u003e) of ergosterol synthesis expressed downregulation for the inhibitory group compared to A10 and non-inhibitory group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed), and this may give rise to yeast growth slowdown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCarotenoid cleavage dioxygenases are involved in plant growth, development, and stress response thought their catalytic products apocarotenoids (Moreno et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 2-Phenylethanol is a kind of quorum sensing compound in \u003cem\u003eS. cerevisiae\u003c/em\u003e, which is controlled by cell density (Chen and Fink \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In this study, an interesting phenomenon was found that some \u003cem\u003eCCDs\u003c/em\u003e (\u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e) from plants inhibited 2-phenylethanol production when they were expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e, but others (\u003cem\u003ePhCCD1 and NtCCD1-3\u003c/em\u003e) cannot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). And the biomass of yeast was consistent with the production of 2-phenylethanol (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Thus, we propose that the expression of inhibitory group genes (\u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e and \u003cem\u003eCaCCD4C\u003c/em\u003e) in yeast leads to biotic stress, which inhibits host growth and ultimately reduces the production of 2-phenylethanol, whereas the non-inhibitory group genes (\u003cem\u003ePhCCD1 and NtCCD1-3\u003c/em\u003e) do not have such an effect. This discovery has not been previously reported in the literature.\u003c/p\u003e \u003cp\u003eThe non-inhibitory CCDs (PhCCD1 and NtCCD1-3) are closely clustered in the CCD1 subclade, while the inhibitory CCDs (NtCCD1-2, NtCCD10, ZmCCD1 and CaCCD4C) distributed in three subclades (CCD1, CCD10, and CCD4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Accordingly, CCDs inhibition of 2-phenylethanol generation is more common in the CCDs family. We propose that the CCDs originally possessed the ability to inhibit the synthesis of 2-phenylethanol in \u003cem\u003eS. cerevisiae\u003c/em\u003e. However, some members of the CCD1 subclade underwent mutations, resulting in CCDs that no longer inhibited the synthesis of 2-phenylethanol, such as PhCCD1 and NtCCD1-3. Hence, 20 amino acid sites that may be the mutations were screened based on amino acid sequence alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApocarotenoids such as \u003cem\u003eβ\u003c/em\u003e-cyclocitral, dihydroactinidiolide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), \u003cem\u003eβ\u003c/em\u003e-ionone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and geranylacetone (data not shown) were not responsible for inhibiting yeast production of 2-phenylethanol. Therefore, the biotic stress that inhibits yeast growth may be not triggered by the apocarotenoids produced through the cleavage of carotenoids by the inhibitory \u003cem\u003eCCDs\u003c/em\u003e. This contrasts with the CCDs in plants, which exert their effects through the catalytic production of apocarotenoids (Moreno et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe transcriptome profiles of N1, N10, Zm1, and Ca4 differ from those of A10, whereas the profiles of Ph1 and N3 are similar to those of A10. The result is consistent with both the biomass and 2-phenylethanol production data, suggesting that the transcriptional changes contributed to the biomass and production of 2-phenylethanol. Just as a paper remarked, the transcriptome can affect cellular phenotypes (Pelechano \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ribosomes act as key stress sensors, triggering signaling pathways that influence cell fate (Iordanov et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Iordanov et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). MAPK signaling pathway play a crucial role in fungal signal transduction, mediating responses to various stresses and regulating developmental processes (Gonz\u0026aacute;lez-Rubio et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the study, the ribosome pathway was significantly enriched in the first place and downregulated in N1, N10, Zm1, and Ca4 compared to A10, and many metabolic pathways were also enriched and downregulated, while MAPK signaling pathway was upregulated. These results indicate that the expression of the inhibitory CCDs may trigger biotic stress, which lead to downregulation of ribosome pathway and some metabolic pathways, as well as upregulation of MAPK signaling pathway. Therefore, the ergosterol synthesis is downregulated and the growth of yeast slow in the inhibitory group, resulting in cell biomass reduction which means low cell density. Transcription factor Aro80 inducing by high cell density (Chen and Fink \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) is downregulated in the inhibitory group. Aro80 positively regulates the expression of \u003cem\u003earo9\u003c/em\u003e and \u003cem\u003earo10\u003c/em\u003e (Iraqui et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, the gene \u003cem\u003earo10\u003c/em\u003e was downregulated in the inhibitory group with the downregulation of \u003cem\u003earo80\u003c/em\u003e. In the inhibitory group, the expression levels of key enzymes involved in the biosynthesis of phenylpyruvate (PPA) from glucose, such as Aro1, Aro2, Aro3, and Aro4, as well as the enzymes catalyzing the decarboxylation of phenylpyruvate to phenylacetaldehyde (Pdc1 and Pdc6), and those catalyzing the reduction of phenylpyruvate to 2-phenylethanol (Adh1, Adh2, and Adh7) were downregulated. The downregulation of the expression of these enzymes greatly weakened the de novo biosynthetic pathway of 2-phenylethanol, resulting in its suppression in the inhibitory group.\u003c/p\u003e \u003cp\u003eAt the same time, the expression levels of a series of thiamine regulon genes (i.e., \u003cem\u003ethi2\u003c/em\u003e, \u003cem\u003ethi4\u003c/em\u003e, \u003cem\u003ethi5\u003c/em\u003e, \u003cem\u003ethi7\u003c/em\u003e, \u003cem\u003ethi11\u003c/em\u003e, \u003cem\u003ethi12\u003c/em\u003e, \u003cem\u003ethi13\u003c/em\u003e, \u003cem\u003ethi20\u003c/em\u003e, and \u003cem\u003ethi73\u003c/em\u003e) were also downregulated in the inhibitory group. These genes are involved in the synthesis of thiamine pyrophosphate (TPP), a cofactor of enzymes that catalyze the decarboxylation of phenylpyruvate (Kneen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In our previous research on the molecular mechanisms regulating yeast tryptophol synthesis, we also found that 13 of these thiamine regulon genes were upregulated in high-yield 2-phenylethanol-producing cells (Gong et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since tryptophol and 2-phenylethanol share the Ehrlich pathway, this result is consistent with our current findings, suggesting that thiamine regulon genes positively regulate 2-phenylethanol synthesis in yeast. Furthermore, studies have shown that mutations in the lipoamide dehydrogenase gene (\u003cem\u003elpd1\u003c/em\u003e) result in a decreased 2-phenylethanol yield, suggesting that 2-phenylethanol synthesis requires the involvement of Lpd1 (Dickinson et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In this study, the expression of the \u003cem\u003elpd1\u003c/em\u003e gene was downregulated in the inhibitory group. Therefore, the downregulation of both the thiamine regulon genes and the \u003cem\u003elpd1\u003c/em\u003e gene in the inhibitory group further weakened the yeast Ehrlich pathway, leading to a further suppression of 2-phenylethanol synthesis.\u003c/p\u003e \u003cp\u003eBased on the above research results, we have preliminarily revealed the potential regulatory mechanism by which the inhibitory \u003cem\u003eCCDs\u003c/em\u003e suppress 2-phenylethanol synthesis in yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Specifically, the inhibitory \u003cem\u003eCCDs\u003c/em\u003e genes inhibit 2-phenylethanol production by suppressing yeast cell growth and downregulating the de novo biosynthetic pathway of 2-phenylethanol. This study provides important insights into the biosynthesis of 2-phenylethanol and \u003cem\u003eβ\u003c/em\u003e-ionone using yeast as a chassis. It also offers a new approach to regulating yeast growth. However, further research is needed, such as identifying the key sites at which inhibitory \u003cem\u003eCCDs\u003c/em\u003e genes suppress yeast growth and 2-phenylethanol synthesis, as well as further exploration of the genes through which inhibitory \u003cem\u003eCCDs\u003c/em\u003e regulate 2-phenylethanol synthesis in yeast.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e XH, MW and XG conceived and designed the study. FL and XG conducted the experiments. XG and GM analysed the data and drafted the manuscript. XH and MW revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (32560036), the Science and Technology Talent and Platform Project of Yunnan Provincial (202405AD350017), the Caiyun Postdoctoral Innovation Project of Yunnan Province (C615300504053), and the Key Scientific Research Project of China Tobacco Yunnan Industrial Co., Ltd. (2025XY01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe data are available within the article and its supplementary file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhrazem O, Rubio MA, Berman J, Capell T, Christou P, Zhu CF, G\u0026oacute;mez GL (2016) The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. 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J Mol B Enzym 134:105\u0026ndash;114. doi: 10.1016/j.molcatb.2016.10.003\u003c/li\u003e\n \u003cli\u003eZhong Y, Pan X, Wang R, Xu J, Guo J, Yang TX, Zhao JY, Nadeem F, Liu XT, Shan HY, Xu YJ, Li, XX (2020) \u003cem\u003eZmCCD10a\u003c/em\u003e encodes a distinct type of carotenoid cleavage dioxygenase and enhances plant tolerance to low phosphate. Plant Physiol 184 (1):374\u0026ndash;392. doi: 10.1104/pp.20.00378\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"Carotenoid cleavage dioxygenase, Saccharomyces cerevisiae, 2-Phenylethanol, Inhibition, Molecular mechanism","lastPublishedDoi":"10.21203/rs.3.rs-9333171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9333171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study reveals for the first time that overexpression of the plant-derived \u003cem\u003eCCD\u003c/em\u003e genes \u003cem\u003eNtCCD1-2\u003c/em\u003e, \u003cem\u003eNtCCD10\u003c/em\u003e, \u003cem\u003eZmCCD1\u003c/em\u003e, and \u003cem\u003eCaCCD4C\u003c/em\u003e significantly inhibits 2-phenylethanol biosynthesis in engineered \u003cem\u003eβ\u003c/em\u003e-ionone-producing \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strains (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001), whereas \u003cem\u003ePhCCD1\u003c/em\u003e and \u003cem\u003eNtCCD1-3\u003c/em\u003eexhibit no inhibitory effect. Using comparative transcriptomics and phenotypic analysis, we identified a novel regulatory mechanism by which these inhibitory \u003cem\u003eCCD\u003c/em\u003egenes suppress 2-phenylethanol production through impairing yeast growth and downregulating its de novo biosynthetic pathway. Mechanistically, inhibitory \u003cem\u003eCCD\u003c/em\u003egenes trigger biotic stress, downregulate the ribosome and multiple metabolic pathways, and upregulate the MAPK signaling pathway, thereby reducing cell biomass. In the inhibitory group, the high cell density-induced transcription factor Aro80 is downregulated, leading to the repression of \u003cem\u003earo10\u003c/em\u003e and a series of genes involved in 2-phenylethanol synthesis, together with \u003cem\u003elpd1\u003c/em\u003eand multiple thiamine regulon genes. These combined effects result in decreased 2-phenylethanol yield. This work deepens our understanding of the crosstalk between 2-phenylethanol and \u003cem\u003eβ\u003c/em\u003e-ionone biosynthesis in yeast, and offers a novel strategy for coordinating yeast growth and terpenoid metabolism.\u003c/p\u003e","manuscriptTitle":"Carotenoid cleavage dioxygenase genes negatively regulate 2-phenylethanol biosynthesis in yeast","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 18:06:49","doi":"10.21203/rs.3.rs-9333171/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","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}}],"origin":"","ownerIdentity":"312848b5-f2aa-4534-bff1-89ea06062557","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-08T13:26:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T22:30:51+00:00","index":15,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T13:48:33+00:00","index":14,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T13:44:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 18:06:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9333171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9333171","identity":"rs-9333171","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}