Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications.The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, β-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of β-d-xylose, β-d-glucose, β-d-galactose, α-l-arabinofuranose, α-l-rhamnose, β-d-glucuronic acid, and N-acetyl-β-d-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and β-d-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57–76 °C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by α--arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single- as well as double-arabinosylated xylooligosaccharides.To reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose.The obtained results lead to a better understanding of the hemicellulose-degrading capacity of The online version of this article (10.1186/s13068-018-1228-3) contains supplementary material, which is available to authorized users. Hemiic acid) , 4. Accoic acid) . Becauseic acid) .Effective enzymatic degradation of hemicelluloses is of great interest for various applications in industrial processes, such as efficient conversion of lignocellulosic biomass to fuels and chemicals, bleaching of paper pulp, and improvement of food properties . The majClostridium stercorarium [C. stercorarium degrades polysaccharides with a variety of hemicellulolytic enzymes, ferments a wide range of sugars, hexoses as well as pentoses, and produces acetate, ethanol, CO2, and H2 [C. stercorarium to the new genus Ruminiclostridium and to rename it Ruminiclostridium stercorarium [Saprophytic bacteria degrade lignocellulosic biomass by secreting glycoside hydrolases active on cellulose and hemicellulose . One of corarium , 11. The, and H2 –14. Rececorarium .C. stercorarium enzymes involved in the degradation of polysaccharides have already been characterised [C. stercorarium produces two cellulases that form a two-component cellulase system: Cel9Z  and Cel48Y . Cel9Z, which combines endoglucanase and exoglucanase activities, and the exoglucanase Cel48Y act synergistically to degrade crystalline cellulose [C. stercorarium have been described: Xyn11A, Xyn10B, and Xyn10C (sometimes referred to as Xyn10B) [l-arabinofuranosidases , and two β-d-xylosidases  [l-rhamnosidase Ram78A hydrolyses naringin to prunin and rhamnose and was applied for the production of rhamnose from citrus peel naringin [A number of cterised , 16, 17.ellulose –22. Thre Xyn10B) –25. The , Bxl3B) . All sev, Bxl3B) –30. The naringin . In addinaringin –34.C. stercorarium is one of the model organisms for carbohydrate-degrading bacteria, only a few individual enzymes and the enzyme systems for hydrolysis of cellulose and xylan have so far been described in detail. In addition, the bacterium produces a number of putative hemicellulolytic enzymes with still unidentified activity awaiting further characterisation for potential applications [Although ications .C. stercorarium DSM 8532 to reach a deeper insight into the polysaccharide-degrading machinery of this bacterium. A total of 50 proteins potentially involved in hemicellulose hydrolysis were screened for activity on numerous substrates, including hemicellulosic polysaccharides and related oligosaccharides, as well as various aryl-glycosides. We characterised the most promising enzymes for biotechnological applications and present a set of new enzymes for the degradation of xylan, xyloglucan, mannan, galacto- and glucomannan, arabinan, and galactan. The presented enzymes may function as a technology platform for utilisation of these polysaccharides through modification, partial or complete degradation.In this study, we investigated the majority of glycoside hydrolases from the genome of C. stercorarium cells grown on filter paper or oat spelt xylan as carbon source (5 g/L) in 500 mL GS2 medium until substrate depletion. The main culture was inoculated with 25 mL of a 50-mL starter culture [4)2SO4 solution [g, 20 min) and precipitated protein was dissolved in 200 µL 0.1 M MOPS reaction buffer . Protein concentration was determined by using a Bradford Protein Assay kit (Thermo Fisher Scientific).Culture supernatants were prepared from  culture . Extracesolution . For enzC. stercorarium DSM 8532 (NC_020134.1) and cloned into pET-24c(+) vectors (Merck) using the Gibson assembly method  [Escherichia coli BL 21 Star™ using ZYP-5052 auto-induction medium [E. coli proteins were removed by heat denaturation . During the purification, samples were taken to confirm the size and purity of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [Glycoside hydrolases (GH) of interest were identified using the classification of the Carbohydrate-Active Enzymes database (CAZy) , 38. Gen32 NC_02034.1 and y (IMAC) –43. Enzyn medium . Proteinn medium . After pDS-PAGE) . The ExPDS-PAGE) , 48.C. stercorarium. C. stercorarium was grown in 50 mL GS2 medium with 0.5% w/v cellobiose until early logarithmic phase. Bacterial cells were harvested by centrifugation . The cell pellet was resuspended in 5 mL 20 mM MES buffer (pH 6.5). Extracellular protein was precipitated overnight as described above. The precipitate was resuspended in 150 µL supernatant. Proteins were isolated and digested as described by Hornburg et al. [We employed a quantitative mass spectrometry-based proteomics strategy to identify secreted and intracellular proteins of g et al. . PeptideLC–MS/MS: The peptides were separated via a nano HPLC (Thermo Fisher Scientific) employing a C18 column. The HPLC was directly coupled to quadrupole-Orbitrap mass spectrometer via a nano electrospray ion source . HPLC and Q Exactive™ were operated as described previously , 50.C. stercorarium database containing 2688 forward entries as well as a list of common contaminants included in MaxQuant. Enzyme specificity was set to trypsin allowing cleavage N-terminally to proline and up to two miscleavages. The minimum length for peptides to be considered for identification was set to seven amino acids. Carbamidomethylation was defined as a fixed modification, and acetylation (N-terminus) and methionine oxidation were set as variable modifications. As false discovery rate (FDR) a cutoff of 1% was applied at the peptide and protein level. The median of replicates for protein length normalised log2 protein intensities  were used to compare protein abundances.We processed the MS raw data with MaxQuant (v. 1.3.8.2) . MS/MS spara-nitrophenyl (pNP) glycosides and polysaccharide substrates . Determined activities were quantified with 0.05–10 mg/L enzyme and reaction times between 5 min and 2 h. All reactions were carried out at 60 °C and pH 6.0 in a total volume of 50 µL or 500 µL using standard reaction buffer . The reaction was stopped by addition of two volumes of 1 M Na2CO3 and absorption of pNP was measured at 405 nm. Activity was calculated by comparison to pNP as standard.Activity determination was performed with g, 5 min, 4 °C) and reducing sugars were determined in a 50-µL sample. For qualitative activity determination, 5 mg/L of secreted protein or recombinant enzyme was incubated with the polysaccharides in standard reaction buffer at pH 6.5 and 60 °C for 16 h. Quantitative determination of the enzyme activity was realised using selected polysaccharides with 0.05–24 mg/L of recombinant enzyme and reaction times between 30 min and 2 h. The pH dependence of enzyme activity was determined in 100 mM citrate-phosphate buffer  with pH between pH 4.0 and pH 8.0 (pH at 60 °C) in steps of 0.5 at 60 °C. Temperature dependence of enzyme activity was also determined in 100 mM citrate-phosphate buffer at each enzyme’s optimal pH. 10 different points were measured over a temperature gradient spanning 30 °C. The gradient was chosen depending on the temperature optimum of the enzyme. For the determination of the specific activity of the enzymes, hydrolysis was performed in 100 mM MOPS buffer  at pH 6.0 or 6.5 (Abn43A) and optimal temperature. All enzymatic assays were performed in triplicate using a xylose calibration curve.The hemicellulolytic activity of secreted or recombinantly produced enzymes was determined by measuring the increase of reducing groups caused by polysaccharide hydrolysis with the 3,5-dinitrosalicylic acid (DNSA) assay . One uni5) and a number of arabinoxylan oligosaccharides (AXOS): 32-α-l-arabinofuranosyl-xylobiose (A3X), 23-α-l-arabinofuranosyl-xylotriose (A2XX), 23,33-di-α-l-arabinofuranosyl-xylotriose (A2+3XX), 23,33-di-α-l-arabinofuranosyl-xylotetraose (XA2+3XX), 33-α-l-arabinofuranosyl-xylotetraose (XA3XX), and a mixture of XA3XX and 23-α-l-arabinofuranosyl-xylotetraose (XA2XX) (Megazyme). Hydrolysis was performed overnight at optimal pH and temperature of each enzyme, with 2 mg/L enzyme and 20 mg/L oligosaccharide or oligosaccharide mixture in 500 µL volume. The degradation products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as described by Mechelke et al. [Selected enzymes involved in xylan degradation were incubated with xylopentaose  prior to analysis. The volume for 1 µg xylose equivalent, determined by DNSA assay, was separated by TLC as described by Zverlov et al. [d-mannitol as internal standard (ISTD). Degradation products were identified by comparing them to a number of monosaccharide (2 mg/L) and oligosaccharide (10 mg/L) standards: arabinose (A), xylose (X), xylobiose (X2), -triose (X3), -tetraose (X4), -pentaose (X5), and arabinoxylooligosaccharides (AXOS) as listed above.Analysis of poly- and oligosaccharide hydrolysis products was carried out with thin-layer chromatography (TLC) or high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Hydrolysates were centrifuged . To determine their activity, secreted proteins were prepared from C. stercorarium grown on oat spelt xylan or filter paper. Both protein preparations displayed activities on 15 of 26 screened polysaccharides . The polysaccharide substrates for the activity screening of the single recombinant enzymes were selected based on the determined activities of the extracellular enzymes.The activity on insoluble wheat arabinoxylan was 60% lower than on soluble wheat arabinoxylan (set to 100%). Compared with their xylanolytic activity the secreted enzymes had merely low activity on glucomannan, various galactans, xyloglucan, and polygalacturonic acid. Interestingly, the xylan protein preparation displayed higher relative activity on mixed linkage glucans, especially on barley β-glucan (79%) and pullulan (24%), compared to the enzyme preparation from filter paper medium (57% and 5%). While the determined activity of the xylan protein preparation was higher for mixed linkage glucans, the protein preparation from filter paper appeared to be more active on birch and oat spelt xylan. C. stercorarium, distributed over 34 GH families [E. coli for further investigation. Nineteen enzymes of 10 GH families were excluded, because enzymes of these GH families require NAD + (GH4), are mostly parts of starch , chitin (GH18), peptidoglycan (GH23), and cellulose degradation (GH48), or act as phosphorylases. Two enzymes of GH family 36, usually α-galactosidases, were also excluded  lists 71 proteins with glycoside hydrolase (GH) modules for families , 38. BasE. coli culture for BglA to 253.0 mg/L for Bxl39A. One protein, the endo-β-1,4-galactanase Gal53A was produced in a shortened version with only 909 amino acids (AA) instead of the complete 1784 AA, because the full-length gene could not be cloned into the pET-24c(+) expression vector. PCR oligonucleotide primer was designed to bind 1296 bp behind the 5′ end and 1329 bp in front of the 3′ end of the galA gene to include the complete GH53 module while excluding the S-layer homology modules  in the secretome, while they were not detected in the intracellular proteome  or belonged to the 5% (Cel9Z) and 40% (Xyn10C) least abundant proteins of the intracellular proteome. These four enzymes, as well as Gal53A and Man26A, two enzymes ranking middle in the secretome and among the 10% of most rare proteins in the intracellular proteome, are predicted to be produced with N-terminal signal peptides and, therefore, can be regarded as secretory proteins. This indicates that C. stercorarium has an effective secretion system for endo-acting glycoside hydrolases, especially for the most important xylanases. Although 32 of the studied proteins do not appear to have a signal peptide, they were found in the secretome. This might be due to the release of proteins from cells by cell lysis or to alternative secretion pathways.To examine if the heterologously produced enzymes of this study are also produced by C. stercorarium glycoside hydrolases which are here characterised the first time, including three enzymes with non-classified, possibly new GH modules. The recombinant enzymes exhibited activities for the same polysaccharides as the secretome, except for pachyman, pullulan, and polygalacturonic acid, and in addition, the enzymes Abn43A and Man26A hydrolysed arabinan and mannans, respectively. One enzyme with a GH105 module, Xyn105F, displayed xylanase activity, which is the first description of this activity for GH family 105.The activity screenings revealed 20 new pNP-glycosides, 27 of the 32 enzymes with determined activity were able to hydrolyse the glycosidic bond in one or more of the artificial substrates. The assay revealed three α-l-arabinofuranosidases , two β-d-glucosidases , three β-d-galactosidases , two α-l-rhamnosidases , seven β-d-xylosidases , one β-d-glucuronidase (Uid2A), and one N-acetyl-β-d-glucosaminidase (Nag3A), which showed their main activities towards pNP-glycosides . More than half of the enzymes had side activities for further pNP-glycosides. The xylanases Xyn10D, Xyn10E, and Xyn105F, the arabinanase Abn43A, and the enzymes RamB and Xyl43A showed insignificant activities under the employed standard assay conditions. In a further assay the cell extract of E. coli producing RamB was incubated overnight with pNP-α-l-rhamnopyranoside at three different temperatures. Whereas the activity of the RamB preparation proved to be low at 60 °C (0.04 U/mg), it was 2.3-fold (0.10 U/mg) and 9.7-fold higher (0.43 U/mg) at 55 °C and 50 °C, respectively.In activity screenings with d-galactosidases , two β-d-xylosidases , one endo-glucanase (Cel9Z), six xylanases , one mannanase (Man26A), one arabinoxylan arabinofuranohydrolase (Axh43A), two α-l-arabinofuranosidases , one arabinanase (Abn43A), and one endo-galactanase   of each enzyme Table . The galAll six xylanases acted as endo-xylanases and showed their highest specific activity for soluble wheat arabinoxylan. Xyn10B and Xyn10C, both containing a GH10 module, additionally hydrolysed the β--glucan backbone of barley β-glucan and lichenin. While the activity of Xyn10B for arabinoxylan was 4.6 times higher than that of Xyn10C, the activities for β-glucan and lichenin were nearly the same. The xylooligosaccharides released by the xylanases differ in length: Xyn10B and Xyn10C mainly produced xylose and xylobiose while Xyn11A, containing a GH11 module, mainly produced xylobiose and xylotriose. All three enzymes released a number of additional oligosaccharides during the hydrolysis of arabinoxylan -linked backbone of arabinan and released arabinose from galactan and pectic galactan. Three enzymes, Arf51B, Arf43C, and Axh43A, liberated arabinose from arabinan. Arf51B and Axh43A in addition released arabinose from soluble wheat arabinoxylan, and Arf43C released arabinose from galactan and pectic galactan. While Arf51B showed the highest specific activity for arabinan, Axh43A displayed the highest activity for arabinoxylan and only a low activity for arabinan. Arf43C showed the highest activity for potato pectic galactan. The mannanase Man26A degraded the β--linked backbones of mannan, galactomannan and glucomannan. In general, the determined activities for the exo-acting enzymes Axh43A, Arf43A, Arf51B, Arf43C, Bxl3B, Bga35A, and Bga2B, were significantly lower than for endo-acting enzymes.Cel9Z hydrolysed barley β-glucan and lichenin to oligosaccharides and degraded the backbone of glucomannan, which is composed of β--linked C. stercorarium with promising hemicellulolytic activities were determined. All enzymes preferred a slightly acidic pH and showed the highest activity between pH 5.0 and pH 6.5. While the pH optima were nearly identical, the temperature optima differed significantly and ranged from 49 to 81 °C , β-xylosidases  and arabinose-releasing enzymes  in greater depth, purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were hydrolysed and the degradation products were analysed by HPAEC-PAD -linked arabinosylations. Xyn10D cleaved off the terminal xylose at the reducing end of all AXOS with the exception of A3X.The secreted proteins of l-arabinose residue off singly and doubly substituted AXOS releasing xylooligosaccharides and arabinose. Both β-xylosidases, Bxl3B and Arf43A, sequentially degraded xylopentaose to xylose. Arf43A and Bxl3B cleaved off the terminal xylose at the non-reducing end of XA2XX, but only Arf43A was able to also hydrolyse XA3XX and XA2+3XX.Arf51B and Axh43A containing a GH51 and two GH43 modules, respectively, completely cleaved the α- and α--linked 3X, XA2+3XX, and A2+3XX. The amount of released A3XX was approximately twice as high as the amount of XA2+3XX and A2+3XX. Xyn11A produced XA3XX, XA2+3XX, A2+3XX, and an unknown substance with a retention time at 43.1 min. While Xyn10B produced more A2+3XX than XA2+3XX, Xyn11A produced more XA2+3XX. In total, Xyn10B released larger amounts of XOS and AXOS from wheat arabinoxylan, due to its broad substrate specificity, even though the specific activity of Xyn11A was higher.To further investigate the substrate specificities Figs.  and 5 anC. stercorarium, Clostridium thermocellum and closely related species were identified as major hydrolytic bacteria in biogas fermenters [C. thermocellum and C. stercorarium degraded 76% of maize silage and performed only slightly less effectively than the best thermophilic bacterial consortium isolated from a biogas plant [C. thermocellum degrades and utilises cellulose with high efficiency, C. stercorarium seems to be specialised in the degradation of hemicellulose, since this organism harbours a great number of genes involved in the hydrolysis of hemicellulose in its genome and has the capability to utilise the degradation products, hexoses as well as pentoses [Utilisation of renewable biomass for the generation of fuels, electricity, and heat is a climate-friendly addition to the common energy production technologies . In ordermenters . It was as plant . Whereaspentoses –14.C. stercorarium cultures grown on oat spelt xylan or filter paper, degraded 15 of the 26 tested substrates, preferably soluble xylans and barley β-glucan  were not accessible [Thirty-two of the investigated hemicellulolytic enzymes displayed polysaccharide-degrading activities when produced heterologously while no activity could be determined for the remaining 18 proteins. The reasons could be missing substrates or pH and temperature optima that differ significantly from the applied assay conditions  as shown for RamB, which had a significantly higher activity at 50 °C than at 60 °C. The pH optimum of the previously characterised enzyme Xyn43B is pH 3.5, while the activity of the previously characterised α-cessible , 30. Furcessible .Clostridium stercorarium has an effective secretion system for endo-acting glycoside hydrolases, especially for the most important xylanases. However, four of the proteins were apparently not present in the proteome. Whether these proteins are produced in the presence of alternative carbon sources remains to be determined.d-xylopyranose backbone, which is substituted by short glycoside and ester groups. Substitutions comprise l-arabinose (Ara), d-glucuronic acid (GlcA) or 4-O-methyl-α-d-glucuronic acid (MeGlcA), d-xylose (Xyl), galactose, acetic acid and phenolic acid esters. Xylan in hardwood, usually referred to as 4-O-methyl-d-glucurono-d-xylan (MGX), has mainly MeGlcA substitutions attached at position O-2 of the xylose chain with an average Xyl:MeGlcA ratio of 10:1. Arabinoxylan (AX), the major hemicellulose component in grasses and cereals, is substituted by α-l-arabinofuranosyl residues at position O-2 and/or O-3 of the xylose monomers [All xylans share a β--monomers .C. stercorarium is reflected by its multitude of xylanases. Besides the well-characterised extracellular xylanases Xyn11A, Xyn10B, and Xyn10C, the activity screening revealed three new endo-β--xylanases: Xyn10D, Xyn10E, and Xyn105F. All six xylanases were most active on soluble wheat arabinoxylan. Xyn10B and Xyn10C further showed activity for mixed linkage glucans as previously reported for both enzymes [The apparent importance of xylans as growth substrates for  enzymes . Xyn11A  enzymes . One pos enzymes .O-2. Xyn10D differed significantly and hydrolysed the second β--linkage behind the arabinosylation at the reducing end of AXOS. A similar hydrolytic mechanism was described for the xylanase Xyn10B of Herbinix hemicellulosilytica [The substrate and product specificities of Xyn11A and Xyn10B are in accordance with the product pattern described for GH11 and GH10 xylanases. GH11 xylanases hydrolyse only unsubstituted xylan regions producing longer XOS and AXOS, while GH10 xylanases are able to hydrolyse the xylan backbone at the non-reducing end next to an arabinosylated xylose. GH10 xylanases produce mainly xylose, xylobiose and short AXOS , 64. Unlsilytica . This hiTo our knowledge, Xyn105F is the first xylanase with a GH105 module. A relatively low but significant activity was measured for soluble arabinoxylan, while Xyn105F was also active on xylopentaose, releasing xylose and xylobiose. Therefore, we discovered a new activity for GH family 105, which until now had exclusively comprised unsaturated rhamnogalacturonyl and β-glucuronyl hydrolases which also might be the main activity of Xyn105F , 65. HowC. stercorarium under the growth conditions of this study.The pH and temperature optima of the four xylanases Xyn11A, Xyn10B, Xyn10C, and Xyn10D are around pH 5.5–6.0 and 65–76 °C, in accordance with previous work , 66. ThepNP-glycosides, as described previously [pNP-β-d-xylopyranoside and, like Bxl3B, released only xylose units from XOS and AXOS [Arf51B, Bxl3B, and Arf43A had little activity on xylans and showed α-arabinofuranosidase and β-xylosidase activity towards eviously . Arf43A and AXOS , 26. As and AXOS . Arf51B and AXOS . Axh43A and AXOS .C. stercorarium. The new enzymes and the detailed information about their substrate and product specificities are helpful for improving the reconstituted enzyme system for xylan hydrolysis. A substitution of Arf51B with Axh43A, for example, could accelerate the debranching of arabinoxylan while Xyn10B and Xyn10D are able to hydrolyse more AXOS than Xyn11A and Xyn10C.Xylanases, α-arabinofuranosidases, including arabinoxylan arabinofuranohydrolases, and β-xylosidases act synergistically to completely hydrolyse arabinoxylan. Adelsberger et al.  showed td-glucose oligomers, which are separated by single β--linkages. Xyloglucans (XG) have a β--d-glucopyranose backbone branched with α--d-xylopyranoside units. The xylopyranoside residues can be substituted by β--linked d-galactopyranose, a further xylopyranose, or other sugars [d-glucopyranose and β-d-mannopyranose. The main polymeric chain is acetylated and substituted by α--d-galactopyranosyl residues [d-mannopyranose units and are also substituted with galactosyl residues. Slightly galactosylated (4–15%) polymers are referred to as glucomannans and mannans [Mixed linkage glucans are linear polysaccharides comprised of β-1,4)-linked ,4-linkedr sugars . Galactoresidues . Galactolated 4–1% polymerC. stercorarium and showed activity towards mixed linkage glucans and glucomannan. In contrast to previous characterisations, only insignificant activity towards microcrystalline cellulose (Avicel) was determined [d-galactopyranosyl substitution of xyloglucan. Man26A contains a CBM35 module that commonly binds to mannan and mannooligosaccharides [The endo-glucanase Cel9Z was secreted by termined , 20. Thetermined . The dettermined . Unlike charides .l-arabinofuranoside backbone branched with α-- and/or α--linked l-arabinosyl oligosaccharides at position O-2 or O-3. Galactans are linear β--d-galactopyranoside polymers substituted by single α-l-arabinofuranoside units at position O-3 [Arabinans and (arabino)galactans are side chains of the pectic polysaccharide rhamnogalacturonan I. In addition, α--linked arabinan structures may also exist as free polymers unattached to the pectic domains . Arabination O-3 , 73.As described above, Arf51B released arabinose from arabinan, probably by cleaving off the α-- and α--linked arabinosylations . Four neC. stercorarium and probably binds to the cell surface [Gal53A showed endo-β-galactanase activity towards galactan and pectic galactan in accordance with the common activity for GH family 53 . The nat surface . CBM61 b surface , indicat surface .pNP-glycoside-cleaving activities were determined. Beside the previously characterised enzymes Bxl39A, Bgl3Z, Xyl43A, Xyl43B, and Ram78A, the activity screening revealed ten new enzymes: β-d-galactosidases Bga2C, Bga2D, and Bga2E; β-d-xylosidases Bxl43C and Bxl31D; β-d-glucosidase BglA; α-l-arabinofuranosidase ArfD; α-l-rhamnosidase RamB; β-d-glucuronidase Uid2A; and N-acetyl-β-d-glucosaminidase Nag3A [pNP-β-d-xylopyranoside, thereby adding a new activity to the GH family 31 [For 15 glycosidases only se Nag3A , 23, 28.se Nag3A , 69, 76.amily 31 , 69. It Clostridium stercorarium serves as a model organism for the degradation of lignocellulosic biomass, especially for the hydrolysis of hemicellulose. However, only enzymes for the degradation of cellulose and xylan have been well characterised so far [C. stercorarium DSM 8532 and we present 20 new enzymes for the bioconversion of hemicellulose. We characterised the first C. stercorarium enzymes involved in the degradation of xyloglucan, mannan as well as galacto- and glucomannan, arabinan, and galactan. The determined activities of native extracellular and recombinantly produced enzymes lead to a better understanding of the hemicellulose-degrading capacity of the bacterium and the involved enzymes. Additionally, we discovered new activities not reported before for the GH families 31 and 105 and present three enzymes possibly belonging to new GH families. While the optimal temperature for the majority of the characterised enzymes (57–76 °C) matches the optimum growth temperature of C. stercorarium (60–70 °C), the enzymes prefer a slightly more acidic pH than the bacterium, pH 5.0–6.5 instead of pH 7.3 [d so far , 35. In C. stercorarium specific enzyme systems for the complete degradation of defined polysaccharides and hemicellulose. The degradation of xyloglucan or mannan requires at least one xyloglucanase and one β-mannosidase, respectively, activities that have not yet been identified in C. stercorarium [C. stercorarium secretomes secreted in the presence of various polysaccharides could be applied to identify the enzymes the bacterium employs to their degradation. The hydrolysis of complex polysaccharides requires further accessory enzymes like carbohydrate esterases (CE) and polysaccharide lyases (PL), which were not subject of this study [Enzyme combinations selected from the newly characterised glycoside hydrolases, especially of enzymes with endo- and exo-activity, could lead to a complete degradation of mixed linkage glucan, arabinan or galactan to monosaccharides, as previously described for arabinoxylan . Howevercorarium . In combis study .l-arabinofuranosidases with the ability to hydrolyse single and double substitutions, like Arf51B and Axh43A, are of great interest because these substitutions inhibit or even prevent arabinoxylan and pectin from complete degradation to their monomers [Detailed substrate and product specificities as determined for xylanases, α-arabinofuranosidases and β-xylosidases increase the understanding of the reaction mechanism and provide a promising platform for biotechnological applications. Such applications often require highly specific activities. Especially α-monomers , 67.C. stercorarium are a rich source of specific enzyme activities needed for industrial applications. By cloning hitherto unknown gene families, as done in this study, even new protein families can be detected.Bacteria active in hemicellulose degradation such as Additional file 1. This file contains additional tables and figures as follows: (i) Table S1. PCR primer for the amplification of the 50 selected glycoside hydrolase genes of C. stercorarium. (ii) Table S2. Polysaccharide and p-nitrophenyl substrates including the final concentration in enzymatic assays. (iii) Table S3. Glycoside hydrolase (GH) families present in the C. stercorarium genome. (iv) Figure S1. SDS-PAGEs of 10 examples of the 50 C. stercorarium proteins recombinantly produced by E. coli and purified by IMAC. (v) Figure S2. Schematic structure of the glycoside hydrolases with proven activity from C. stercorarium. (vi) Table S4. Studied enzymes in the secretome or intracellular proteome of C. stercorarium analysed by LC-MS/MS. (vii) Figure S3. Hydrolytic products of different polysaccharides analysed by Thin-layer chromatography (TLC). Polysaccharides were hydrolysed by a) Xyn10C, b) Xyn11A, c) Xyn10D, d) Arf51B, e) Arf43C, f) Axh43A, g) Abn43A, h) Cel9Z, i) Man26A, j) Bga35A, k) Bga2B. (viii) Figure S4. Relative activity of characterised enzymes at different pH. pH profiles of a) xylanases Xyn11A & Xyn10B-D, b) Axh43A and α-arabinofuranosidases Arf51B & Arf43C, c) β-galactosidases Bga2B & Bga35A, d) Bxl3B, Cel9Z, Man26A, and Abn43A. (ix) Figure S5. Relative activity of characterised enzymes at different temperatures. Temperature profiles of a) xylanases Xyn11A & Xyn10B-D, b) Axh43A and α-arabinofuranosidases Arf51B & Arf43C, c) β-galactosidases Bga2B & Bga35A, d) Bxl3B, Cel9Z, Man26A, and Abn43A.Additional file 2. This file contains the complete processed data set of the LC-MS/MS proteome analysis: LC–MS/MS data of the secretome (SU) and the intracellular proteome (PE) of C. stercorarium.


Dear Editor,Angiosarcoma is a rare malignancy, which accounts for approximately 1%–2% of soft-tissue sarcomas . Howevern = 62) of the patients were men. Forty-three (48.3%) patients had localized tumor at the time of diagnosis, whereas 46 (51.7%) patients had advanced disease . The locations of the tumors at diagnosis were variable. The head and neck location including the scalp  was the most common primary site. The tumor location of 22 (24.7%) and 15 (16.9%) patients were at the liver and heart, respectively. Twelve (13.5%) cases were at the trunk including three cases each located at the lung, pulmonary artery, and ovary, and one case each at the chest wall, diaphragm, and pleura. There were 7 cases (7.9%) originated from the spleen and 4 (4.5%) breast angiosarcoma. The number of cases of angiosarcoma located at the extremities, bone, and nasal cavity were 2 (2.2%), 2 (2.2%) and 1 (1.1%), respectively  between January 1992 and December 2014were evaluated. The median age at diagnosis was 60 years (range 20–85 years), and 69.7% (Thirty-nine (43.8%) patients were treated with curative intent treatments, including 20 patients with surgery only, 9 patients with surgery and radiotherapy (RT), 7 patients with surgery and CT, and 3 patients with surgery, RT and CT. Thirty-five (39.3%) patients were treated in palliative setting, including 20 patients with palliative CT only , 8 patients with palliative surgery only , 4 patients with surgery and CT, 2 patients with RT only , and 1 patients with CT and RT. Twelve (13.5%) advanced patients were treated with only best supportive care  . In regard to OS, age above 60 years, poor performance status, advanced disease and primary liver angiosarcoma were associated with poor survival . Multivariate analyses showed that poor performance and advanced disease were significant prognostic factors for PFS , while old age, poor performance, and advanced disease were significant prognostic factors for OS   and overall survival (OS) were different for different primary tumor site. The median PFS was longest in angiosarcoma located at the trunk , followed by the breast , heart , scalp or head and neck , spleen , liver , and others. The median OS was longest in patients with angiosarcoma located at the trunk , followed by the breast , scalp or head and neck , heart , spleen , liver , and others. With median follow-up of 10.3 months (range 0.1–179.3 months) in surviving patients, the 1-year OS rate was 43.4%. In univariate analysis, poor performance status, advanced disease, and primary liver angiosarcoma were associated with shorter PFS (n = 3) or difficulty for surgical resection (n = 1). In patients with localized tumors, those who had curative resection demonstrated significantly better PFS  and OS  than those who did not had curative operation. The median PFS and OS of patients who had curative surgery only were 6.9 months (95% CI 2.6–11.3) and 21.6 months (95% CI 5.4–37.8), respectively. The median PFS of patients who had curative surgery and CT were 30.8 months (range 13.2–48.5 months), which was numerically the longest compared to the patients who received other treatment. In these patients, the primary tumor sites were the ovary (n = 3), right atrium of heart (n = 2), lung (n = 1), and pulmonary artery (n = 1). For the 46 patients with advanced disease, their median PFS and OS were 3.4 months (range 2.5–4.3 months) and 4.7 months (range 3.4–6.4 months), respectively  and 21.6 months (95% CI 12.2–30.1), respectively. Among them, 39 (90.7%) patients had curative surgery. Four (9.3%) patients did not undergo surgery because of the patient’s refusal (P = 0.030) and OS (P = 0.001) than those who were treated with best supportive care  and OS (P = 0.221) between the patients who received different first-line palliative CT regimens.A total of 20 patients received palliative CT . Regardless of the regimens prescribed, patients who received palliative CT had significantly longer PFS  and overall survival (B).


Here, we report the design and synthesis of a new thermally activated delayed fluorescence emitter, 3Ph2CzCzBN. A sky-blue device based on 3Ph2CzCzBN exhibits a high external quantum efficiency of 16.6% at 1000 cd m−2. The device shows a sky-blue electroluminescence of 482 nm and achieves Commission Internationale de l’ Eclairage coordinates of . The sky-blue device exhibits a superb LT90 of 38 h. This is the first demonstration of high-efficiency and stable sky-blue devices (CIEy < 0.4) based on delayed fluorescence, which represents an important advance in the field of blue organic light-emitting diode technology.Organic light-emitting diodes utilizing thermally activated delayed fluorescence is a potential solution for achieving stable blue devices. Sky-blue devices  for display and lighting applications is limited by the need for blue emitters that show efficient and stable operation. Here, the authors report stable and efficient thermally activated delayed fluorescence OLEDs based on a sky-blue emitter. In order to commercialize OLEDs for various electronic applications, the color purity11, efficiency15, driving voltage19 and device stability27of OLEDs are some of the important parameters to be considered.Organic light-emitting diodes (OLEDs) are attractive technology for display and lighting due to their high flexibility, high efficiency and light-weighty < 0.4 have been developing over 15 years28, and the resultant PhOLEDs with high efficiencies and long operation times are still difficult to achieve simultaneously at high brightness30. Regarding to the device stability, the LT90s  of blue and sky-blue PhOLEDs are usually less than 10 h, which are insufficient for commercialization30. In 2014, Forrest and coworkers reported an improved stability of a single-unit blue phosphorescent OLEDs (dopant: Ir(dmp)3) with the use of a graded-dopant-concentration technique in the emitting layer (EML)20. The graded device achieved a moderate external quantum efficiency (EQE) of 9.5% at 1000 cd m−2 with a driving voltage of 7.7 V. Such devices exhibited a LT90 of ~40 h at an initial luminance of 1000 cd m−2. Recently, in 2017, Forrest and coworkers further extended the device lifetime by introducing an extra molecule functioning as a hot excited-state manager in the EML of the graded devices21. The managed device achieved a similar EQE of 9.6% with a higher driving voltage of 9.0 V at 1000 cd m−2, and resulted in a lengthened LT90 of ~140 h. Nonetheless, the EQEs of both graded and managed devices were still less than 10% at 1000 cd m−2, rendering them less competitive with the commercial fluorescent blue emitters. Moreover, the driving voltages for both devices at 1000 cd m−2 were around 8 V or above, which were rather high for practical use.Blue and sky-blue phosphorescent OLEDs (PhOLEDs) with CIE31. TADF is believed to be a potential solution to achieve stable blue OLEDs with an internal quantum efficiency (IQE) of 100%. Since then, there are numerous reports on efficient blue and sky-blue TADF OLEDs with CIEy < 0.434. However, reports on stable blue and sky-blue TADF OLEDs are still rare and limited25. Again, the LT90s  of blue and sky-blue TADF OLEDs are always less than 10 h25. Recently, Adachi and coworkers reported a stable sky-blue TADF OLED by the use of a n-type host material (SF3-TRZ)24. The sky-blue TADF OLED was able to achieve a EQE of 7% with a driving voltage of 5.7 V at 1000 cd m−2. The corresponding device exhibited a comparable LT90 of ~20 h  to those reported sky-blue PhOLEDs21. However, same as PhOLEDs21, the EQE of the stable sky-blue TADF OLED was still below 10%. As a result, there is still a room for designing new TADF emitters to concurrently achieve blue or sky-blue OLEDs with a high EQE and a high stability at high brightness.Meanwhile, efficient OLEDs based on thermally activated delayed fluorescence (TADF) were first demonstrated by Adachi and coworkers in 20123Ph2CzCzBN) exhibits a high external quantum efficiency of 16.6% at 1000 cd m−2. The device shows a sky-blue electroluminescence of 482 nm and achieves Commission Internationale de l’ Eclairage coordinates of . The sky-blue device exhibits a superb LT90 of 38 h. This is the first demonstration of high-efficiency ( > 15%) and stable sky-blue devices (CIEy < 0.4) based on delayed fluorescence at 1000 cd m−2.Here we show that a sky-blue device based on a new fluorescence emitter , in which a good mixing of the 3LE state with the lowest charge transfer triplet excited state (3CT) can facilitate a faster reverse intersystem crossing rate (kRISC)38. Moreover, such faster kRISC results in a shorter delayed lifetime of the TADF emitters, which eventually improves the device stability since triplet exciton intensity in devices will be reduced. Herein, we synthesized a new blue benzonitrile-based TADF emitter, 3Ph2CzCzBN, in two steps  should show a shorter delayed lifetime and a longer device lifetime than that of the homo-donor-based reference compound (4CzBN). Quantum-chemical calculations based on 3Ph2CzCzBN and 4CzBN were first performed by time-dependent density functional theory (TD-DFT) at B3LYP/6–31 G(d) level. The calculated highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), singlet (S1) and triplet (T1) energy levels, oscillator strength (f) and ΔESTs were depicted in Fig. 3Ph2CzCzBN and 4CzBN showed similar ΔESTs of 0.13 and 0.14 eV and high oscillator strengths (fs) of 0.0619 and 0.0818, respectively. The calculated HOMOs of both compounds were mainly localized on the carbazole donors with some extensions on the benzonitrile acceptor, whereas the LUMOs are fully localized on the benzonitrile acceptor.The long-lived triplet excitons are believed to be lethal to the device stability for both phosphorescent and TADF OLEDs, because hot triplet excitons are generated by triplet-triplet annihilation or triplet-polaron annihilation processeseps Fig. . 4CzBN w3Ph2CzCzBN and 4CzBN were first examined in toluene solution at the concentration of 10−5 M. Both compounds showed absorption bands from 290 to 450 nm, in which the absorption bands at around 400–450 nm were assigned as the intramolecular charge transfer (ICT) bands  of 3Ph2CzCzBN and 4CzBN were found to be 16 and 9%, respectively, before degassing. After purging N2 into the solutions, the corresponding PLQYs of 3Ph2CzCzBN and 4CzBN were greatly enhanced and found to be 95 and 63% −1,1’-biphenyl (mCBP) resulted in slightly lower PLQYs of 91 and 51%  of > 500 and 455 °C, respectively  at 240 °C  were fabricated using the following structure: Indium–tin oxide (ITO)-coated glass (100 nm)/ HATCN (10 nm)/ Tris-PCz (30 nm)/ mCBP (5 nm)/ 15 wt% of 3Ph2CzCzBN (device A) or 15 wt% of 4CzBN (device B): mCBP (30 nm)/ T2T (10 nm)/ BPy-TP2 (40 nm)/ LiF (0.8 nm)/ Al (100 nm), in which hexaazatriphenylenehexacarbonitrile (HATCN) was the hole-injection layer, 9,9’,9”-triphenyl-9H,9’H,9“H−2,3’:6’,2”-tercarbazole (Tris-PCz) was the hole-transporting layer, 3,3’-di(9H-carbazol-9-yl)-1,1’-biphenyl (mCBP) was theelectron-blocking layer, 15 wt% of 3Ph2CzCzBN (device A) or 15 wt% of 4CzBN (device B) doped in mCBP was the emitting layer (EML), 2,4,6-tris(biphenyl-3-yl)−1,3,5-triazine (T2T) was the hole-blocking layer, 2,7-ditriphenylene (BPy-TP2) was the electron-transporting layer and LiF and Al were the electron injection layer and cathode, respectively. The materials used and the device structure for devices A and B were depicted in Supplementary Figures To evaluate the electroluminescence (EL) properties of A and B were shown in Supplementary Figures  A and B showed sky-blue emission with maximum peaks at 482 and 477 nm, respectively. The CIE coordinates of device A and B were found to be  and , respectively. Device A showed a maximum EQE of 15.9% and dropped to 15.0% at 1000 cd m−2, whereas device B showed a severe rolloff with a maximum EQE of 9.7% and a much lower EQE of only 5.8% at 1000 cd m−2. The driving voltages of devices A and B were found to be 6.3 and 7.6 V at 1000 cd m−2, respectively. Devices A and B were driven at a constant DC current at room temperature for testing the device stability. The LT90s of devices A and B were found to be 4 and 0.1 h, respectively. Hole-only and electron-only devices (HOD and EOD) based on the structures of devices A and B were also fabricated to understand more about the carrier transport properties  than that of 4CzBN (13.7 μs). The shortening of the delayed lifetime of 3Ph2CzCzBN lowered the triplet exciton intensity in EML, in which the rolloff in device A was significantly reduced. Moreover, the diminished triplet exciton intensity also avoided exciton-exciton annihilation and exciton-polaron annihilation, thus enhancing the device stability.The detailed OLEDs performance for devices C and D were fabricated with the following device structure: Indium–tin oxide (ITO)-coated glass (100 nm)/ HATCN (10 nm)/ Tris-PCz (30 nm)/ mCBP (5 nm)/ 15 wt% of 3Ph2CzCzBN (device C) or 20 wt% of 3Ph2CzCzBN (device D): mCBP (30 nm)/ SF3-TRZ (10 nm)/30 wt% of Liq: SF3-TRZ (50 nm)/ Liq (2 nm)/ Al (100 nm), in which HATCN was the hole-injection layer, Tris-PCz was the hole-transporting layer, mCBP was the electron-blocking layer, 15 wt% of 3Ph2CzCzBN (device C) or 20 wt% of 3Ph2CzCzBN (device D) doped in mCBP was the emitting layer (EML), 2-−4,6-diphenyl-1,3,5-triazine (SF3-TRZ) was the hole-blocking layer, 30 wt% of 8-quinolinolato lithium (Liq): SF3-TRZ was the electron-transporting layer and Liq and Al were the electron injection layer and cathode.To further improve the device stability, devices A, device C showed the identical EL peak maximum of 482 nm with the CIE coordinates of . Device C also showed a same maximum EQE of 15.9%. The turn-on voltage of device C was 3.7 V, which was lower than that of device A (4.0 V). Furthermore, the driving voltage of device C was found to be 6.4 V at 1000 cd m−2. Although device C showed a slightly lower EQE of 14.3% at 1000 cd m−2, the LT90 of device C was significantly improved by four times to 16 h. Further optimizing the device performance, the doping concentration of 3Ph2CzCzBN was increased from 15 wt% (device C) to 20 wt% (device D). In device D, the EL peak maximum was mildly redshifted to 486 nm with CIE coordinates of . Device D showed a maximum EQE of 17.9%. Interestingly, device D showed an insignificant rolloff and achieved a EQE of 17.2% at 1000 cd m−2. Meanwhile, a driving voltage of 5.8 V was obtained. Most importantly, the LT90 of device D was lengthened to 32 h, which is a 320-times increase comparing to that of the 4CzBN-based device. The detailed device characteristics of device C and D were depicted in Fig. D for achieving bluer emission is possible by optimizing the thicknesses of each layer or by the use of micro-cavity effect and/or filters.Comparing to device  0.18, 0.7. DeviceD has been performed to study the effect of the thickness of the ETL (10 to 60 nm) on the EL emission color  to 20 nm (device E) was performed. Device E resulted in comparable EQEs of 17.8% and 16.6% at the maximum value and 1000 cd m−2, respectively. Also, device E showed a slightly lengthened stability with a LT90 of 38 h. Most importantly, device E achieved better CIE coordinates of  at 1000 cd m−2, which is bluer than that of  in device D  with a high external quantum efficiency (EQE~16.6%) and a long stability (LT90 ~38 h) at high brightness (1000 cd m−2), which represents a big milestone to the field of the blue OLED technology.A summary of the best reported blue and sky-blue OLEDs based on delayed fluorescence and phosphorescence was listed in Supplementary Table 3Ph2CzCzBN, has been synthesized and characterized. It showed a higher PLQY and a shorter delayed lifetime than that of the reference compound, 4CzBN. Devices based on 3Ph2CzCzBN showed a sky-blue EL of 482 nm and CIE coordinates of . Such device achieved a high EQE of 16.6% at 1000 cd m−2. Most importantly, 3Ph2CzCzBN-based TADF OLEDs showed superior device stability. With an initial luminance of 1000 cd m−2, an exceptional LT90 of 38 h was achieved. In conclusion, a stable sky-blue OLED (CIEy < 0.4) and a high external quantum efficiency (>15%) at high brightness (1000 cd m−2) were achieved simultaneously with the use of a newly synthesized TADF emitter, 3Ph2CzCzBN. This outstanding result definitely suggests the possibility for achieving efficient and stable blue OLEDs by TADF technology.A new TADF emitter, 1H, 13C and 19F nuclear magnetic resonance (NMR) spectra were obtained in CDCl3 or acetone-d6 with a Bruker Biospin Avance-III 500 NMR spectrometer at ambient temperature. Chemical shifts (δ) are given in parts per million (ppm) relative to tetramethylsilane  as the internal reference. Mass spectra were measured in positive-ion atmospheric-pressure chemical ionization (APCI) mode on a Waters 3100 mass detector. Elemental analyses  were carried out with a Yanaco MT-5 elemental analyzer.All reagents were used as received from commercial sources and were used without further purification. Chromatographic separations were carried out using silica gel (200–300 nm). The two materials investigated in this paper were synthesized by following the procedures described below. All compounds were purified twice by temperature gradient vacuum sublimation. N,N-dimethylformamide (30 mL) in a two-neck round-bottom flask equipped with a condenser. The reaction mixture was cooled to 0 °C, then NaH  was added. The reaction mixture was slowly warmed to room temperature and stirred for half an hour. After that, 2-(9H-Carbazol-9-yl)-3,5,6-trifluorobenzonitrile  was added and the reaction was heated to 150 °C for 16 h. The reaction was quenched with water and the precipitate was filtered off. The crude product was purified by column chromatography. Yield: 976 mg (80%). 1H NMR : δ = 9.07 , 8.36 , 8.29 , 7.95–8.00 , 7.55–7.70 , 7.30–7.45 , 7.18 . 13C NMR : δ = 143.1,143.1, 142.1, 142.0, 141.6, 141.5, 141.0, 140.7, 140.2, 140.0, 136.2, 135.9, 135.8, 130.6, 130.5, 128.8, 128.8, 128.7, 128.5, 128.4, 127.6, 126.8, 126.7, 126.7, 126.5, 126.42, 125.8, 122.9, 122.0, 120.5, 120.4, 120.4, 113.4, 112.9, 112.7, 112.7.MS (APCI) calcd. for C91H57N5: m/z = 1220.5; found: 1221.0 [M]+. Elemental analysis calcd. (%) for C91H57N5: C 89.55, H 4.71, N 5.74; found: C 89.51, H 4.65, N 5.72.Under nitrogen atmosphere, 3,6-diphenyl-9H-carbazole  was dissolved in dry H-carbazole  was dissolved in dry N,N-dimethylformamide (30 mL) in a two-neck round-bottom flask equipped with a condenser. The reaction mixture was cooled to 0 °C, then NaH  was added. The reaction mixture was slowly warmed to room temperature and stirred for half an hour. After that, 2,3,5,6-tetrafluorobenzonitrile  was added and the reaction was heated to 150 °C for 16 h. The reaction was quenched with water and the precipitate was filtered off. The crude product was purified by column chromatography. Yield: 557 mg (73%). 1H NMR : δ = 8.43 , 7.77–7.79, 7.35 , 7.30–7.32 , 7.18 , 7.09–7.17 . 13C NMR : δ = 139.2, 138.8, 137.8, 136.9, 136.3, 125.7, 124.3, 123.9, 121.3, 121.0, 120.4, 120.2, 118.3, 113.0, 109.9, 109.2. MS (APCI) calcd. for C55H33N5: m/z = 763.3; found: 763.3 [M]+. Elemental analysis calcd. (%) for C55H33N5: C 86.48, H 4.35, N 9.17; found: C 86.50, H 4.29, N 9.18.Under nitrogen atmosphere, 9S0 → S1 and S0 → T1 transitions using the B3LYP functional were then performed according to the optimized geometries of the lowest-lying singlet and triplet states, respectively.All calculations were carried out using the Gaussian 09 program package. The geometries in the ground state were optimized via TD-DFT calculations at the B3LYP/6-31 G(d) level. TD-DFT calculations for the −5M) were prepared to investigate their absorption and photoluminescence characteristics in the solution state. Thin-film samples (15% wt- doped in mCBP 100 nm) were deposited on quartz glass substrates by vacuum evaporation to study their exciton confinement properties in the film state. Ultraviolet–visible absorption (UV-vis) and photoluminescence (PL) spectra were recorded on a Perkin-Elmer Lambda 950 KPA spectrophotometer and a JobinYvon FluoroMax-3 fluorospectrometer. Phosphorescent spectra were recorded on a JASCO FP-6500 fluorescence spectrophotometer at 77 K. Absolute PL quantum yields were measured on a Quantaurus-QY measurement system  under nitrogen flow and all samples were excited at 360 nm. The transient PL decay characteristics of solution samples were recorded using a Quantaurus-Tau fluorescence lifetime measurement system . The prompt and delayed PL spectra of the samples were measured under a vacuum using a streak camera system  equipped with a cryostat . A nitrogen gas laser  with an excitation wavelength of 337 nm was used.Toluene solutions containing these two materials  analysis was performed by Netzsch DSC204 Phoenix calorimeter at a scanning rate of 5 °C min−1 under N2 atmosphere.Thermal gravimetry-differential thermal analysis (TG-DTA) was performed by Bruker TG-DTA 2400SA with a heating rate of 10 °C min+/Fc) as the internal standard. Argon-purged N,N-dimethylformamide was used as solvent for scanning the oxidation with tetrabutylammonium hexafluorophosphate (TBAPF6) (0.1 M) as the supporting electrolyte. The cyclic voltammograms were obtained at a scan rate of 100 mV s−1.Cyclic voltammetry (CV) was carried out on a CHI600 voltammetric analyzer at room temperature with a conventional three-electrode configuration consisting of a platinum disk working electrode, a platinum wire auxiliary electrode and an Ag wire pseudo-reference electrode with ferrocenium–ferrocene . For all OLEDs, the emitting areas were determined by the overlap of two electrodes as 0.04 cm2. The J-V-luminance characteristics were evaluated using a Keithley 2400 source meter and an absolute external quantum efficiency (EQE) measurement system . Device operational stability was measured using a luminance meter  at a constant DC current at room temperature.The OLEDs were fabricated through vacuum deposition of the materials at ca. 2.0 × 10The optical simulation of OLED devices was performed by using SETFOS 4.6 simulation program, in which the corresponding refractive index (n) and extinction coefficient (k) of each layer, dipole orientation factor of emitter, thickness of each layer and emission spectrum of EML were input. The optical constants of all the organic layers were measured using ellipsometer . The dipole orientation factor of emitter was set to be isotropic. During the optical simulation, the thicknesses of all layers were fixed, except the thickness of ETL was varied from 10 to 60 nm.Supplementary InformationPeer Review File


E.coli. However, the mechanism underlying how cortisol regulates E.coli-induced inflammatory response in bovine endometrial epithelial cells (BEEC) remains elusive.Bacterial infections are common in postpartum dairy cows. Cortisol level has been observed to increase in dairy cows during peripartum period, and is associated with the endometrial innate immunity against pathogens like E.coli, but up-regulated these gene expressions in BEEC stimulated by live E.coli.Cortisol decreased the expressions of IL1β, IL6, TNF-α, IL8, and TLR4 mRNA in BEEC treated with LPS or heat-killed E.coli-stimulated BEEC, but the pro-inflammatory action on live E.coli-induced BEEC.Cortisol exerted the anti-inflammatory action on LPS- or heat-killed  Escherichia coli (E.coli) precedes infection by Trueperella pyogenes and a range of anaerobic bacteria that include Fusobacterium, Prevotella and Bacteriodes species [Endometritis is the inflammation of the endometrium which usually occurs in postpartum dairy cows. There are two main types of bovine endometritis, including clinical endometritis with purulent (> 50% pus) discharge in uterine lumen between 20 and 33 days postpartum or mucopurulent (approximately 50% pus and 50% mucus) discharge after 26 to 33 days postpartum , 2, and  species .E.coli [Uterine defenses rely initially on classical innate immunity. The bovine endometrial epithelial cells (BEEC) are the first to make contact with potential pathogens that enter the uterus . One of E.coli , 8. The E.coli , 10.E.coli has not yet been clarified. The effect of cortisol on E.coli-stimulated BEEC is worthy of being investigated.As a kind of glucocorticoids, cortisol is widely considered as an anti-inflammatory steroid hormone –13, whicE.coli or LPS. Quantitative PCR method was used to detect the expressions of IL1β, IL6, TNF-α, IL8, and TLR4 mRNA in BEEC co-treated with cortisol and LPS, heat-killed E.coli or live E.coli.Here we reported the different effects of cortisol on the gene expressions of pro-inflammatory cytokines induced by E.coli challenge. As shown in Fig. E.coli (1 × 108 CFU/mL) from 0 to 60 h. Compared to the control group, the cell viability was not influenced by live E.coli (1 × 106 CFU/mL) at 6 h  and trypan blue assay were used to evaluate the viability of BEEC after heat-killed or live p < 0.01) levels of LPS-induced IL1β, IL6, TNF-α, and IL8 mRNA expressions were observed as compared with the control group at all time points, whereas the TLR4 mRNA increased (p < 0.01) only at 6 h after challenge. Compared with E.coli treated group, cortisol  decreased (p < 0.01) the mRNA expressions of TLR4 at 6 h, and the IL1β, IL6, TNF-α, and IL8 at most observed time points.The effect of cortisol on the pro-inflammatory and TLR4 mRNA expressions in BEEC stimulated with LPS were shown in Fig. E.coli and cortisol were shown in Fig. E.coli upregulated (p < 0.01) the mRNA expressions of IL1β, IL6, IL8, and TLR4 at all indicated time points, and TNF-α at 6 and 18 h. Compared with E.coli treated group, cortisol  treatment generally down-regulated (p < 0.05) these gene expressions at 6 and 18 h, and the IL1β and TLR4 mRNA at 2 h.The results of IL1β, IL6, TNF-α, IL8, and TLR4 mRNA expressions in BEEC co-treated with heat-killed p < 0.01) by live E.coli challenge at all time points. The TLR4 mRNA in E.coli group decreased (p < 0.01) at 4 and 6 h, and unchanged at 2 h. Compared with E.coli treated group, co-treatment of cortisol and E.coli generally increased the mRNA expressions of IL1β, IL6, IL8, and TNF-α, which was most pronounced (p < 0.05) at 4 h by 5, 15 and 30 ng/mL cortisol, at 2 h by 15 ng/mL cortisol, and at 6 h by 30 ng/mL cortisol. The mRNA levels of TLR4 in the co-treatment group were higher (p < 0.05) than the E.coli group at 2 and 4 h.The results of IL1β, IL6, IL8, TNF-α, and TLR4 mRNA expressions were shown in Fig. E.coli-induced expressions of pro-inflammatory genes  in BEEC. However, in cells challenged by live E.coli, we detected the reduced TLR4 mRNA expression and increased mRNA levels of IL-1β, IL-6, IL-8, and TNF-α. Co-treatment of cortisol and live E.coli further up-regulated the expressions of these pro-inflammatory genes.In this study, we demonstrated that cortisol inhibited the LPS- and heat-killed E.coli-stimulated BEEC by Chapwanya et al. [E.coli activated TLR4 receptor in HEK293 cells [E.coli could up-regulate TLR4 mRNA. Binding of LPS to TLR4 increases the secretion of inflammatory cytokines [E.coli induced the protein productions of IL-1β, IL-6, IL-8, and TNF-α [E.coli.Increased mRNA expressions of TLR4 have been reported in LPS-stimulated BEEC by Shen et al. , Herath a et al. . Similar93 cells .. This cytokines , 22. IL1ytokines . IL8 is ytokines . Braunernd TNF-α . SimilarE.coli, the live E.coli down-regulated TLR4 expression. However, the levels of IL-1β, IL-6, IL-8, and TNF-α mRNA increased after the live E.coli stimulation. Previous study indicated that the pathogenic mechanism of E.coli is complex. Some pathotypes of E.coli have a large repertoire of effectors that are translocated into host cells by the type III secretion system (T3SS) and affect host cell activities [E.coli group. Therefore, we speculated that the underlying mechanism of the increased expressions of inflammatory genes was not mediated by TLR4. We were unable to explain why the TLR4 gene expression decreased after the stimulation of live E.coli, which may require further investigation on TLR4 at posttranscriptional level and on the interaction between TLR4 and live E.coli.In contrast with increased TLR4 mRNA expression induced by LPS or heat-killed tivities , 27. T3Stivities , resultitivities . This poE.coli. However, cortisol increased the levels of IL-1β, IL-6, IL-8, TNF-α, and TLR4 mRNA in BEEC treated with live E.coli, exacerbating the pro-inflammatory effects. It has been reported that glucocorticoids have not only universally anti-inflammatory actions, but also pro-inflammatory effects in acute stress situations [E.coli stimulation may cause acute stress of cells, resulting in the pro-inflammatory effects of cortisol. There is a substantial space for further research to determine the condition and underlying mechanism of how the pro-inflammatory effects occur.It has been accepted that glucocorticoids induce anti-inflammatory effects by inhibiting the expressions of many pro-inflammatory genes  and the tuations , 33. Thetuations , 32. We E.coli or live E.coli. Cortisol inhibited the LPS- or heat-killed E.coli-induced inflammatory response, but up-regulated the live E.coli-induced inflammatory response.The present study demonstrated a two-side effect of cortisol in BEEC stimulated with LPS, heat-killed Streptomyces griseus  diluted in DMEM/F-12  at 4 °C for 18 h. Then the uterine horns were incised to facilitate scraping the endometrium. The scraped materials were washed, collected and centrifuged at 1000×g for 5 min. The cells were resuspended in DMEM/F-12 containing 15% fetal bovine serum  and 50 U/mL of penicillin/streptomycin. These cells were seeded in 25 cm2 flasks and cultured at 37 °C containing 5% CO2. The medium was changed when the cells reached 90% confluence. The purification of BEEC was confirmed to be above 99% by detecting CK-18 using immunohistochemistry.The cells were isolated as described from Dong et al.  and fromE.coli O55 was obtained and isolated from a cow with endometritis (purulent uterine discharge) at experimental farm of Yangzhou University. The strain was routinely cultured overnight  in nutrient LB broth , and the bacterial fluid was pipetted into sterile LB for incubating  until to an optical density (OD 600 nm) of 0.6 to have a final density to 4 × 108 ± 1 × 108 CFU/mL. Both active and heat-inactive E.coli were used in this study. For live E.coli, the bacterial suspension was centrifuged at 4000×g for 10 min and followed twice further washes with PBS, then resuspended in DMEM/F-12 to a dose of 1 × 106 CFU/mL. To inactive bacteria, the bacterial suspension was heat killed for 60 min at 70 °C after washed with PBS as described. Then it was centrifuged at 4000×g for 10 min and resuspended in DMEM/F-12 to have a final inoculum corresponding to 1 × 108 CFU/mL. The doses of active and heat-inactive E.coli were selected based on a preliminary study and on studies of Korzekwa et al. [a et al.  and Braua et al. , respectE.coli or LPS, the cells plated on 6-well plates at a density of 1 × 106 cells per well were challenged with 1 × 106 CFU/mL live E.coli, 1 × 108 CFU/mL heat-killed E.coli, or 1 μg/mL LPS in the control medium or the medium containing cortisol . The concentrations of cortisol from 5 ~ 30 ng/mL were within the physiological levels in cows. The selection of 5, 15, and 30 ng/mL cortisol was based on a previous report of our lab [E.coli challenge or 2, 6, and 18 h after heat-killed E.coli or LPS stimulation.To evaluate the impact of cortisol on the inflammatory response of BEEC induced by  our lab . For detE.coli on cell viability, the Cell Counting Kit-8 (CCK-8) obtained from Dojindo Molecular Technologies, Inc.  was used. The cells were plated on 96-well plates at a density of 1 × 103 cells per well and grown to 80% confluence in a 37 °C, 5% CO2 incubator. Then the cells were treated with 1 × 108 CFU/mL heat-killed E.coli for 0, 2, 6, 9, 12, 16, 18, 24, 48, and 60 h, followed by the addition of CCK-8 to each well. After incubation for 2 h at 37 °C, the optical density (OD 450 nm) was detected with a microplate reader .The previous study in our lab demonstrated that 5, 15, and 30 ng/mL cortisol had no effect on BEEC viability . To deteE.coli on cell viability was evaluated by trypan blue assay . The cells were incubated in the 6-well plates (1 × 106 cells per well) and grown to 80% confluence. After treatment with 1 × 106 CFU/mL E.coli for 6 h, the cell suspension was mixed with trypsin blue according to the manufacturer’s protocol. Cells were counted using a cytometer .The effect of live -△△Ct method was used to analyze the relative gene expression. The β-actin was used as the internal control. The sequences of the primers were shown in Table After treatment, cells were washed with 1 mL of PBS. Total RNA was extracted using the TRIzol reagent  according to the manufacturer’s protocol. The extracted RNA was quantified using a Nanodrop 2000 spectrophotometer . Total RNA (1 μg) was reverse transcribed into cDNA with the PrimerScript RT regent Kit gDNA Eraser . Real-time PCR was carried out by using SYBR Premix Ex Taq™ II  on the CFX 96 Real-Time PCR Detection System  as previously described . The 2-△p-value was less than 0.05. Each experiment was repeated three times.Data analysis was performed using IBM SPSS Statistics 21.0 . All data were expressed as the means ± standard error (SEM). The significance of differences between groups was evaluated by one-way ANOVA, followed by Dunnett’s test. Significance was attributed when a two-sided


Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective mannerR-manoyl oxide  was produced by expressing CfTPS2 and CfTPS3 from Coleusforskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 . The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor.In this study, 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.In this study, an efficient 13The online version of this article (10.1186/s12934-019-1123-z) contains supplementary material, which is available to authorized users. Plants have evolved to produce diverse secondary metabolites in order to adapt to their living environment and protect themselves . TerpenoColeusforskohlii and has been used to treat hypertension, heart complications, and asthma [R-manoyl oxide (13R-MO), which was found in the root cork cells of C.forskohlii, was found to be the precursor of forskolin [C.forskohlii, CfTPS2 which synthesizes the intermediate copal-8-ol diphosphate from GGPP and CfTPS3 which catalyzes the stereospecific formation of 13R-manoyl oxide were confirmed to stereospecifically biosynthesize 13R-MO from geranylgeranyl diphosphate (GGPP) [Forskolin, a labdane-type diterpenoid, was discovered in the roots of the medicinal shrub d asthma . Forskold asthma . NKH477,d asthma . Howeverd asthma . Recentlorskolin . By minie (GGPP) . The lare (GGPP) , 12.Saccharomyces cerevisiae, acetyl-CoA is sequentially catalyzed by seven MVA pathway enzymes to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the two five-carbon building blocks required for the synthesis of all terpenoids. Erg20p then catalyzes the condensation of IPP and DMAPP to produce the monoterpenoid and sesquiterpenoid precursors geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively [S. cerevisiae GGPP synthase (Bts1p) showed a higher Km value (3.2 μM) for FPP than for squalene synthase (2.5 μM) [ERG9 expression with a PMET3 promoter [S. cerevisiae.Yeast is a particularly good host for terpenoid production as it has a native mevalonate (MVA) pathway and is robust for industrial fermentation and genetic engineering . In Saccectively . GGPP isectively . A yeastectively ; howeverectively , 18. How(2.5 μM) . Therefo(2.5 μM) , convert(2.5 μM) , and reppromoter . These eS. cerevisiae, regulating the overall metabolic pathway, and not just the rate-limiting steps, is important for maximizing the synthesis potential of the target product [CfTPS2 and CfTPS3 were expressed in S. cerevisiae W303-1a, and 13R-MO was detected (2.31 mg/L). The 13R-MO titer was increased by 142-fold to 328.15 mg/L via regulation of the key genes of the MVA pathway and truncation of diterpene synthase , which was constructed for the production of the sesquiterpene α-humulene in our previous study, resulting in strain LZJ1 [R-MO, which was slightly higher than that produced by the original strain GW-1. In order to reduce squalene accumulation, the strain LZJ2 was constructed by fusing a PEST  sequence [R-MO, which was 1.5-fold higher than that produced by the original strain GW-1.FPP and GGPP are the two crucial precursors for diterpenoid production in ain LZJ1 . LZJ1 cosequence  to the CS. cerevisiae strain LZJ2. The co-expression of BTS1 and ERG20F96C at the HO site of strain LZJ2 resulted in the strain LZJ3, which produced 7.78 mg/L of 13R-MO. A GGGS linker was then used to fuse Bts1p with Erg20p or Erg20F96Cp in LZJ2, resulting in strains LZJ4 and LZJ5, which were able to produce 9.75 mg/L and 23.31 mg/L of 13R-MO, respectively.GGPP production was then optimized in the https://www.cbs.dtu.dk/services/ChloroP/) software and were removed to obtain tCfTPS2 and tCfTPS3. The coding sequences of the plastid transit peptides are shown in Additional file tCfTPS2 and tCfTPS3 were co-expressed in the δ sites of strain LZJ5 under the strong promoters TEF1p and TDH3p, respectively. The resultant strain LZJ6 could produce 176.8 mg/L of 13R-MO, which was 6.6-fold higher than that produced by its parental strain. The key rate-limiting enzymes of the MVA pathway, tHmg1p and Bts1-GGGS-Erg20F96Cp, were over-expressed by the rDNA site insertion to obtain strain LZJ7, further increasing the production of 13R-MO to 328.15 mg/L. The stepwise increase in the production of 13R-MO is shown in Fig. To generate pseudo-mature variants, the N-terminal plastid transit peptides of CfTPS2 and CfTPS3 were predicted using ChloroP  and geranylgeraniol (GGOH) content are listed in Table tHMG1, ERG20, BTS1 and ERG9) in the MVA pathway, with the highest titer (33.62 mg/L) detected in the strain LZJ3. GGOH also gradually accumulated to 40.91 mg/L via the stepwise MVA pathway engineering. Bts1-GGGS-Erg20p/Erg20F96Cp fusion  successfully drew FPP into the GGPP pool, and the highest GGOH accumulation, 93.12 mg/L, was detected in strain LZJ5. The N-terminal truncation of CfTPS2 and CfTPS3 in LZJ6 increased the 13R-MO titer by 7.5-fold compared to LZJ5, and decreased the GGOH titer to 51.13 mg/L from 93.12 mg/L, and decreased the FOH titer from 15.2 to 5.54 mg/L. The accumulation of GGOH and FOH were further decreased in the final strain LZJ7  during fermentation anaphase, although ERG9 was controlled by HXT1p, an efficient promoter for repressing ERG9 transcription under glucose-limiting conditions [R-MO titer remained low and the FOH and GGOH titers increased by 3.53-fold and 1.87-fold, respectively. The level of FOH and GGOH accumulation often reflects the intracellular content of the FPP and GGPP pools [To date, 15,804 diterpenoid compounds have been discovered in plants . However sterols . In our the cell , 29. In nditions . This stnditions . Indeed,S. cerevisiae, the diterpenoid precursor GGPP is synthesized by the condensation of FPP and IPP by Bts1p. The coupling of FPP and GGPP synthesis was found to be an important limitation of diterpene production [BTS1 were all attempted to improve the supply of GGPP [F96Cp) was constructed to produce GGPP, and was found to efficiently produce diterpenes and carotenoids [BTS1 and ERG20F96C were co-expressed in LZJ3 causing an increase in FOH, GGOH, and 13R-MO production compared with the parental strain LZJ2. Although Erg20F96Cp produced GGPP, no obvious effect on FPP formation was observed in vitro [F96Cp; the two fusion proteins were able to effectively draw FPP to GGPP, resulting in a decrease in FOH accumulation of approximately 50% compared with the parental strain LZJ3. The Bts1-GGGS-Erg20F96Cp fusion protein was found to be more effective for GGPP production and led to about threefold GGOH production when compared with Bts1p and Erg20F96Cp co-expression. The 13R-MO titer was also increased to 23.21 mg/L. Erg20p and Bts1p fusion is an efficient method of improving diterpene production [F96Cp) and Bts1p enabled FPP and GGPP to be coupled more efficiently.In oduction . Selecti of GGPP . An Erg2otenoids . To drawoduction , 31, andS. cerevisiae [δ sites of LZJ6 [R-MO production increased by 7.6-fold (176.8 mg/L) compared with the level produced by the parental strain LZJ5. The accumulation of GGOH and FOH decreased significantly, which indicated that the precursor pool was successfully converted to 13R-MO. The two limiting-nodes, tHMG1 and BTS1-GGGS-ERG20F96C, were further over-expressed in LZJ7, resulting in a higher level of 13R-MO production (328.15 mg/L).In plants, GGPP is synthesized in plastids through the methylerythritol 4-phosphate pathway . A plastrevisiae . In this of LZJ6 . 13R-MO R-MO yeast cell factory was constructed in this study. Stepwise metabolic engineering strategies were used to modify the MVA pathway leading to 13R-MO production. The highly efficient production of GGPP from FPP and the high activity of diterpene synthase were the two limiting steps for diterpenoids production. Transcription and protein level regulation were combined to transfer the metabolic flux from squalene to FPP and then to GGPP, which was an efficient way of modifying the Erg9p node. This strategy may also work well for sesquiterpene production in yeast. In summary, the engineered S. cerevisiae strain LZJ7 produced 602.05 mg/L of the diterpene 13R-MO in batch culture with 40 g/L of glucose, and the titer reached 3 g/L with fed-batch fermentation. This study provides a new strategy for the highly efficient production of diterpenoids and potentially even tetraterpenes by using the microbial cell factory.An efficient 13Saccharomyces cerevisiae W303-1a  was used as the original strain and was maintained in our laboratory. YPD medium  was used for yeast cultivation, and SC medium  was used for recombinant yeast strains selection. For shake flask cultivation, a single yeast colony was inoculated in 3 mL of YPD medium and cultivated overnight; the overnight culture was transferred to 250 mL shake flasks containing 30 mL of YPD medium with an initial OD600 of 0.05 and incubated for 96 h at 30 °C, 220 rpm. Dodecane (10%) was added for biphasic fermentation at 6 h after inoculation. YPD medium with different glucose concentrations  was used to optimize the fermentation of the strain LZJ7 in shake flasks. All fermentations were performed in triplicate.CfTPS2 (GenBank: KF444507) and CfTPS3 (GenBank: KF444508) were synthesized and codon-optimized for S. cerevisiae by Jinsirui Biotechnology Co., Ltd. , and the sequences are listed in Additional file S. cerevisiae W303-1a genomic DNA, and the fragments were purified for gene expression module construction via fusion PCR. The Bts1-Erg20p fusion protein was constructed with a widely used linker, “Gly-Gly-Gly-Ser”. The primer pairs ERG20-F/F96C-R and F96C-F/ERG20-R were used to construct Erg20pF96C. The constructed gene expression modules, homologous arms of the integration sites and selection markers were transformed into S. cerevisiae, according to the methods of Zhao et al. [The strains established in this study are listed in Table o et al. .Table 2SR-MO quantification, according to the method reported by Elias et al. [The yeast cells were harvested by centrifugation, then the cell pellets and supernatants were extracted with hexane. The hexane phase was filtrated and analyzed using GC or GC–MS. For biphasic-fermentation, the dodecane layer was separated by centrifugation  and subsequently analyzed with GC or GC–MS. The samples (1 μL) were analyzed using a Shimadzu GCMS-TQ8030 instrument equipped with an HP-5ms GC column  and with helium as the carrier gas. The following temperature gradient program was used: injection temperature, 250 °C; 60 °C for 1 min, 15 °C/min to 200 °C, 10 °C/min to 280 °C, and hold for 2 min; 20 °C/min to 300 °C; and hold for 2 min. The ion source temperature was set to 300 °C, and the spectra were scanned from 30 to 550 m/z. 1-Eicosene was used as the internal standard for 13s et al. .Glucose was quantified with an SBA-40C bio-analyzer , according to the manufacturer’s instructions. The yeast cells were collected by centrifugation, washed three times and OD600 was measured using a spectrophotometer . Squalene was extracted and quantified using HPLC , according to our previously described methods . FOH in 2SO4 and NH3·H2O. The temperature was maintained at 30 °C, and the air flow rate was set at 2 vvm. The dissolved oxygen (DO) content was maintained above 35% by adjusting the agitation speed between 300 and 600 rpm. For two-phase fermentation, 10 vol % of dodecane was added after 6 h of cultivation.For batch and fed-batch fermentation, OYPD medium  was used as the seed and fermentation medium. The strain LZJ7 was prepared in a 500 mL shake-flask containing 100 mL of OYPD medium at 30 °C and 220 rpm for 16 h. The pre-culture was then inoculated at 10 vol % in a 5 L bioreactor (ShangHai BaiLun Bio) containing 3 L (batch) or 2 L (fed-batch) of OYPD medium. The pH was automatically maintained at 5.5 using 5 N H3·H2O was used as the nitrogenous source and was added automatically when the pH dropped below 5.5.The feed solution was prepared according to our previously reported methods and was added automatically according to the pH and DO after 18 h [Additional file 1: Table S1. Primers used in this study. Figure S1. Expression cassettes construction and insertion. Table S2. Synthesized DNA sequences.