Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16α-hydroxytestosterone. Since the hydroxylation at 16α-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids.Cytochrome P450 monooxygenases – able to regio- and stereoselectively hydroxylate non-activated carbon atoms – are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes . Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16α-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 μmol substrate consumed min-1 μmol-1 CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 μmol substrate consumed min-1 μmol-1 CYP154C5.CYP154C5 was coexpressed in N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16α-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (μmol substrate consumed μmol-1 CYP154C5) exceeding 2000.CYP154C5 from  Cytochrome P450 enzymes (CYPs) form a huge superfamily of heme-containing monooxygenases present in all domains of life, where they play important roles in the detoxification of xenobiotics, drug metabolism, the assimilation of carbon sources and the formation of secondary metabolites . They caet al. examined a library consisting of 213 different bacterial CYPs for their ability to convert testosterone [Nocardia farcinica IFM 10152. Agematu et al. reported that this enzyme was able to convert 100 μM testosterone (5) selectively into 16α-hydroxytestosterone (11) using whole cell catalysis with an overall conversion >10% [N. farcinica is a human pathogen causing a disease called nocardiosis by infection of lung, central nervous system, brain and cutaneous tissues. The bacterium belongs to the phylum of Actinobacteria and is naturally found in soils. The genome sequence of N. farcinica IFM 10152 was recently determined revealing the presence of 21 genes coding for putative cytochrome P450 monooxygenases, of which two were shown to hydroxylate steroids [Pseudomonas putida[Rhodococcus sp. NCIMB 9784 [Because of their natural role in steroid synthesis, today a lot more eukaryotic CYPs  capable of steroid hydroxylation are known compared to prokaryotic ones. However, the biotechnological use of eukaryotic P450 monooxygenases is significantly hampered by the fact that they are membrane bound within the cells . Despiteosterone . Of thesosterone  using whsteroids . One of osterone  using whIMB 9784  can be aSteroids are structurally derived from the four-ring system sterane and vary in the presence of functional groups and the oxidation state of the rings. The type, number and position of functional groups in the steroid backbone directly influence the physiological activity  . Therefoet al., we herein report the characterization and catalytic potential of CYP154C5 from Nocardia farcinica IFM 10152 for its application in regio- and stereoselective steroid hydroxylations as well as the optimization and up-scaling of respective biocatalytic reactions using different androstanes and pregnanes as substrates.Based on the initial findings of Agematu N. farcinica IFM 10152 [P. putida DSM50198 served as electron transfer components in bioconversions [E. coli C43(DE3) - a mutant of E. coli BL21(DE3) - was used [E. coli as a host for P450 expression and biocatalysis offers the advantage that E. coli naturally does not contain any endogenous P450 enzyme which could result in background activity. Protein expression was induced by IPTG addition and δ-aminolevulinic acid was added for enhanced heme synthesis. Additionally, trace element solution was used to supplement the media especially with iron. Protein production at 10 L fermentor scale with controlled oxygen supply and pH resulted in a higher expression level compared to shake flasks (0.5 L). In particular, expression of the redox partners Pdx and PdR was more efficient. The obtained specific activity of the electron transfer components (ETC activity) was in the range of 1.1 to 1.4 U/mg total soluble protein at fermentor scale in comparison to only 0.6 to 0.8 U/mg obtained in shake flasks. With regard to P450 expression, approximately 2.4 mg of CYP154C5 per g of wet cells  were produced during 24 h cultivation in the fermentor.The gene of CYP154C5 was amplified from vector pKNL031_M17 from a genomic library of FM 10152  using PCFM 10152 . Putidarversions . For effE. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) were compared. Additionally, the influence of increasing concentrations of steroid substrate  was investigated. In the whole-cell system an OD600 of 40 was used, which is equivalent to 3 μM CYP154C5 and an ETC activity of 9 U/mL (corresponding to a specific activity of 1.4 U/mg) as determined by analysis of cell-free extract that was obtained after cell disruption. In the case of bioconversions using CFE 3 μM P450 and 6 U/mL  ETC activity were employed. Since the three proteins were coexpressed in one host , only the level of one of them was kept constant in the biocatalytic reactions (3 μM CYP154C5 in this case). In bioconversions using CFE efficient regeneration of the required cofactor NADH was realized by addition of a cofactor regeneration system consisting of formate dehydrogenase from Candida boidinii and sodium formate. In contrast, glucose was added for NADH regeneration within the E. coli metabolism in case of whole-cell reactions. As controls, either whole cells or CFE of E. coli C43(DE3) (pACYCcamAB), not expressing the cytochrome P450, were employed in steroid biotransformations. Steroid conversions were determined by GC or HPLC based on substrate depletion and total turnover numbers  were calculated. For all tested substrates application of whole cells resulted in higher conversions and higher TTN than cell-free extract . In all cases, except for testosterone (5), the difference in conversion and TTN between whole cells and CFE increases with increasing substrate concentration. Hence, the whole-cell biocatalytic system is especially more suitable for reactions with high initial steroid concentration. Highest TTN were obtained for progesterone  and androstenedione  for 10 and 15 mM substrate concentration, respectively, using whole cells. In contrast, TTN in steroid transformations using CFE hardly exceeded 1000. However, it has to be stressed that despite the high TTN full substrate conversion was not achieved within 20 h reaction time using high initial substrate concentrations (≥ 8 mM). In control reactions with E. coli C43(DE3) (pACYCcamAB) not expressing CYP154C5 no product formation was observed  or cell-free extract (CFE) of 1, 2, 3 and 4 are still fully converted for initial steroid concentrations of up to 4 mM, whereas conversions obtained for substrates 5 and 6 are already significantly reduced at substrate concentrations above 0.5 mM. Hence, achieved TTN for 5 and 6 are also significantly lower as compared to steroids 1–4. Additionally, the influence of cell density on the performance of the whole-cell biocatalytic reactions was investigated. For that, E. coli cells containing CYP154C5, Pdx and PdR were resuspended to obtain OD600 of 20, 30, 40 and 60 and tested in bioconversions with up to 8 mM 1, 3 and 5. For all substrates, an optical density of 40 yielded the highest conversions (data not shown). Other than expected, product formation was not further increased in whole-cell reactions with OD600 of 60. This might be attributed to an insufficient oxygen input and distribution in reactions with high cell mass since oxygen availability plays an important role in monooxygenase-catalyzed reactions [Comparing reactions of the different substrates using whole cells, it becomes obvious that eactions .3) and testosterone (5) using also different substrate concentrations (from 0.5 to 8 mM). For this, concentrated CFE was diluted with different volumes of buffer in order to obtain 0.5, 5, and 18 μM final P450 concentration. The specific activity of the electron transfer components PdR and Pdx stayed constant at 0.4 U/mg total protein (corresponding to 3.2 U per μM CYP154C5). As expected, the more enzyme present in the reaction mixture the more substrate was converted  and nandrolone (6), small amounts of a secondary product were formed. However, the amounts were so low that they could be easily removed by column chromatography purification. As mentioned before, 16α-hydroxylation of testosterone (5) by CYP154C5 had already been demonstrated by Agematu et al. on analytical scale and was now confirmed for five other steroid substrates by our NMR results [After investigation of important parameters for steroid bioconversion on analytical scale, best reaction conditions for each steroid were chosen for scale-up. Thus, an optical density of 40 and 2 mM substrate concentration were chosen for the whole cell steroid bioconversions at 30°C, except for substrates  results .1, 2, 3 and 4 were fully converted within 24 h of reaction whereas only one third and one half of the initial substrate concentration of 5 and 6, respectively, were hydroxylated under the applied reaction conditions. Furthermore, isolated yields of the purified steroid products without optimization of the purification conditions ranged between 70-80% (40–130 mg hydroxylated steroid).Conversions and TTN achieved on preparative-scale were similar to the results of the analytical-scale biotransformations. Hence, scale up did not negatively influence the performance of the whole-cell biocatalyst. Substrates E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) were performed with an OD600 of 40 (corresponding to 3 μM CYP154C5) and 2 mM substrate concentration. As shown in Figure 1, 2, 3 and 4 reached full conversion within the first 4–6 hours of reaction, whereas in bioconversions of 5 and 6 roughly only 50% of the substrate was converted within the first 8 hours  for all six steroid substrates were determined. In this way the efficiency of the catalytic system for the different substrates could be analyzed and compared. Bioconversions using whole cells of 1), was rather low during the first hour  and sufficient activity of the electron transfer components .Industrial steroid synthesis involves sophisticated chemical and enzymatic steps including the selective incorporation of hydroxyl groups in the steroid backbone. For this reason, cytochrome P450 monooxygenases, which are able to directly hydroxylate non-activated carbon atoms, are attractive enzymes for the production of steroid hormones in the pharmaceutical industry. In the present paper, the recombinant expression and biocatalytic characterization of bacterial CYP154C5 for the regio- and stereoselective hydroxylation of different steroid compounds is reported. Since the natural electron transfer partners of this P450 are unknown, Pdx and PdR from 1–6) we could show that CYP154C5 exhibits very high regio- and stereoselectivity not only for testosterone but also for all other tested steroids always giving 16α-hydroxylated products (7–12). Such a high specificity in the hydroxylation of various steroids carrying different functional groups and differing in the position of double bonds is remarkable since often more than one product is formed for the same steroid substrate by a P450 monooxygenase [4 and 6 low quantities of a secondary product were observed in whole-cell transformations, which, however, were not formed using purified proteins. Hence, they might not be direct products of CYP154C5 catalysis. Steroid hydroxylation in 16α-position was also described in literature for a P450 monooxygenase from Streptomyces roseochromogenes NCIB 10984 as well as CYP123 from Rhodococcus erythropolis PR4 and CYP110A1 from Nostoc sp. PCC7120 [S. roseochromogenes) and further oxyfunctionalization of the product at additional positions was reported. In contrast, the here obtained TON with CYP154C5 are already by a factor of 40 to 160 higher depending on the used steroid substrate.Applying this three-component system in bioconversions of six different steroid substrates 1– we couldxygenase ,19,20 an PCC7120 . HoweverE. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) clearly revealed an advantage of our whole-cell system, especially at high initial substrate concentrations. Not only significantly higher steroid concentrations (up to 15 mM) were still efficiently converted using whole cells, also substantially higher TTN and product yields could be achieved applying only low P450 concentrations. Thus, space-time yields of several grams of hydroxylated steroid per liter and day could be obtained. However, when comparing both systems it has to be taken into account that in the case of CFE reactions (i) a different cofactor regeneration system was used and (ii) the applied ETC activity was slightly lower as compared to the whole-cell system. Nevertheless we could prove that NADH regeneration by formate dehydrogenase and sodium formate in the CFE system was not limiting the overall performance of the P450 reaction. Furthermore, similar product yields for both systems could only be obtained using six times higher P450 concentration and three times higher volumetric ETC activity in CFE reactions as compared to whole cells. Thus, the slightly lower ETC activity in CFE reactions alone cannot account for the significantly reduced conversions and product yields obtained with the CFE system as compared to whole cells. Hence, the experimental results clearly prove the superior performance of our whole-cell system over reactions using CFE. In contrast, Zehentgruber et al. [Bacillus megaterium in E. coli together with bovine adrenodoxin and adrenodoxin reductase as redox partners resulted in significantly higher initial reaction rates as compared to whole cells in testosterone and progesterone bioconversions. However, the authors applied only low substrate concentrations (0.5 mM) since activity decreased significantly at higher steroid concentrations. On the other hand, the selectivity of their P450 system decreased to 80% when using CCE in comparison to whole cells due to unwanted formation of secondary hydroxylation products.Comparing the efficiency of steroid bioconversions using either resting whole cells or cell-free extract of r et al.  reportedE. coli-based whole-cell biocatalyst containing CYP154C5, Pdx and PdR still efficiently hydroxylated steroids at concentrations of 4 mM or higher. Thus, productivities of 2–3 g/L could be achieved in case of pregnenolone (1), dehydroepiandrosterone (2), progesterone (3) and androstenedione (4). The use of hydroxypropyl-β-cyclodextrin in our reactions clearly facilitated the solubilization and thus conversion of the required steroid amounts without exhibiting any destabilizing effects on cells or enzymes. Only for testosterone (5) and nandrolone (6) much lower yields were obtained at initial steroid concentrations of 1 mM or higher. Likewise also the measured TON for the two steroids using our whole-cell system are by a factor of 2–3 lower compared to the other steroids. This might be due to a different substrate specificity of the enzyme or even an inhibitory effect of the two steroids at higher concentrations. Structurally, testosterone (5) and nandrolone (6) are very similar, both carrying a hydroxyl group at carbon 17, which might explain the similar behavior. Currently we are trying to solve the crystal structure of CYP154C5 in order to reveal structural determinants of the enzyme’s high regio- and stereoselectivity in steroid hydroxylation and to understand on a structural level the difference in reactivity of CYP154C5 towards the different steroids.In literature, generally only low initial steroid concentrations (<1 mM) are applied in biotransformations of steroids using bacterial or eukaryotic P450 systems (whole-cell as well as isolated enzyme approaches). This might be explained by the very low solubility of steroids in aqueous solution , typicalN. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of various pregnanes and androstanes as well as its high-level expression in E. coli. Using this enzyme, 16α-hydroxylated steroids, which are important precursors in the synthesis of high value steroidal drugs such as glucocorticoids in the pharmaceutical industry, can be produced. Furthermore, an E. coli-based whole-cell biocatalyst – recombinantly expressing CYP154C5 together with putidaredoxin and putidaredoxin reductase as redox partners – was generated that allowed efficient conversion of the different steroid substrates even at high initial steroid concentration with the sole addition of glucose for cofactor regeneration.Bacterial CYP154C5 from Pregnenolone, testosterone, progesterone and cholesterol were purchased from Sigma-Aldrich, Germany. Dehydroepiandrosterone, androstenedione, nandrolone, 16α-hydroxypregnenolone, 16α-hydroxyprogesterone, 16α-hydroxydehydroepiandrosterone and 16α-hydroxyandrostenedione were purchased from Steraloids Inc., USA. All solvents and chemicals necessary for the experiments were obtained from Applichem, Roth or Sigma-Aldrich and used without further purification.Escherichia coli TOP10 strain  was used for genetic manipulations, while E. coli C43(DE3)  was used for expressions.The Nocardia farcinica containing the gene of CYP154C5  was kindly provided by Jun Ishikawa . Preparation of plasmid pACYCcamAB for coexpression of putidaredoxin reductase (CamA) and putidaredoxin (CamB) from Pseudomonas putida was described elsewhere [Plasmid pACYC-Duet1 was purchased from Novagen  while the broad host-range expression vector pIT2-MCS was prepared as described elsewhere . Plasmidlsewhere .cyp154C5 into vector pIT2-MCS. The resulting vector was named pIT2cyp154C5.The gene of CYP154C5 was amplified by PCR from vector pKNL031_M17 using primers NF-P-NdeI-FW: 5′-AAAAAAAACATATGAACGCCTGCCCCCATTCGG-3′ and NF-P-HindIII-RV: 5′-TTTTTTAAGCTTCACCTGCCCAGGTGGATGGG-3′. Thereby restriction sites NdeI and HindIII were introduced for subsequent cloning of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) was used for coexpression of CYP154C5 together with CamA and CamB in one host. For that, 5 mL liquid LB media supplemented with tetracycline  and chloramphenicol  was inoculated from a frozen stock of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB). After overnight incubation at 37°C and 250 rpm, a 200 mL TB preculture supplemented with tetracycline (10 μg/mL) and chloramphenicol (25 μg/mL) was inoculated with 1% v/v of the overnight culture and incubated for 24 h under the same conditions. Finally, a 16 L fermentor containing 9.8 L of TB media supplemented with tetracycline (10 μg/mL), chloramphenicol (25 μg/mL) and 0.1% v/v trace element solution  was inoculated with 2% v/v preculture. The pH was set to 7.0 and adjusted during the expression with 5 M KOH and H3PO4 solutions. Dissolved oxygen was controlled with a pO2 electrode. Foam formation was controlled using commercial Antifoam from Sigma. At an optical density (OD600) of 1 the system was induced with isopropyl-β-D-1-thiogalactopyranoside  and δ-aminolevulinic acid  was added as a precursor for heme synthesis. After 24 hours of expression at 30°C and 300 rpm the cells were harvested by centrifugation , washed with 50 mM potassium phosphate buffer pH 7.4, centrifuged again  and the pellets were stored at −20°C.E. coli C43(DE3) (pACYCcamAB) harbouring the camA and camB gene but not the gene of CYP154C5 was used as control strain in bioconversions. In this case, expression was performed according to the procedure described for E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) in 500 mL scale with 2 modifications: only chloramphenicol (25 μg/mL) was used for selective growth and δ-aminolevulinic acid was not added during the expression.E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) or E. coli C43(DE3) (pACYCcamAB). Thus, frozen cells were resuspended in 50 mM potassium phosphate buffer pH 7.4 in order to reach an optical density of 40 and phenylmethylsulfonyl fluoride  was added as protease inhibitor. Cell disruption was performed in an EmulsiFlex-Homogenizer  with 5 cycles of 1500 psi. After centrifugation  the resulting CFE was immediately used for the assays. CYP154C5 concentration was measured by CO-difference spectra [450=91 mM-1 cm-1). The activity of the electron transfer components (ETC) Pdx and PdR in CFE was determined by the cytochrome c reduction assay [550=19.1 mM-1 cm-1).All assays were measured with cell lysate of  spectra  and calcon assay , monitorE. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) overexpressing Pdx, PdR and CYP154C5 were resuspended in 50 mM potassium phosphate buffer pH 7.4 to the desired final OD600. All bioconversions were carried out in 1 mL scale at 30°C and 250 rpm with addition of glucose  for cofactor regeneration within the E. coli metabolism. Substrate stock solutions of steroids were prepared in 36% w/v hydroxypropyl-β-cyclodextrin in water. For substrates 3–6, stocks of 100 mM were prepared whereas only 50 mM stocks could be used for substrates 1 and 2. From the prepared stocks, the respective amounts were added to the reactions in order to achieve final steroid concentrations of 0.5 to 15 mM. Hence, the ratio between substrate and hydroxypropyl-β-cyclodextrin stayed constant in each reaction. Control reactions were carried out in parallel with E. coli C43(DE3) (pACYCcamAB) which contained only Pdx and PdR. After 20 hours of reaction the bioconversions and controls were extracted for subsequent HPLC and GC analysis. For that, the complete sample was extracted twice with ethyl acetate (700 μL and 500 μL) and once with chloroform (500 μL), the organic phases were combined, dried with sodium sulfate and the solvent was removed under reduced pressure.In case of whole-cell bioconversions, frozen cells of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) were resuspended in 50 mM potassium phosphate buffer pH 7.4 and phenylmethylsulfonyl fluoride  was added as protease inhibitor. Cell disruption was performed in an EmulsiFlex-Homogenizer  with 5 cycles of 1500 psi. After centrifugation  the resulting CFE was immediately used for bioconversions. In 1 mL scale bioconversions, NADH  was used as electron donor, sodium formate  and formate dehydrogenase from Candida boidinii  were added as NADH regeneration system in biocatalyses. Steroid substrates were also added as stock solutions in 36% w/v hydroxypropyl-β-cyclodextrin in water as described for whole-cell reactions. After 24 hours of reaction at 30°C and 250 rpm, the bioconversions and controls were extracted as described for the whole-cell system.In case of bioconversions using cell-free extract (CFE), frozen cells of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) (from frozen stock) at 30°C and 250 rpm, with the addition of glucose (0.54 mg/mL) in shake flasks. In all cases, resting cells with an optical density of 40 were used which is equivalent to approximately 3 μM CYP154C5 and an ETC activity of 1.1 U/mg of total protein, as determined by CO-difference spectra and cytochrome c assay, respectively. Substrate stock solutions were prepared in 36% w/v hydroxypropyl-β-cyclodextrin in water. In general 2 mM substrate concentration was used, except for progesterone (3) and testosterone (5) where 4 and 1 mM were applied, respectively. After 22–24 h of reaction, the complete reaction volume was extracted twice with ethyl acetate (50 and 40 mL) and once with chloroform (30 mL), the organic phases were combined, dried with sodium sulfate and the solvent removed under reduced pressure. Hydroxylated steroid products were afterwards purified by silica gel column chromatography  with a gradient of ethyl acetate in n-heptane to pure ethyl acetate (starting with ethyl acetate / n-heptane ratios of 6:4 for pregnenolone (1), 4:6 for dehydroepiandrosterone (2), 9:1 for progesterone (3) and testosterone (5), 7:3 for androstenedione (4) and 95:5 for nandrolone (6)) as mobile phase.Preparative-scale bioconversions were carried out in 100–200 ml of 50 mM potassium phosphate buffer pH 7.4 using resting whole cells of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) were resuspended in 50 mM potassium phosphate buffer pH 7.4 to an optical density (OD600) of 40. This cell density was equivalent to a P450 concentration of 3 μM and a specific activity of the ETC of 0.7 U/mg of total protein, as determined by CO-difference spectra and cytochrome c assay, respectively. All bioconversions were carried out at 25 mL scale in shake flasks for 26 h at 30°C and 250 rpm with addition of 30 mM glucose for cofactor regeneration. Substrate stock solutions were prepared in 36% w/v hydroxypropyl-β-cyclodextrin in water. In all cases 2 mM initial steroid concentration was used. During bioconversions, samples were taken every hour for subsequent HPLC or GC analysis. For that, 1 mL of reaction was extracted and the sample prepared for HPLC or GC analysis as previously described. Turnover numbers were calculated based on substrate consumption and for the period of time where the highest substrate consumption rate was observed  and nandrolone (6) were the period between 180 and 360 minutes was used). All bioconversions were done in triplicate.Frozen cells of 1) and dehydroepiandrosterone (2) the solid residues were redissolved in chloroform containing 30 mM cholesterol as internal standard. The samples were analyzed on a GC2010 gas chromatograph (Shimadzu) equipped with an OPTIMA 17ms column  with a linear gradient starting at 250°C and heating with 10°C/min until 300°C. Injector and detector temperature were set to 350 and 300°C respectively. Substrates and products were detected by FID. Both substrates, pregnenolone (1) and dehydroepiandrosterone (2), eluted at a retention time of 9.69 min and 7.94 min respectively, whereas their products eluted at 9.27 (7) and 10.18 min (8) respectively.In the case of pregnenolone (3), androstenedione (4), testosterone (5) and nandrolone (6) were dissolved in acetonitrile: water (60:40) and injected on a high performance liquid chromatograph  equipped with a Nucleosil 100–5 C18, 250 x 4,5 mm column  at 50°C. Acetonitrile: water (60:40) was used as mobile phase with a flow rate of 1 mL/min. Detection of substrates and their respective products was performed by UV absorbance at 254 (5 and 11), 230 (3 and 9) and 245 nm . The substrates eluted at 9.42 (3), 6.68 (4), 7.73 (5), and 5.93 (6) min, whereas the main products were detected at 4.95 (9), 4.47 (10), 4.12 (11), and 3.95 (12) min. Secondary products of androstenedione (4) and nandrolone (6) eluted at 5.68 and 5.22 min, respectively. In all cases conversions were calculated based on substrate consumption.The dried residues of bioconversions with progesterone  or Supreme 5ms (products 7 and 10) column  with a linear gradient starting at 250°C and heating with 10°C/min until 300°C. Injector and interface temperature were set to 300°C. For products 7, 10 and 11, derivatization using N-methyl-N-(trimethylsilyl) trifluoroacetamide  was necessary in order to facilitate GC-MS analysis.Preliminary product identification was performed with a gas chromatograph - mass spectrometer  equipped with an OPTIMA 17ms (products 7–11) was performed by 1H, 13C, COSY and HSQC NMR analysis on a Bruker AV400 instrument (1H-NMR 400 MHz and 13C-NMR 100 MHz). Measurement of product standards and structure elucidation of product 12 was performed by 1H, 13C, COSY, HSQC and NOESY NMR on a Bruker AV600 (1H-NMR 600 MHz and 13C-NMR 150 MHz). In all cases deuterated chloroform was used as solvent with TMS as internal standard except for product 7 were deuterated DMSO with TMS was used. Chemical shifts (δ) are given in ppm and coupling constant (J) in Hz  (pIT2cyp154C5) (pACYCcamAB) and E. coli C43(DE3) (pACYCcamAB) as control. Figure S2. Comparison of whole cells (WC) and cell-free extract (CFE) of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) in steroid bioconversions each containing 3 μM CYP154C5; Figure S3. Comparison of TTN achieved in steroid bioconversions by E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) using cell-free extract containing 18 μM CYP154C5 and whole cells containing 3 μM CYP154C5 (OD600 = 40). Furthermore, a detailed description of product structure elucidation by GC-MS and NMR analysis is given.Representative HPLC chromatograms of steroid bioconversions using Click here for file


Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR–Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR–Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.Prokaryotic CRISPR–Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR–Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from  Transcription of CRISPR spacer-repeat arrays and subsequent processing of the precursor transcripts yields individual CRISPR RNAs (crRNAs)  provide adaptive and heritable immunity against mobile genetic elements such as viruses and plasmids . These R(crRNAs) . These c(crRNAs) . Five tyrference , 2015.Staphylococcus epidermidis  targets . Upon tadermidis , 2014. De phages . Purifie complex . The S. tivities . The comtivities . These rtivities .csm6 genes are frequently associated with type III CRISPR–Cas systems  at 2.3 Å resolution and show that the protein is a ssRNA-specific endoribonuclease. The HEPN domains in the TtCsm6 dimer form a composite ribonuclease active site whose architecture and function is likely conserved in other Csm6 proteins. These results suggest that besides the intrinsic RNA cleavage activity of the Csm effector complex mediated by its Csm3 subunits, type III-A CRISPR–Cas systems harbor an additional ribonuclease module—Csm6—whose activity may play an important role in the interference mechanism.In this study, we sought to shed light on the biochemical function of Csm6 proteins in type III-A CRISPR–Cas systems. We report the crystal structure of 4-6-H  and probed its nuclease activity using fluorescently labeled RNA and DNA oligonucleotides. A randomly selected RNA oligonucleotide sequence was efficiently cleaved by TtCsm6 at 37°C to yield discrete shorter oligonucleotide products . Incubation of all three substrates with TtCsm6 resulted in the formation of anomalously migrating short oligonucleotide products, including the terminal dinucleotides  in the presence of ATP. Treatment with T4 PNK resulted in a marked shift in electrophoretic mobility, indicating that the 3′ products of Csm6-catalyzed phosphodiester bond hydrolysis carry a free 5′-hydroxyl group . This suggests that RNA substrate cleavage by Csm6 yields products containing a 5′-hydroxyl and a 2′–3′ cyclic phosphate groups, as shown for other HEPN domain RNases .2+ ions, which allowed us to solve the structure using single-wavelength anomalous diffraction (SAD) phasing. The atomic model was refined at a resolution of 2.30 Å with Rwork of 21.9% and Rfree of 24.9%  and Sulfolobus solfataricus  contain both the N-terminal CARF and the C-terminal HEPN domains but lack the middle 6H region. Structural alignment performed using the DALI server  superimposes with an rmsd of 4.0 Å over 198 Cα atoms. In this case, the structural homology is restricted to the C-terminal HEPN domain because SmCsm6 lacks the N-terminal CARF domain . The 6H and HEPN domains dimerize via hydrophobic interactions involving amino acid residues provided by helices α12 and α14 and by the loop connecting helices α18 and α19, which contains the R-X4-6-H motif . Overall, the architecture of the TtCsm6 dimer is similar to the crystallographic dimers of PfCsx1 and SsCsx1 -X-(S/T)-X3-(R/K) motif that maps to the canonical ligand-binding face of the Rossmann fold  chloride. In SmCsm6, the R-X4-6-H motif residues reside in an α helix, whereas the TtCsm6 R-X4-6-H motif adopts an irregular loop conformation, suggesting that the TtCsm6 active center might be distorted as a result of Ni2+ binding  as well as a nearby glutamate (Glu332) were substituted by alanine. As a control, we tested TtCsm6 proteins in which the conserved CARF domain residues Thr133 and Lys137 were mutated. All mutant proteins were dimeric, as judged by size-exclusion chromatography . Individual substitutions of the HEPN domain residues led to near-complete loss of ribonuclease activity, whereas mutations in the CARF domain had little effect (4-6-H motif in the HEPN domain while the CARF domain (D/N)-X-(S/T)-X3-(R/K) motif is not directly involved in RNA hydrolysis. Additionally, to test whether the HEPN domain is sufficient for ribonuclease activity in TtCsm6, we generated a truncated TtCsm6 protein lacking the N-terminal CARF domain (Δ1-190). This protein was as active as full-length TtCsm6, showing that the CARF domain is not required for enzymatic activation . The observation that TtCsm6 is a metal-independent ribonuclease that generates products containing a free 5′-hydroxyl group suggests that RNA hydrolysis involves a nucleophilic attack by the 2′-hydroxyl group to yield a 2′,3′ cyclic phosphate, as shown for other HEPN domain ribonucleases such as Ire1 and PrrC  as well as Csm6 from Pyrococcus horikoshii (PhCsm6), a hyperthermophilic euryarchaeon, to probe whether these orthologs also possess ribonuclease activities. Both proteins contain the canonical R-X4-6-H HEPN domain motif and were therefore predicted to be enzymatically active. We heterologously expressed and purified Csm6 proteins and assayed their activities alongside TtCsm6. Both SeCsm6 and PhCsm6 proteins were able to degrade a 24-nt ssRNA substrate at 37°C, confirming that they are active ribonuclease enzymes  cells. Cultures were grown at 37°C in the presence of 100 µg/mL ampicillin and 33 μg/mL chloramphenicol to an optical density (OD600nm) of ∼0.7. Expression was induced by adding IPTG to a final concentration of 200 µM and the cultures were grown at 18°C for 16 h. Harvested cells were lysed by sonication in lysis buffer containing 20 mM HEPES pH 8.0, 500 mM KCl, 5 mM imidazole, supplemented with protease inhibitors. The clarified lysate was applied to a 10-mL HIS-Select Nickel Affinity Gel (Sigma) column and the column was subsequently washed with five column volumes of the same buffer. Bound proteins were eluted with lysis buffer supplemented with 250 mM imidazole. TEV protease was added to the eluted fraction and the sample was dialyzed against 20 mM HEPES 7.5, 500 mM KCl at 4°C for 16 h. To remove the hexahistidine tag and protein contaminants, the dialyzed proteins were re-applied to a HIS-Select Nickel Affinity Gel column. The flow-through fraction was collected, concentrated and further purified by size exclusion chromatography using a Superdex 200 (26/600) column  in 20 mM HEPES 7.5, 500 mM KCl. Eluted Csm6 proteins were concentrated to 15–50 mg mL−1.The sequences encoding TtCsm6 (TTHB152), PhCsm6 (PH_RS00765), and SeCsm6 (SERP_RS12035) were PCR-amplified from their respective genomic DNAs and inserted into a pET-based expression vector  using ligation-independent cloning. The resulting fusion protein constructs contained an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease cleavage site and the full-length protein sequence. Point mutations were introduced using inverse PCR and verified by DNA sequencing. All proteins were expressed in 2, 5 mM CoCl2, 5 mM NiCl2, and 5 mM MgCl2 at protein concentrations ranging from 6 to 25 mg mL−1. The optimal crystal growth condition was subsequently refined to 0.1 M HEPES pH 7.5, 14% (w/v) PEG 3400 and 35 mM NiCl2 at protein concentrations ranging from 5 to 10 mg mL−1. Iterative rounds of microseeding yielded large single crystals that typically formed within few days and were fully grown within 1–2 wk. For cryoprotection, crystals were transferred into 0.1 M HEPES pH 7.5, 12% (w/v) PEG 3400, 35 mM NiCl2 and 35% (v/v) ethylene glycol and were flash-cooled in liquid nitrogen. The crystals belonged to space group P21212 and contained two TtCsm6 molecules in the asymmetric unit. X-ray diffraction data were measured at beamline X06DA (PXIII) of the Swiss Light Source . The data were indexed, integrated and scaled in XDS  PEG 3350, 5 mM CdCld in XDS . Native d in XDS . Phasingd in XDS , resultid in XDS . The fin−1 cm−1), RNA2 (223 700 M−1 cm−1), DNA1 (236 100 M−1 cm−1) and DNA2 (234 100 M−1 cm−1) and at 280 nm for TtCsm6 (105 660 M−1 cm−1 for homodimer), PhCsm6 (109 780 M−1 cm−1) and SeCsm6 (107 080 M−1 cm−1). To determine TtCsm6 substrate specificity 200 nM RNA1 were incubated with 400 nM TtCsm6 homodimer in 20 mM HEPES pH 7.5 and 50 mM KCl and the reaction was incubated for 1 h at 37°C. To prepare dsRNA and dsDNA substrates, RNA1 and RNA2 as well as DNA1 and DNA2 oligonucleotides were preincubated in a 1:2 stoichiometry, heated to 75°C and subsequently slow-cooled to room temperature. Cleavage reactions using dsRNA, ssDNA, and dsDNA were set up the same way as for ssRNA. All reactions were quenched by addition of 0.5 volumes formamide supplemented with 0.005% (w/v) bromophenol blue. Twenty microliters of the sample were loaded on a 15% denaturing (7 M urea) polyacrylamide gel and resolved in 0.5× TBE at 55 W for 3 h. The gels were visualized using a Typhoon FLA 9000 laser gel scanner .All synthetic RNA and DNA oligonucleotides were purchased from Microsynth AG unless stated otherwise. Cleavage assays to determine substrate specificity were performed with 24 nt 3′-end Cy5 labeled synthetic RNA1 (ACUGCAACGCAAUAUACCAUAGCU) and its nonlabeled complementary strand RNA2 (AGCUAUGGUAUAUUGCGUUGCAGU). Corresponding DNA substrates were 3′-end Cy5 labeled DNA1 (ACTGCAACGCAATATACCATAGCT) and its nonlabeled complementary strand DNA2 (AGCTATGGTATATTGCGTTGCAGT). DNA, RNA, and proteins were quantified with a NanoDrop spectrophotometer using the calculated extinction coefficients at 260 nm for RNA1  were mixed with 200 nM TtCsm6 homodimer in 20 mM HEPES pH 7.5 and 50 mM KCl  and the reactions were incubated at 37°C. Twenty microliter samples were taken at indicated time points and reactions were quenched by addition of 0.5 volumes formamide supplemented with 0.005% (w/v) bromophenol blue. An RNase T1 control digest was performed under the same conditions by substituting TtCsm6 with 0.06 U of RNase T1 (Thermo Scientific). The samples were resolved and visualized as above.12, C12, and U12, respectively, obtained from Integrated DNA Technologies) labeled with Cy5 at their 3′ termini. Two-micromolar substrates were incubated with 8 µM TtCsm6 at 37°C in 20 mM HEPES pH 7.5 and 50 mM KCl in a total volume of 25 µL. Three-microliter samples were taken at indicated time points and the reactions were quenched by addition of 1 volume formamide supplemented with 0.005% bromophenol blue. The samples were resolved on a 20% denaturing (7 M urea) polyacrylamide gel and visualized as above.Ribonuclease activity assays to determine substrate preference of TtCsm6 were conducted with synthetic homo-oligomeric RNAs  polyacrylamide gel at 25 W for 4.5 h.In assays to determine the chemistry of RNA cleavage by TtCsm6, 4.5 µM RNA1 was mixed with 20 µM TtCsm6 in 2 mM HEPES pH 7.5 and 50 mM KCl in a total reaction volume of 15 µL. The reaction was incubated at 37°C for 15 min, stopped by heat inactivation at 95°C for 15 min and centrifuged to remove precipitated denatured TtCsm6 protein. The supernatant was equally split into three 4-µL aliquots. The first sample remained untreated. The second sample was mixed with 1 mM ATP and 1× T4 Polynucleotide Kinase buffer in a total volume of 10 µL . The third sample was treated the same way, but 0.5 U/µL T4 Polynucleotide Kinase were added to the reaction (Thermo Fisher Scientific). All samples were then incubated at 37°C for 1 h and subsequently mixed with 0.5 volumes of formamide supplemented with 0.005% bromophenol blue. The alkaline hydrolysis ladder was generated as follows. 2.5 µM RNA1 was incubated with 150 mM NaHCOTo perform TtCsm6 mutant cleavage assays, 1 µM RNA1 was incubated with 2 µM TtCsm6 homodimer in 20 mM HEPES, pH 7.5, and 50 mM KCl at 37°C . Ten-microliter samples were taken at indicated time points and reactions were quenched by addition of 0.5 volumes formamide supplemented with 0.005% (w/v) bromophenol blue. The samples were resolved on a 16% denaturing (7 M urea) polyacrylamide gel and visualized as above.Nuclease activity assays of Csm6 orthologs were performed by mixing 250 nM RNA1 with 500 nM TtCsm6, 500 nM SeCsm6 or 5 µM PhCsm6 in 20 mM HEPES, pH 7.5, and 50 mM KCl in a total reaction volume of 80 µL. Reactions were then incubated at 37°C. Ten-microliter samples were taken at indicated time points and quenched by adding 0.5 volumes of formamide supplemented with 0.005% bromophenol blue. The samples were resolved on a 20% denaturing (7 M urea) polyacrylamide gel and visualized as above.Atomic coordinates and structure factors of TtCsm6 have been deposited in the Protein Data Bank under PDB code 5FSH.Supplemental material is available for this article.


Hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibitors have been recently developed to inhibit NS5A activities and have been approved for the treatment of HCV infection. However the drawback of these direct acting antivirals (DAAs) is the emergence of resistance mutations. The prevalence of such mutations conferring resistance to HCV-NS5A inhibitors before treatment has not been investigated so far in the Tunisian population. The aim of this study was to detect HCV variants resistant to HCV-NS5A inhibitors in hepatitis C patients infected with HCV genotype 1 before any treatment with NS5A inhibitors.Amplification and direct sequencing of the HCV NS5A region was carried out on 112 samples from 149 untreated patients.In genotype 1a strains, amino acid substitutions conferring resistance to NS5A inhibitors (M28V) were detected in 1/7 (14.2 %) HCV NS5A sequences analyzed. In genotype 1b, resistance mutations in the NS5A region  were observed in 17/105 (16.2 %) HCV NS5A sequences analyzed. R30Q and Y93H  predominated over P58S  and L31M .Mutations conferring resistance to HCV NS5A inhibitors are frequent in treatment-naïve Tunisian patients infected with HCV genotype 1b. Their influence in the context of DAA therapies has not been fully investigated and should be taken into consideration. Hepacivirus genus of the Flaviviridae family . 49 % of patients had liver cirrhosis.The NS5A gene was successfully sequenced in 112 out of 149 (75.1 %) samples that were amplified by nested PCR. Among the 112 NS5A sequences obtained, 7 sequences (6.2 %) were HCV genotype 1a and 105 (93.8 %) HCV genotype 1b.Sequencing and alignments of the 112 NS5A sequences allowed the identification of amino acid substitutions implicated in resistance to HCV NS5A inhibitors . This sequence showed the M28V substitution associated with very low-level resistance to DCV and displayed 58-fold resistance to OBV.In genotype 1b strains, amino acid substitutions associated with resistance to HCV NS5A inhibitors were observed in 17/105 (16.2 %) sequences analyzed. Three sequences  exhibited the substitution L31M and six sequences  showed the Y93H variant. These mutations are considered as primary resistance mutations. The L31M substitution is known to confer 3-fold resistance to DCV and 2.09-fold resistance to LDV. The Y93H mutation is known to confer 24-fold resistance to DCV, 77-fold resistance to OBV and very high-level resistance to LDV yielding 1319-fold resistance. Six sequences  exhibited the R30Q substitution and P58S was present in four sequences . These two substitutions are known as secondary resistance mutations that increase the resistance of primary mutations but do not confer resistance by themselves.None of the 112 NS5A sequences from either subtype contained the P32L mutation, which is known to be a primary resistant variant associated with high-level resistance to HCV NS5A inhibitors.Combinations of viral variants in the NS5A region were rare. No combinations of viral variants were observed in genotype 1a. Among the 105 NS5A 1b sequences analyzed, only two sequences  showed combinations of two resistant variants R30Q + Y93H. When present on the same genome, this combination is known to confer 284-fold resistance to OBV.In this study, the first in the Tunisian population, 149 treatment-naïve patients infected with HCV genotype 1 were evaluated to determine the natural prevalence of HCV variants resistant to NS5A inhibitors. The sequencing technology used was the Sanger method which is known to allow detection of dominant variant populations above 20 %. We cannot eliminate minor variant populations that might have been detected by ultra-deep pyro-sequencing, whose threshold for detecting minor variants is estimated to be fairly lower than the Sanger threshold.Baseline identification of pre-existing mutations resistant to anti-HCV inhibitors in treatment-naïve patients is essential for defining new DAAs therapies . Some stIn this work some resistance mutations conferring resistance to HCV NS5A inhibitors such as DCV, LDV and OBV were observed. One out of 7 (14.2 %) patients with subtype 1a and 17 out of 105 16.2 %) with subtype 1b showed amino acid substitutions within the NS5A inhibitor resistance sites. In this study, the number of patients infected with HCV genotype 1b was very large compared to the number of patients infected with HCV genotype 1a, which is due to the high 80 % prevalence of genotype 1b in Tunisia [.2 % withSome studies have described the natural prevalence of substitutions conferring resistance to HCV inhibitors in patients naïve to treatment with NS5A inhibitors. In Italy, Paolucci et al. reported a frequency of 12.5 % and 53.3 % of substitutions involved in resistance to HCV NS5A inhibitors in DAA treatment-naïve patients infected with genotype 1a and 1b, respectively . Suzuki In subtype 1b strains, the primary resistance mutations L31M conferring low-level resistance to DCV  and Y93HMoreover, other substitutions like R30Q (5.7 %) and P58S (3.8 %) were found. These variants are known as secondary resistance mutations that increase the resistance of primary mutations but do not confer resistance by themselves . None ofIn subtype 1a, a primary resistance mutation in the NS5A region M28V conferring low-level resistance to DCV  and highIn conclusion, the present study in treatment-naïve Tunisian patients infected with HCV genotype 1b demonstrates a significant proportion of substitutions conferring resistance to HCV NS5A. Their influence in the context of DAAs therapies has not yet been fully investigated and should be taken into consideration. Further studies are needed to determine the impact of the NS5A baseline resistance mutations on the treatment outcome in clinical trials investigating combination therapies of NS5A inhibitors with other classes of DAAs, although it can be predicted that combination therapies would raise the resistance barrier and reduce the importance of natural substitutions at resistance positions to NS5A inhibitors.The current study included 149 Tunisian patients infected with HCV genotype 1 who were referred to the gastroenterology department in three Tunisian University Hospitals between 2009 and 2011: Habib Bougatfa University Hospital (Bizerte), La Rabta University Hospital (Tunis) and Mohamed Tahar Maamouri University Hospital (Nabeul). Plasma samples were collected from each patient after obtaining written informed consent.Total RNA was extracted from 140 μl of plasma using the QIAamp viral RNA method  according to the manufacturer’s recommendations. After reverse transcription (RT) using the SuperScript® III One-Step RT-PCR system with Platinum® Taq High Fidelity DNA Polymerase , a nested-polymerase chain reaction (nested-PCR) was performed with AmpliTaq Gold DNA Polymerase .HCV RNA was amplified as previously described using primers 1a-NS5A-F0  and 1a-NS5A-R0  for subtype 1a and 1b-NS5A-F0  and 1b-NS5A-R0  for subtype 1b. The PCR product obtained was 1345 bp in size . BrieflyFor the nested PCR, two overlapping fragments of DNA were amplified. For subtype 1a, the first fragment was amplified as previously described  using thFor subtype 1b, the first fragment was amplified using the primers 1b-NS5A-F1  and 1b-NS5A-SeqR  and the second fragment was amplified using the primers 1b-NS5A-SeqF  and 1b-NS5A-R0 as previously described . For botTwo microliters from the first PCR reaction were used in the nested PCR. The amplification conditions were the same for both subtypes: initiation of denaturation at 95 °C for 10 min followed by 40 cycles consisting of denaturation at 95 °C for 30 s, primer annealing at 56 °C for 30 s, and primer extension at 72 °C for 2 min, followed by a final extension of 10 min at 72 °C. The PCR amplification products were visualized on 1.5 % agarose gel stained with ethidium bromide under UV transilluminator after electrophoresis.The nested PCR products were purified using the Microspin™ S-400 HR Columns, GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. DNA sequencing was performed using the Big Dye® Terminator v 3.1 Cycle Sequencing Kit with a DXL 3500 DX sequencer . The same primers used in the two semi-nested PCR for the amplification of the NS5A region for both subtypes 1a and 1b were used for sequencing in this region.Nucleotide sequences were aligned with reference sequences of different subtypes with SeqScape v 2.7  and analyzed for the presence of previously identified mutations conferring resistance to NS5A inhibitors. GeneBank accession numbers for NS5A reference sequences are AF009606 for subtype 1a and AY045702 for genotype 1b. The threshold of nucleotide mixture detection during sequencing of samples is estimated to be around 20 %.NS5A sequences were screened for amino acid substitutions conferring resistance to NS5A inhibitors. For genotype 1b, sequences were screened for variants L31F/M/V, P32L and Y93C/H/N, which act as primary resistance mutations and L23F, R30Q and P58S, which act as secondary resistance substitutions, thus enhancing the resistance of primary mutations but not conferring resistance by themselves. For subtype 1a, sequences were screened for substitutions M28T/V, Q30H/E/R/K, L31M/V, P32L and Y93C/H/N, which act as primary resistance mutations , 25, 27.


Human herpesvirus 6B (HHV6B) infects over 90% of the population, and normally establishes a latent infection, where episodes of reactivation are asymptomatic. However, in immunocompromised patients HHV6B reactivation is associated with high morbidity and mortality. Cellular immunotherapy has been utilised against other herpesvirus in immunocompromised settings. However, limited information on the immune response against HHV6B has hampered the development of immunotherapy for HHV6B-driven disease. In this study, we have analysed the cellular immune response against four HHV6B antigens in a panel of 30 healthy donors. We show that the base-line level of T cell reactivity in peripheral blood is very low to undetectable. A short-term reactivation step enabled expansion of T cell responses, and all donors responded to at least 1 antigen, but more commonly 3 or 4. A hierarchy of immunogenicity was determined with antigens U90 and U54 being co-dominant, followed by U11 and U39. Putative CD8+ T cell epitopes were mapped to U90 and U11, predicted to be presented in the context of HLA-A1, A29, B39 and C6. T cells reactive against these novel epitopes were able to recognise virus-infected cells. Our data is supportive of the application and on-going development of T cell immunotherapy against HHVB-driven disease in the immunocompromised host. Human herpesviruses are ubiquitous pathogens that generally cause self-limited disease in healthy individuals. However, in the context of an immunosuppressed host herpesvirus infection can lead to serious, often fatal disease HHV6 exists as two species, HHV6A and HHV6B, with a high level of nucleotide similarity in vitro by short-term stimulation with appropriate antigenic peptides. Indeed, of 30 donors analysed all were able to mount responses to at least one of the four target antigens, with the majority of donors responding to three or all four. We identify three novel putative CD8+ T cell epitopes in U90, predicted to be restricted through HLA-A1, -A29 and -B39, and one epitope in U11, restricted through HLA-C6. Importantly, T cells reactivated with these peptides were able to recognise HHV6B-infected target cells highlighting their potential clinical utility. The continual identification and characterisation of the targets of HHV6-specific T cells is important for the future development of T cell therapies against HHV6B driven disease, and the data presented here is an important addition.In the current study, we have analysed a cohort of healthy donors for T cell responses to HHV6B antigens U90, U11, U54 and U39, representing an immediate early antigen, two viral tegument proteins and a viral envelope glycoprotein, respectively ex vivo against HHV6B antigens corresponding to known immunogenic HCMV proteins. We focused on four antigens from HHV6B, namely U11, 39, U54 and U90, corresponding to HCMV antigens pp150, gB, pp65 and IE1. PBMCs were isolated from a panel of 30 donors, with a broad range of HLA backgrounds, stimulated for 16 h with single tube 15-mer PepMixesTM for each HHV6B antigen, and analysed for the frequency of CD8+ve, IFN-γ+ve and CD4+ve, IFN-γ+ve cells by ICS. A representative example of the flow cytometry analysis of HHV6 antigen-specific CD8+ve, IFN-γ+ve cells is shown for donor HD05 in n = 30). Only seven donors had levels detectable at 0.1% or above after subtraction of the unstimulated background, six for U90  and one for U54 . To ensure that this cohort of donors could respond to herpesviruses, 15 HCMV seropositive donors were also analysed for responses to four immunogenic antigens from HCMV . Good responses were detected against IE1, pp65 and gB, with IE2 being recognised at low levels by a small number of donors in this group. Overall median values for the HCMV antigens were IE1 0.13%, IE2 0.00%, pp65 0.10% and gB 0.04% . Responses to HCMV IE1, IE2, gB and pp65 were also measured, and found to be present at detectable levels in the majority of HCMV-seropositive donors tested, median values for IE1 0.07% (0–0.52%), IE2 0.02% (0–0.25%), pp65 0.4% (0.08–3.13%) and gB 0.24% , with median responses to U54, U11 and U39 of 95 (range 0–346), 90 (range 0–372) and 50 (range 0–186) SFC/105 input cells, respectively (n = 19). We were able to expand HHV6B-specific T cells from the HCMV-seronegative donors supporting the conclusion that we are expanding authentic HHV6B response rather than cross-reactive HCMV T cells. There was a slight difference in the strength of responses detected in these donors, with U39 showing the strongest response with median value of 110 SFC/105 input cells (range 0–120), followed by U54 106 (range 39–149), U90 69 (range 10–286) and U11 53 (range 0–260) SFC/105 input cells (n = 6). However, when data from all donors were combined the median responses were U90 96 (range 0–376) SFC/105 input cells followed by U54 95 (range 0–346), U39 77 (range 0–186) and U11 66 (range 0–372) SFC/105 input cells. When analysed individually all donors responded to at least one antigen: 14 donors responded to all 4 antigens, 3 donors responded to 3 antigens, 6 responded to 2 and only 2 donors recognised only 1 . Correspondingly, lower values of CD3+CD8+ cells were seen in the expanded populations for each antigen, U11 21.5 ± 11.0%; U39 20 ± 8.9%; U54 27.9 ± 8.9%, U90 20.1 ± 9.1%  also identified novel CD8+ T cell epitopes. A mini-pool screen of expanded T cells, enriched for CD8+ cells, from donor HD10  identified a number of potential stimulating pools. The positive pools 5 and 31 were selected for further analysis, where the common 15-mer was peptide 226, EANHCFINHFVPIKT , using the U11 PepMixpeptides . A numbepeptides . This 15peptides . Responspeptides , but thipeptides . Based oWith the ultimate objective of using these cells clinically, it was important to confirm that these new HHV6B-specific T cell epitopes could recognise endogenously expressed antigen. Thus, T cell populations were expanded using individual 9-mers representing U90 PSK, LNI and NHC, and U11 LKT and the expanded T cells were tested for recognition of HHV6B-infected autologous cells lines. HHV6B reactivation is a significant clinical problem after HSCT and SOT, and is associated with GvHD, encephalitis and increased mortality. Manipulation of cellular immune responses against other herpesviruses, such as EBV and HCMV, has proved to be successful in treating clinical disease driven by reactivation of these viruses in HSCT and SOT patients. This suggests that T cell immunotherapy should also be considered as a treatment option for patients with HHV6B reactivation after transplant. Currently, this is limited by a paucity of detailed information on the cellular immune response to this virus, such that although HHV6B is known to induce immune responses, there is little detailed information on target antigens, T cell epitopes or HLA restriction elements. This detail is a key requirement to understand the role of cellular immunity in controlling HHV6B reactivation, and will feed into development of future immunotherapy against this virus. In this report, we have investigated the T cell response to HHV6B infection in a group of healthy individuals, and mapped four putative novel CD8+ antigenic peptides to U90 and U11 predicted to be presented in the context of HLA alleles A1, A29, B39 and C6 . In contrast, significant levels of HCMV IFNγ+ CD4+ and CD8+ responses to comparable antigens were observed in HCMV seropositive donors within this group. Thus, confirming we are able to detect herpesvirus-specific responses in our donors directly ex vivo, and that the very low levels of HHV6B-specific response are a feature of the immune response to HHV6B in these donors. Indeed, the levels of HHV6 T-cell reactivity detected in peripheral blood in study is in agreement with previous publications using viral infected cell lysates TM stimulation approach TMin vitro followed by a short-term culture. Using this approach we did not differentiate between CD4+ and CD8+ responses; however, all 30 of the donors responded to at least one antigen, with most (57%) responding to three or all four. Based on the percentage of donors responding and median frequency of IFNγ+ T cells, we observed a hierarchy of immunodominance amongst these antigens, with U90 and U54 being co-dominant followed by U11 and then U39. This is in general agreement with a previous report where responses against five antigens  were analysed in 14 donors, and U90 was found to be the most immunogenic, followed by U14 and then U54 TM expanded cultures were analysed for the percentage of CD3+ CD4+ and CD3+ CD8+ cells, we found that for all four antigens the majority for cells were CD4+. Our analysis was limited to measuring total CD4+ and CD8+ T cells in the expanded population, rather than also determining antigen specificity, and it could be argued that the CD4/8 T cell ratio observed in the expanded population reflects a non-specific expansion maintaining the same approximate ratio that is present in PBMC. However, the mainly CD4+ T cell response was similar to that seen when PepMixTM expanded cells were analysed for frequency CD3+CD4+ and CD3+CD8+ cells based on production of IFNγ after antigenic stimulation We started by addressing the level of HHV6-specific T cells in peripheral blood, against four antigens, U11, U39, U54 and U90. These antigens were chosen to represent antigens expressed immediately upon infection (U90) or present as part of the virion , and being functional homologues of immunogenic HCMV antigens  Why the cellular immune response to this herpesvirus is so low in peripheral blood of latently infected individuals is unclear. It is likely that, in common with other herpesvirus TM, we depleted CD4+ T cells and screened for peptide reactive CD8+ T cells using overlapping 15-mer libraries. We identified three putative novel CD8+ T cell epitopes in U90, termed PSK, NHC and LNI. These epitopes were predicted to be restricted through HLA-A29, B39 and A1 respectively. The B39-restricted NHC epitope sequence conforms to the motif of peptides known to bind to HLA-B*3901, which have a highly conserved His at position 2, an aliphatic anchor at position 9 Identifying the immunogenic viral antigens is an important first step for development of T cell immunotherapy. However, it is also important to map and characterise T cell epitopes within these viral antigens, particularly MHC Class I restricted peptides. Thus, we extended our study to begin identifying HHV6B CD8+ T cell epitopes. We focussed on donors known to respond to U90 and U11, and used an unbiased approach with regard HLA background. After stimulation with the relevant PepMixFor the development and enhancement of adoptive T cell therapy approaches for diseases driven by HHV6B, a comprehensive understanding of the overall immune response to this virus and details such as target antigens and peptides, and HLA-restricting alleles is vital. The data presented in this report adds to and expands the existing information, and supports the on-going efforts to develop T cell immunotherapy for HHV6 reactivation in transplant settings.TM Plus . Donors were HLA typed using a PCR-based protocol by the Clinical Immunology Tissue Typing Laboratory, Royal Liverpool and Broad Green Hospital Trust, Liverpool, UK. In most cases, HLA types were obtained for A, B and C alleles, and provided to two-digit resolution. PHA blasts were generated from PBMC by treatment with 5 μg/mL PHA and culturing in T cell media . HHV6B virus (strain Z29) was generated as culture supernatant from SupT1/Z-29 cells g for 5 min to remove cells, before being filtered through a 0.45 μm Minisart® NML Syringe Filter . Virus containing supernatant was stored in single use aliquots at −80°C.PBMCs were obtained from healthy adult volunteers, ranging in age from 22 to 63 years (median 39 years). The study was approved by the Liverpool Adult Research Ethics Committee and informed consent was obtained from all participating donors according to the Helsinki Declaration. PBMCs were isolated by Ficoll Hypaque density gradient centrifugation using Ficoll-PaqueTM, containing 15-mer peptides (overlapping by 11aa) spanning the whole protein for HHV6B antigens U11, U39, U54 and U90, HCMV antigens IE1, IE2, pp65 and gB, and BK polyomavirus VP1 were obtained from JPT technologies . Individual 15-mer peptides, overlapping by 11aa, for HHV6B antigens U11 and U90, were provided by Dr Ann Leen . The individual U11 and U90 15-mer peptides were used to generate mini-pools of 13–17 peptides, such that there were 30 mini pools for U11 and 33 for U90, with each 15-mer represented in 2 mini pools Single tube PepMixes6/mL) were incubated with PepMixesTM  in a 5 ml polystyrene tube at 37°C for 2 h in T cell media, before addition of Brefeldin-A , and incubation continued for a further 14 h. Controls were unstimulated PBMC and PBMC stimulated with ConA (5 mg/mL). PBMCs were then washed twice with PBS at 4°C, pelleted and incubated with a PC5-conjugated anti-CD8 mAb  for 20 min at 4°C before being fixed with 4% paraformaldehyde. Cells were then permeabilised by incubation for 5 minutes with permeabilization buffer , followed by staining with a FITC-conjugated anti-IFN-γ mAb (BD Biosciences) for 30 min at 4°C in the dark. Cells were then washed twice with PBS, resuspended in 0.5 mL PBS, before 0.5 × 106 were analysed for CD8+, IFN-γ+ve cells. Data was acquired on a BD FACSCalibur using Cell Quest Software, and analysed using FlowJo software (Treestar).PBMCs  was then determined manually. Controls in ELISPOT included unstimulated cells (DMSO treated) and cells stimulated with the BKV VP1 PepMixTM.ELISPOT analysis was carried out using the Human IFN-γ ELISPOT kit from MABTECH . T cell populations were added to wells at known cell numbers in duplicate or triplicate in the presence of either PepMixesELISA for human IFN-γ was carried out using an IFN-γ ELISA Ready-Set-Go® set (eBioscience), according to the manufacturer's guidance. Supernatants from T cell assays were measured directly or diluted as appropriate. Absorbance was read using a Multiskan Spectrum microplate reader  using SkanIt software 2.2. The concentration of IFN-γ (pg/mL) in each sample was calculated against the standard curve.6) were incubated with PepMixesTM  or individual peptides at a final concentration of 10 μg/mL in 100 μL RPMI for 60 min at 37°C. Cells were resuspended in T cell media and seeded in a 24-well plate. T cell cultures were supplemented on day 3 with 100 IU/mL IL-2 (Sigma), which was replenished every 3 days. On day 10 onwards T cells were harvested, counted and used in subsequent assays. CD8-enriched polyclonal T cell lines were generated from 10 days polyclonal T cell cultures by depletion of CD4+ T cells using an EasySep™ Human CD4 Positive Selection Kit  according to the manufacturer's instructions. The efficiency of CD8+ T cell enrichment was analysed by flow cytometry, and monitoring CD4+ depletion which ranged between 95% and 99%.Polyclonal HHV6-specific T cell populations were generated as follows. PBMCs  were added in triplicate to wells of a 96-well V-bottom plate and 100 μl of T cells from short-term in vitro reactivations were added at an Effector-to-Target (E/T) ratio of 10:1. The effectors and targets were co-cultured overnight at 37°C, before 100 μL of supernatant was harvested and monitored for IFN-γ by ELISA.Autologous, partially HLA-matched and HLA mis-matched PHA blasts or PBMCs were used as targets in T cell assays. Target cells were loaded with individual peptides (2 µg/mL) for 2 h in volume of 100 μL RPMI at 37°C, then washed 3 times with RPMI. Peptide-pulsed cells were then resuspended in T cell media at 1 × 106) in 500 μL T cell media were infected with 500 μL of HHV6B virus-containing supernatant for 90 min at 37°C. A further 1 mL T cell media was added and the infection continued. Six days after infection, virus-infected PHA blasts and comparable mock-infected cells were counted, resuspended in T cell media at 1 × 105 cells/mL and used as targets in a T cell assay. Target cells (1 × 104 cells in 100 μL) were added in triplicate to wells of a 96-well V-bottom plate and 100 μl of T cells from short-term in vitro reactivations were added at E/T ratios between 20:1 and 5:1. The effectors and targets were co-cultured overnight at 37°C, before 100 μL of supernatant was harvested and monitored for IFN-γ by ELISA. Infection of PHA blasts by HHV6B was confirmed by monitoring visually for cytopathic effects and by immunofluorescence staining with a mouse–antihuman HHV6B mAb . The HHV6B mAb was used at a dilution of 1:50 in PBS, and incubated with cells for 90 min at 37°C in 5% CO2, before being stained with a secondary antibody (Goat anti-mouse IgG-FITC). Slides were counter stained DAPI then analysed with a Nikon fluorescence microscope.PHA blasts (2 × 10


Buthus martensi Karsch) venom, is a potent and selective Kv1.3 channel blocker. However, the mechanism through which BmP02 recognizes and inhibits the Kv1.3 channel is still unclear. In the present study, a complex molecular model of Kv1.3-BmP02 was developed by docking analysis and molecular dynamics simulations. From these simulations, it appears the large β-turn (residues 10–16) of BmP02 might be the binding interface with Kv 1.3. These results were confirmed by scanning alanine mutagenesis of BmP02, which identified His9, Lys11 and Lys13, which lie within BmP02’s β-turn, as key residues for interacting with Kv1.3. Based on these results and molecular modeling, two negatively charged residues of Kv1.3, D421 and D422, located in turret region, were predicted to act as the binding site for BmP02. Mutation of these residues reduced sensitivity of Kv 1.3 to BmP02 inhibition, suggesting that electrostatic interactions play a crucial role in Kv1.3-BmP02 interaction. This study revealed the molecular basis of Kv 1.3 recognition by BmP02 venom, and provides a novel interaction model for Kv channel-specific blocker complex, which may help guide future drug-design for Kv1.3-related channelopathies.The potassium channel Kv 1.3 plays a vital part in the activation of T lymphocytes and is an attractive pharmacological target for autoimmune diseases. BmP02, a 28-residue peptide isolated from Chinese scorpion ( EM) cells, and constitutes the predominant K+ conductance for these cells, and provides the electrochemical driving force for Ca2+ influx which is necessary for both T-cell proliferation and cytokine secretion135Kv1.3 is a voltage-gated potassium channel that is crucial for T cell development and activation. Kv1.3 is highly expressed in effector memory T (TLeiurus quinquestriatus and the Kv1.2–2.1 paddle chimera channel9111213α-KTxs are the most well studied and abundant short-chain toxins in scorpion venomButhus martensi Karsch): it is comprised of 28 amino acids, whose tertiary structure is stabilized by 3 disulfide bonds151716BmP02, also referred to α-KTx9.1, is a peptide toxin venom from the Chinese scorpion . At higher concentrations (1 μM), sBmP02 inhibition of Kv3.1a and KCa1 was decreased  was capable of inhibiting Kv1.3 channels, HEK293T cells expressing the Kv1.3 channels were treated with increasing concentrations of sBmP02. 100 nM sBmP02 exhibited significant inhibition of Kv1.3 channel conductance, decreasing it by 50% during the depolarization at +40 mV  and Kbot1 (from Buthus occitanus tunetanus) are dyad free toxins, which have been demonstrated to be potent Kv1.3 blockers1619BmP02 and two other toxins from α-KTx9 subfamily, BmP03 , KCa1.1  and Kv3.1a  were gifts from Yingliang Wu (Wuhan University), Noel Davies (University of Leicester) and Ping Song , respectively. Point mutations were generated using sequential PCR using hKv1.3 channel as a template. The resulting mutants are as follows: D433A, D421A, D422A and H451V. Primers were designed by Primer 5.0  at a ratio of 1 μL reagent with 1 μg total plasmid per well. Electrophysiological experiments were performed 1~2 days after transfection.All experiments were performed on HEK 293T cell lines. HEK 293T cells were obtained from Shanghai cell bank of Chinese Academy of Science. The cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% heat–inactivated fetal bovine serum . Culture dishes were incubated at 37° in a humidified atmosphere containing 5% COWhole-cell voltage-clamp experiments were performed by using an Axon Multiclamp 700B Microelectrode Amplifier  at room temperature (21–25 °C). Patch pipettes were fabricated from glass capillary tubes by PC-10 Puller  with the resistance of 2~3 MΩ. Data acquisition and stimulation protocols were controlled by a Pentium III computer  equipped with pCLAMP10.3 . Capacitance transients were cancelled. Cells with a seal resistance  below 1 GΩ were omitted. Series resistance (Rs) was compensated (80%) to minimize voltage errors, and cells with an uncompensated series resistance (Rs) above 10 MΩ were omitted. Leak subtraction was performed using P/4 protocol. Data were lowpassed at 10 kHz. The rate of solution exchange was studied using solutions with different KCl concentrations and found to be about 95% complete within 20 s. The holding potential was −100 mV. Unless stated specially, all the recordings were done with the pulse of +40 mV.2 1, CaCl2 1.8, HEPES 10, glucose 10 (pH 7.4 titrated with NaOH). Pipette solutions for both Kv4.2 and Kv1.3 were composed of the following (in mM): KCl 130, MgCl2 0.5, MgATP 2, EGTA 10, HEPES 10 (pH 7.3 titrated with KOH).In the patch-clamp recordings, the standard bath solution channels was consisted of the following (in mM) NaCl 135, KCl 5, MgClSynthetic BmP02 and its mutants  were purThe toxin was dissolved in the bath solution, supplemented with 1 mg/mL bovine serum albumin (BSA) in order to prevent adherence of the toxin to the vials and the perfusion apparatus. Application of 1 mg/mL BSA alone did not alter Kv1.3, Kv3.1a and KCa1.1 channel function. Unless otherwise stated, all reagents were purchased from Sigma.Utilizing the X-ray crystal structures of KcsA Potassium channel (PDB: 1BL8)CHARMm forcefield was added to the best Kv1.3-BmP02 complex. The “Standard Dynamics Cascade” was used to run the MD simulations. 1000 steps steepest descent minimization and 2000 steps adopted bassis NR were performed. Then the system was heated from 50 K to 300 K in 50 ps and keep 300 K for 500 ps in order to equilibration under an isothermal-isobaric (NPT). The conformations were saved every 2 ps. Finally, 5000 conformations were got in 10000 ps.The energetic effect of each mutation on the binding affinity is calculated as the difference between the binding free energy in the mutated structures and the wild type protein. All interaction energy terms are calculated by CHARMm using a Generalized Born implicit solvent model and contain empirically scaled contributions of van der Waals and electrostatic interactions and a non-polar solvation energy term. The input structure of Kv1.3-BmP02 complex was obtained from the cluster analysis of the MD simulation trajectory.Data were analyzed by ClampFit 10.3  and Origin 8.5 . Results of data analysis were expressed as mean ± S.E.M. and n represents the number of the cells examined. The Statistical significance was determined using the unpaired Student’s t-Test or one-way ANOVA, and an asterisk denotes P < 0.05 unless otherwise stated.m/(1 + ([toxin]/EC50)n), where Im is maximum of the current, and [toxin] is the concentration of sBmP02. EC50  and n denote the toxin concentration of half maximal effect.Dose-response curve was drawn according to the Hill equation I = IKv1.3 channel currents was elicited by the step pulses ranging from −100 to +60 mV for 200 ms with the increments of 10 mV . For determining the voltage dependence of activation, the conductance was calculated using the formula: G(V) = I(V)/(V − ErK), where I(V) is the currents of Kv1.3 channel at the command voltage V, and ErK is the reversal potential. The conductance were normalized to the maximal value and the voltage dependence for activation of Kv1.3 channel fitted to a Boltzmann equation: G/Gmax = 1/[1 + exp(V-V1/2)/k], where V1/2 is the voltage for half-maximum activation and k is the slope factor.How to cite this article: Wu, B. et al. Mapping the Interaction Anatomy of BmP02 on Kv1.3 Channel. Sci. Rep.6, 29431; doi: 10.1038/srep29431 (2016).


The integration of multiple materials with complementary absorptions into a single junction device is regarded as an efficient way to enhance the power conversion efficiency (PCE) of organic solar cells (OSCs). However, because of increased complexity with one more component, only limited high-performance ternary systems have been demonstrated previously. Here we report an efficient ternary blend OSC with a PCE of 9.2%. We show that the third component can reduce surface trap densities in the ternary blend. Detailed studies unravel that the improved performance results from synergistic effects of enlarged open circuit voltage, suppressed trap-assisted recombination, enhanced light absorption, increased hole extraction, efficient energy transfer and better morphology. The working mechanism and high device performance demonstrate new insights and design guidelines for high-performance ternary blend solar cells and suggest that ternary structure is a promising platform to boost the efficiency of OSCs. Ternary blend solar cells offer the potential for high-power conversion efficiencies (PCEs); however their performances can be limited by design complexity. Here, the authors integrate multiple materials into a single junction device with 9.2% PCE and elucidate the mechanisms of enhancement at play. Jsc) in the devices. A major research effort in the field has been focused on developing new polymers with tunable optoelectronic properties, optimizing device morphology as well as interfacial engineering to enhance exciton dissociation and reduce charge recombination156789101112131415Impressive advances in bulk heterojunction (BHJ) polymer solar cells (PSCs) have been made via material synthesis, device designs, physical measurements and theoretical understanding, which lead to much improved power conversation efficiency 123Recently, the use of ternary structure-active layer is emerging as a promising strategy to enhance the performances in binary polymer:fullerene BHJ devices1718192218241771 butyric acid methyl ester (PC71BM). We achieve a PCE as high as 9.20% for the ternary device with 20% PID2 content without further device engineering. To our best knowledge, this is the best PCE reported for ternary PSCs to date. Detailed studies show that the enhancement results from a synergistic effect of enhanced light absorption, improved hole mobility, energy transfer between PID2 and PTB7-Th, reduced trap-assisted recombination and improved morphology. The results indicate that the mechanism of the enhancement is completely different from our recent work based on donor polymer PTB7 TB7 ref. .71BM used in the ternary structure are shown in 71BM, respectively, which are summarized in −1  characteristics of devices with different PTB7-Th:PID2 weight ratios  under simulated AM 1.5 G illumination at 100 mW cm−2. Devices with different PID2 concentrations showed consistent thickness around 100 nm. PTB7-Th:PC71BM control device started with a Jsc at 14.92 mA cm−2, an open circuit voltage (Voc) at 0.75 V, a fill factor (FF) at 70.3% and a PCE of 7.88%. By adding 10% of PID2 into the host binary blend, PCE was enhanced to 8.51% with a Jsc at 15.60 mA cm−2, a Voc at 0.77 V and a FF at 70.9%. Devices with 20% PID2 incorporation exhibited the best solar cell performance with a Jsc at 16.68 mA cm−2, a Voc at 0.78 V and a FF at 70.8%, resulting in a promising PCE of 9.20%. This is more than 15% enhancement in PCE compared with the reference device. We would like to note that the 9.20% PCE is the highest value reported for ternary blend PSCs so far. An average PCE of 8.97±0.16% was achieved over 10 identical devices at this condition with a mean Jsc at 16.22±0.35 mA cm−2, a Voc at 0.78±0.01 V and a FF at 70.8±0.14%. PCE of the ternary device was still comparable to the reference cell even if PID2 content was enhanced to 50%. FF remained larger than 70.0% even when PID2 content was increased to 70% and Voc increased monotonically with increased PID2 content at all ratios because of deeper HOMO energy levels of PID2 than PTB7-Th. This phenomenon was also observed for many other ternary systems reported203031Voc, such as the electronic alloy charge-transfer state reported in ref. Voc with higher PID2 contents is from the electronic alloy charge-transfer stateVoc changes in the future. Increasing the loading of PID2 to 90% led to dramatically decreased Jsc and FF because of the poor performance of PID2 itself. We used devices with 10 and 20% PID2 contents in the following discussions to clarify the effect of PID2 on the ternary system because of their much improved photovoltaic performance than the control device.The photovoltaic performance of the above ternary system was investigated on the basis of the following simple device structure: indium tin oxide (ITO)/poly:poly(styrenesulphonate) (PEDOT:PSS)/PTB7-Th:PID2:PCJsc values from EQE spectra for PTB7-Th:PC71BM (1.0:1.5), PTB7-Th:PID2:PC71BM (0.9:0.1:1.5) and PTB7-Th:PID2:PC71BM (0.8:0.2:1.5) devices are 14.52, 15.37 and 16.55 mA cm−2, respectively. This is similar to the Jsc values obtained from J–V measurement (the error is within 3%), indicating a good consistency of our solar cell measurements. By comparing with UV–vis absorption spectra in 71BM constructed a cascade structure. As a result, PID2 could act as a hole relay to facilitate hole extraction from PC71BM to PTB7-Th after photoexcitation and charge separation192271BM absorbs.External quantum efficiency (EQE) spectra of the ternary devices are shown in 71BM/MoO3/Au. Hole mobility increased from 3.58 × 10−4 cm2 V−1 s−1  to 6.18 × 10−4 cm2 V−1 s−1  and finally to 8.82 × 10−4 cm2 V−1 s−1 , (5.96±0.32) × 10−4 cm2 V−1 s−1 (10% PID2 loading) and (8.46±0.30) × 10−4 cm2 V−1 s−1 (20% PID2 loading), respectively. The improved mobilities helped to partially explain the better Jsc in the ternary system.Hole mobilities of the three devices were then evaluated by space charge-limited current method with the architecture: ITO/PEDOT:PSS/PTB7-Th:PID2:PCJsat) and exciton dissociation probabilities ) of the ternary solar cells without and with 10 and 20% PID2 incorporation. Jph) versus effective voltage (Veff) characteristics for the three devices. Jph is determined as Jph=JL−JD, where JL and JD are the photocurrent densities under one sun illumination and in the dark, respectively. Veff is determined as Veff=V0−Va, where V0 is the voltage at which Jph is 0 and Va is the applied bias voltage35Veff (2 V in this case) and Jsat is then only limited by maximum exciton generation rate (Gmax). As a result, Jsat=qLGmax, where q is elementary charge and L is the thickness of the active layerGmax values for the three devices were 9.79 × 1027 m−3 s−1 , 1.01 × 1028 m−3 s−1  and 1.08 × 1028 m−3 s−1 , respectively. The average Gmax values over 10 identical devices for the three conditions are (9.69±0.01) × 1027 m−3 s−1 (0% PID2 loading), (9.93±0.01) × 1027 m−3 s−1 (10% PID2 loading) and (1.06±0.01) × 1028 m−3 s−1 (20% PID2 loading), respectively. The increased Gmax with PID2 addition suggested increased overall exciton generations in the ternary devices. This is because of the complementary absorption with a small amount of PID2 incorporation and higher absorption coefficient of PID2 compared with PTB7-Th in the wavelength region below 625 nm. P is determined from the ratio of Jph/Jsat  values at short circuit condition for the three devices were 94.8% (0% PID2 loading), 95.3% (10% PID2 loading) and 94.6% (20% PID2 loading), respectively. The incorporation of 10% and 20% PID2 into PTB7-Th:PC71BM did not change the overall charge dissociation process. This should be ascribed to the higher LUMO energy level of PID2 than PTB7-Th, which impeded the dissociation of excitons generated by PTB7-Th at PTB7-Th:PID2 interface.More insight into the effect of PID2 on charge generation and dissociation process was gained by determining the saturation current density  Jsc in two possible pathways. Excitons generated by PID2 could directly dissociate at the PID2:PC71BM interface and be collected by the electrodes. This is confirmed by the significantly enhanced EQE for ternary devices with (10 and 20%) PID2 content from 500 to 625 nm where PID2 showed improved absorption. Alternatively, energy transfer from photoexcited PID2 to PTB7-Th occurs, followed by charge separation at the PTB7-Th:PC71BM interface. This explains the increased EQE from 625 to 750 nm for the ternary devices. Photoluminescence spectra of PTB7-Th only, PID2 only and PTB7-Th with 10 and 20% PID2 films provide evidence for the energy transfer process  value at 65.6%, energy transfer from PID2 to PTB7-Th helped exciton dissociation because of a high P value at 94.8% for PTB7-Th:PC71BM device.The increased exciton generation from PID2 can enhance  process . The pho process , making Jsc and Voc at various light intensities offers deeper insight into the influence of PID2 on recombination process in our ternary system. In principle, Jsc shows a power-law dependence on light intensity for organic solar cells, which can be expressed as Plight is light intensity and S is the exponential factor. Weak bimolecular recombination in the device would result in a linear dependence of Jsc on light intensity with S value close to 1. Jsc as a function of Plight for PTB7-Th:PC71BM (1:1.5), PTB7-Th:PID2:PC71BM (0.9:0.1:1.5) and PTB7-Th:PID2:PC71BM (0.8:0.2:1.5) devices. The extracted S values are 0.99, 0.98 and 0.98 for the three devices, respectively. The similar S values indicated that low loading of PID2 has negligible impact on bimolecular recombination process for the ternary system. This should be attributed to the fact that the control device already exhibited very weak bimolecular recombination with an S value at 0.99.The dependence of as  refs , 39. HerVoc and Plight in our ternary devices. The slope of Voc versus log(Plight) helps us to determine the degree of trap-assisted recombination in the devices. A slope at kBT/q implies that bimolecular recombination is the dominating mechanism, where kB is Boltzmann's constant, T is temperature and q is elementary charge. As for trap-assisted or Shockley–Read–Hall recombination, a stronger dependence of Voc on light intensity with a slope of 2 kBT/q is observed383971BM device showed a slope of 1.91 kBT/q while for PTB7-Th:PID2:PC71BM (0.9:0.1:1.5) and PTB7-Th:PID2:PC71BM (0.8:0.2:1.5) devices, smaller slopes of 1.57 and 1.77 kBT/q were attained. The results indicated that addition of PID2 into the PTB7-Th:PC71BM blend reduced interfacial surface trap densities between the active layer materials in the devices, suppressed trap-assisted recombination and contributed to enhanced Jsc.qy=0.301±0.009 Å−1 and a distinct out-of-plane π–π stacking peak appears at qz=1.60±0.06 Å−1, suggesting the preferential face-on conformation, whereas the PID2 bilayer is observed to order in a preferential edge-on orientation with the Bragg diffraction of periodic PID2 layers at qy=0.346±0.009 Å−1 and two off-axis scattering spots located at  Å−1, similar to the observation in PCDTBTqy and qz∼1.3−1.4 Å−1 corresponding to the Bragg diffraction of PC71BM. Because the full-width at half-maximum (FWHMs) of scattering peak correlates to the nanocrystallite size4271BM could form larger nanocrystallite sizes than PTB7-Th , the PID2 polymers had little influence on the co-crystal structures of polymers, which remain similar to PTB7-Th polymers except a slight decrease in the nanocrystallite sizes. Meanwhile, the incorporation of PID2 polymers into the ternary films increases the PC71BM nanocrystallite sizes. On further introducing PID2 polymers , a new scattering peak occurs at qy=0.316±0.009 Å−1 in between the scattering of PTB7-Th and PID2 layers, rather than two individual peaks, indicating the intermixing of PTB7-Th and PID2 at the molecular level. The formation of co-crystals would facilitate energy transfer between PTB7-Th and PID2 since the rate constant of fluorescence resonance energy transfer is proportional to (1/r)6, where r is the distance between PTB7-Th and PID2. In addition, the growth of larger PC71BM nanocrystallites could benefit charge transport within PC71BM domains.Influence of PID2 on the morphology of the ternary system was determined by grazing incidence wide-angle X-ray scattering (GIWAXS) and resonant soft X-ray scattering (RSoXS). In the two-dimensional (2D) GIWAXS pattern for each individual polymer film , a broad PTB7-Th . It is i PTB7-Th . By incoI(q), (solid lines) and their corresponding pair distance distribution functions (PDDFs), P(r), of the ternary PTB7-Th:PID2:PC71BM BHJ films, respectively. These ternary PTB7-Th:PID2:PC71BM BHJ films show hierarchical nanomorphologies at multiple length scales, in consistence with the previous observations in PTB7:PC71BM (ref. P(r) (71BM (1:1.5) film exhibits much larger domain sizes than the PTB7-Th:PC71BM (1:1.5) film, which correlates with their unsatisfied device performance. When incorporating a small amount of PID2 polymers into PTB7-Th:PC71BM films , such hierarchical nanostructural characteristics are retained with reduced domain sizes. A kink of P(r) at the length scales of tens of nanometres indicates that the fine domains inside phase-separated domains with hundreds of nanometres scale in these two ternary systems are even smaller than those of PTB7-Th:PC71BM (1:1.5). These smaller domains would increase the area of the interfaces between polymer donors and fullerene acceptors, thus promoting exciton dissociation and leading to improved performance. However, because of the poor performance of PID2, overall exciton dissociation probabilities in the ternary blends at 10 and 20% of PID2 incorporation are similar to that in the PTB7-Th:PC71BM binary blend. On further increasing the amount of PID2 polymers, larger domains are formed in the ternary blend films and, thereby, result in decreased device efficiency at high PID2 contents. Considering the complicated underlying working mechanism of the ternary BHJ solar cell, morphological characterization helps us to understand the tendency of performance change as a function of PID2 content in our devices.Besides the molecular ordering, another important morphological factor that will determine the photovoltaic performance is how these localized molecular crystals and aggregates form phase-separated domains in the BHJ blend. 1BM ref. . As indief. P(r) , the PID71BM binary device. A PCE of 9.20% was achieved with 20% of PID2 incorporation, which is the highest PCE reported for ternary PSCs. Voc of the ternary blends was enhanced with increased PID2 content because of the deeper HOMO energy levels of PID2 compared with PTB7-Th. A high FF at 70.0% was maintained with PID2 content up to 70%. In addition, the combined effects of a complementary light harvesting, efficient energy transfer from PID2 to PTB7-Th, enhanced hole mobility, reduced trap-assisted recombination, increased interfacial areas and better crystallinity of PCBM dramatically improved Jsc in the ternary systems. Our results demonstrate that the use of ternary structure helps to re-evaluate the potential of many low-performance donor polymers and is a promising way towards developing high-performance PSCs.A highly efficient ternary structure PSC is developed by adding PID2 as the additional donor polymer into the PTB7-Th:PC−1 with the dispersity index at 2.9 and 2.6, respectively. PTB7-Th, PID2 and PC71BM were dissolved in chlorobenzene and 1, 8-diodooctane . The overall donor polymer concentration (with or without PID2) was kept constant at 10 mg ml−1. The mixed solution was heated at 90 °C overnight. ITO glasses were purchased from thin film devices and sonicated in water, acetone and isopropyl alcohol for 15 min each, followed by ultraviolet ozone irradiation for 60 min. Later, an ∼40-nm-thick PEDOT:PSS layer was spin-coated at 6,000 r.p.m. on ITO glasses and dried at 80 °C in oven for 30 min. Polymer:PCBM solutions were spin-coated with the as-prepared solutions at 2,000 r.p.m. inside glove box. Ca (20 nm) and Al (80 nm) cathodes were thermal-evaporated.PTB7-Th and PID2 are synthesized according to previous procedures28J–V measurement was determined by masks with well-defined area of 3.14 mm2. UV–vis spectra were taken from a UV-2401PC model UV–vis spectrophotometer. EQE was measured with a 250-W Quartz Tungsten Halogen (QTH) lamp as the light source.A solar simulator with a xenon arc lamp (Oriel model 69920) was used to provide 1-sun, AM 1.5G irradiation for the measurement of current density–voltage characteristics. Light intensity of the measurement was calibrated with an NREL-certified monocrystaline silicon reference cell . The effective area of the λ=1.6868 Å and a beam size of ∼200 μm (h) and 20 μm (v). RSoXS measurements were achieved at beamline 11.0.1.2 .Supplementary Figures 1-3 and Supplementary Methods and Supplementary References.