Rifted margins mark a transition from continents to oceans and contain in their architecture a record of their rift history. Recent investigations of rift architecture have suggested that multiphase deformation of the crust and mantle lithosphere leads to the formation of distinct margin domains. The processes that control transitions between these domains, however, are not fully understood. Here we use high-resolution numerical simulations to show how structural inheritance and variations in extension velocity control the architecture of rifted margins and their temporal evolution. Distinct domains form as extension velocities increase over time and deformation focuses along lithosphere-scale detachment faults, which migrate oceanwards through re-activation and complex linkages of prior fault networks. Our models demonstrate, in unprecedented detail, how faults formed in the earliest phases of continental extension control the subsequent structural evolution and complex architecture of rifted margins through fault interaction processes, hereby creating the widely observed distinct margin domains. Continental rifting and break up processes are poorly constrained in the early stages. Here, the authors using high-resolution numerical simulations to show how early formed faults in continental extension can then control subsequent structure evolution of rifts. Offshore geophysical data and drill cores acquired over the past 30 years have, however, systematically revealed a structural complexity that challenges such simple models of continental rifting. Tectonic interpretations11 based on these recently acquired data sets suggest that rifting occurs through multiple distinct phases of deformation that migrate oceanwards and are characterized by different structural, sedimentary and magmatic processes. Although rifted margins show significant along-strike geometrical variations and differ from each other in many details, observations show that they also contain numerous first-order similarities that point to a common set of evolutionary phases12 , respectively, with viscous creep and plasticity (see Methods and Supplementary Information). Three elements are key to our models: First, the numerical resolution (250 m) is 2–8× higher than most margin studies. Second, deformation localizes on distributed heterogeneities with rapid strain weakening instead of being forced with a single zone of weakness as commonly prescribed. This allows us to achieve the distributed faulting of the initial ‘stretching’ phase of continental extension. Third, we include the commonly observed increase in extension rates towards continental breakupThe models also reveal deformation in the thinning and hyperextension-exhumation phases is accommodated by both new faults and inherited stretching phase faults, which evolve through a combination of fault reactivation, abandonment, development, and incision. This process of complex fault interaction and the resulting patterns of finite-deformation are consistent with detailed observations of rifted margin structure and provide new constraints for the interpretation of rifted margin evolution.−1 permit uniform lithospheric thinning accompanied by distributed faulting in the brittle domains of the crust, which are decoupled by weak ductile crustal layers . Notably, the associated strain rates ( < 10−8 s−1) are consistent with the range of observed strain rates in areas of distributed continental extension23. At 1 mm yr−1, distributed faulting in the decoupled upper crust, lower crust, and mantle continues for 50 Myr  in the thinning phase , initially decoupled zones of normal faults in the upper crust, lower crust, and mantle link near the model center  fault structures, preserved primarily in the necking zone, correspond directly to similar structures observed in the necking and outer domains of the mid-Norwegian margin24 Fig. . As in i. 9 Fig. , the ram. 9 Fig.  form as . 9 Fig. . Notably. 9 Fig. , active . 9 Fig. . However. 9 Fig. . The num25. However, interpretation of these results with respect to the debate on low-angle normal fault seismicity26 should be considered within the context of our numerical assumptions. For example, weakening the friction angle to lower values16, including elastic effects27 or the presence of fluids26 may also promote accommodation of deformation on lower angle structures. Similarly, higher numerical resolutions and constitutive relationships associated with dynamic friction may also promote deformation on lower-angle structures. However, from a long-term tectonic perspective our results clearly suggest the majority of deformation during the thinning and hyperextension-exhumation phases is accommodated on high-angle normal faults that incise abandoned faults rotated to low angles.The process of fault rotation and abandonment outlined in our experiments at first-order matches one proposed model for low-angle normal fault development12. From our models, we can deduce that the use of present-day observed structures to reconstruct continental extension is complicated by the non-linear character of finite-deformation  evolve through a complex pattern of fault linkage, abandonment, reactivation, and incision as the rift zone narrows. These findings provide new constraints on the interpretation of observed rifted margin structures and their relationship to the temporal and structural evolution of continental extension.30. SULEC solves for incompressible viscous flow following the equations for conservation of mass (1) and momentum (2):u is velocity, σ′ is the deviatoric stress tensor, P is pressure, ρ is density and g is gravitational acceleration (gx = 0 and gy = 9.8 m s−2). Density variations follow the Boussinesq approximation:ρo is the reference density, α is thermal expansivity, T is temperature and To is the reference temperature.We model extensional deformation of the lithosphere and asthenosphere with v6.0 of the finite-element, particle-in-cell code SULECT) evolves according to the advection-diffusion equation:c is heat capacity, k is thermal conductivity, H is heat production and S is shear heating. We use time steps of 50,000 and 10,000 years, respectively, when the applied velocity is 1 and 5 mm yr−1. Shear heatingTemperature  drive extensional deformation, while vertical inflow (Vin) at the model base exactly balances material outflow such that the average surface topography remains constant through time. The model surface and base maintain fixed temperatures of, respectively, 0 and 1330 °C, while the sides are insulating boundaries (no heat flux). These thermal boundary conditions combined with the distribution of radioactive elements and variations in thermal conductivity , measured as the second invariant of the brittle (plastic) finite strain tensor, accumulates between values of 0 and 0.1. Notably, this relatively rapid rate of strain weakening helps facilitates distributed normal fault development in the initial stretching phase  is determined by the internal friction angle from the prior time-step (ϕi−1) and a randomization factor (F):r is a random number between 0 and 1. Notably, this formulation may produce values of the internal friction angle higher than the initial value (20°), although this is unlikely in any regions that have accumulated plastic strain. More significantly, adding or subtracting the random value does not lead to excessive overall weakening of the materials and simulates that strength heterogeneities in the lithosphere this method aims to capture  manifest as relative changes in strength rather than only decreases. The majority of models  of 10, while values of 2, 5, and 20 produce similar results that reflect the same underlying processes outlined in this manuscript  not captured by our system of equations. Such heterogeneities are pervasive in the lithosphere as a result of accumulated deformation  and exist at a wide variety of length-scales in both the crust and mantle lithosphere. The randomized internal friction angle at a given time step . The grid resolution is constant at 250 m Figs.  and 14 oTo validate the robustness of our results we performed a wide range of additional numerical experiments. These experiments detail the effects of numerical resolution, rates of strain weakening, brittle randomization, mantle rheology, initial stretching phase velocity, and the ‘fast’ phase velocity. As documented in the Supplementary Materials and main text, the key conclusions of our study are consistent across the examined parameter space.Data from this study are available from the corresponding author on request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Prokaryotic AcrB-like proteins belong to a family of transporters of the RND superfamily, and as main contributing factor to multidrug resistance pose a tremendous threat to future human health. A unique feature of AcrB transporters is the presence of two separate domains responsible for carrying substrate and generating energy. Significant progress has been made in elucidating the three-dimensional structures of the homo-trimer complexes of AcrB-like transporters, and a three-step functional rotation was identified for this class of transporters. However, the detailed mechanisms for the transduction of the substrate binding signal, as well as the energy coupling processes between the functionally distinct domains remain to be established. Here, we propose a model for the interdomain communication in AcrB that explains how the substrate binding signal from the substrate-carrier domain triggers protonation in the transmembrane domain. Our model further provides a plausible mechanism that explains how protonation induces conformational changes in the substrate-carrier domain. We summarize the thermodynamic principles that govern the functional cycle of the AcrB trimer complex.The online version of this article (doi:10.1007/s41048-017-0042-y) contains supplementary material, which is available to authorized users. In Gram-negative bacteria, the periplasm functions as a protective buffer zone against cytotoxic substances from the extracellular environment , to drive their substrate-transport processes  to drive cross-membrane transport of substrates. In contrast, AcrB-like transporters utilize PMF across the bacterial inner membrane to transport substrates, either within an aqueous environment or at the aqueous–membrane interface.The AcrAB–TolC tripartite complex, a prototypical member of the RND family, is constitutively expressed in many Gram-negative bacteria, is responsible for efflux transport of lipophilic and/or amphipathic compounds (a common property of many cytotoxic substances such as antibiotics), and thus plays important roles in multidrug resistance , unless stated otherwise.Structures, substrate recognition mechanisms, and the functional cycle of AcrB-like transporters have been extensively reviewed previously , consisting of 1,049 amino acid residues, is the most extensively studied protein both structurally and functionally. Currently, the Protein Data Bank (PDB) database  and C-terminal subdomain , with the rotation axis perpendicular to the membrane. Around the symmetry axis, TM4 and TM10 form a parallel central helix-pair. Functionally important residues D407, D408, and K940 (numbered as in Ec-AcrB) are located in the TM4–TM10 interface , a long amphipathic helix  represents a conserved structural feature of RND transporters  The The et a et al.  et al.  et al. Based on asymmetric tripartite structures of AcrB reported from multiple laboratories , reliable communication and energy coupling between the two corresponding layers are essential for ensuring proper energy-transduction efficiency and thus high transport rate. Questions remaining to be addressed concerning the peristaltic mechanism include “How does substrate binding in the porter domain activate protonation of the TM domain?” and “How does protonation in the TM domain in turn drive the conformational change in the porter domain?” Finding answers to these questions will allow for a better understanding of the mechanistic details of RND transporters. et al. i.e., pKa) of the acidic pair. In both the access and binding protomers, the positive charge of the K940 side chain is located between the two negatively charged side chains of D407 and D408, preventing the acid residues from being protonated. The equilibrium conformation of such a deprotonated state (including its relative position to the membrane) will be considered as a reference for conformational changes upon protonation (see below). In contrast, in the extrusion protomer, the lysine side chain moves away from the acidic residues  will become protonated , to the proton binding site at the D407–D408 pair. As illustrated in the 1.9 Å-resolution crystal structure of AcrB (PDB ID: 4DX5), the proton-transfer wire, through which protons move, is likely to be formed by side chains of polar residues in TM helices as well as by membrane-embedded water molecules  moves from the periplasmic side of the inner membrane, following the negative-inside electrostatic membrane potential . Thus, many members from this transporter family have only one acidic residue as the protonation site. As suggested in the above YbgH case, a single acidic residue may function effectively as a protonation site, provided that some electro-negative groups are present in its vicinity. In addition, as shown in Fig. S1, the presence of a basic residue in TM10 is not absolutely essential for alternating the protonation status of TM4. Nevertheless, removing a positive charge  is likely accompanied by eliminating a negative charge , and thus the protonation ability of the polar cluster is maintained. In such a case, the pKa value of the remaining buried acidic residue might be reduced by being approached by an H-bond donor. A similar deprotonation mechanism has been proposed recently for activation of class-A GPCRs   results in loss of transport activity  has been shown to be very sensitive to point mutations. Any mutation that causes a charge change  half of TM4 relative to the C-terminal (cytosolic) half, resulting in a helix bulge near the conserved G403 (of the GX3DX6E motif) in the extrusion phase. As a consequence of this deformation, the side chain of D407 interacts with the backbone carbonyl oxygen of G403, presumably increasing the pKa value of D407. Interestingly, both the short β-strands in the NTM-PN1 and CTM-PC1 loops are flanked by two conserved proline residues  regions of TMs 8, 10, and 12, respectively) . In addition, the amphipathic helix α6-7, which is strategically located in the peripheral of the CTM-subdomain, keeps CTM aligned with the membrane surface upon movement of the NTM-subdomain towards the cytosol. Thus, α6-7 serves as a pivot point and may facilitate tilting of the TM domain as a whole. Disrupting this amphipathic helix (including the anchoring tyrosine residue) results in loss of the AcrB activity , the TMs 1–4 in NTM form a groove facing the lipid bilayer. A detergent molecule was found to bind in this groove, suggesting that this site is a ligand- or inhibitor-binding site that may potentially interfere with the coupling between the TM domain and PN1. In the same crystal structure, a similar ligand binding site was also observed in the symmetric position in CTM. Interestingly, in a recently reported crystal structure of human cholesterol transporter, NPC1 , a sterol-sensing domain (SSD) is formed by helices TMs 3–5 . Both genetic and structural analyses suggest that this region is important for NPC1 function. The head group of the cholesterol molecule bound to the SSD may interfere with the putative signaling β-sheet between the TM domain and the exo-membrane domain. Therefore, the signaling motifs that we proposed here for Ec-AcrB may indeed be a general feature for RND transporters.In the high-resolution structures of asymmetric AcrB trimer   > 0)  to bind to the access state and (2) to induce subsequent transitions. These rather general criteria allow AcrB to accommodate a broad substrate specificity, to bind ons Fig. B. Furtheons Fig. B, but poons Fig. .Box 1DefGC1, ∆GC2, and ∆GC3, which are intrinsic property of the transporter and are independent of the substrate binding. As a hypothetical example, as shown in Fig. GC1 > 0). The positive ∆GC1 suggests that in the absence of external energy, the A-B transition is thermodynamically unfavorable. There are many factors that may contribute to these differential conformational energy terms. For instance, the abovementioned hydrophobic mismatch may be considered as a contributor of the conformational energy in the extrusion phase, thus affecting both ∆GC2 and ∆GC3. Since the promoter must return to its initial state after a complete functional cycle, the sum of these energy terms associated with the three conformational changes should be zero:C1, ∆GC2, and ∆GC3) may not be determined experimentally for an AcrB-like transporter.The three transitions between successive phases are associated with conformational energy changes, termed ∆ et al. i.e., ∆µpH +F∆Ψ, where F is the Faraday constant and ∆µpH is explained in Box WC2) may be considered as output work for “cooperativity.” In other words, input energy generated by proton influx in one protomer is partially used to allow the other two protomers to overcome their transition barriers as well as thermodynamically unfavorable ∆GC terms. Like ∆GC1/2/3 terms, the energy terms associated with cooperativity is also in balance:One characteristic feature of the AcrB trimer is the absolute requirement of functional cooperation between the three protomers , rates at the three transitions are maintained to be the same. For instance, if the substrate concentration at the loading site increases, the pseudo first-order rate constant ([S]Lkon0) will increase  in order to maintain all three transitions at the same rate. In contrast, in the absence of any substrate at the loading site, the A-B transition as well as the functional cycle stops. In short, the input PMF energy will be distributed dynamically among the three cooperative promoters of the transporter trimer, depending on the types and concentrations of substrate molecules associated with each protomer. For a given type of substrate at a fixed concentration gradient, the cooperativity works WC1, WC2, and WC3 from all three protomers cancel each other out at any given moment. Furthermore, according to the Arrhenius theorem, the pseudo first-order rate constant of a cooperative transporter complex will change by a factor of exp(−WiC/RT) , relative to the hypothetical rate of an isolated monomeric transporter  in thermodynamic terms and to overcome the transition state energy barrier in kinetic terms.For a hypothetical, isolated, monomeric transporter, relative free-energy levels (and the associated rate constants) of each state together would determine the population probability among different states. This distribution in turn would affect the overall transport rate of the transporter Hill . The ratase Hill . This in et al. The United Nations General Assembly recently unanimously approved a declaration committing nations to a more active battle against threat of microbial resistance to existing drugs, which is one of the most severe and rapidly growing threats to contemporary public health. Massive intergovernmental efforts will be required to achieve such an ambitious undertaking. Since bacteria develop drug resistance naturally in an environment containing antibiotics, studies on the mechanism of multidrug resistance, including those of AcrB-like transporters, are an essential part of our fight against multidrug resistance Below is the link to the electronic supplementary material.


Discovered in 1994, this conserved, triple helical globular domain has been characterised in various coactivator proteins of yeast, mammals and plants, including the p300/CBP (a histone acetyl transferase), MED15 (a subunit of the mediator complex of RNA polymerase II), and RECQL5 helicases. In this work, we describe the first rigorous meta analysis of KIX domains across all forms of life, leading to the development of KIXBASE, a predictive web server and global repository for detection and analysis of KIX domains. To our knowledge, KIXBASE comprises the largest online collection of KIX sequences, enabling assessments at the level of both sequence and structure, incorporating PSIPRED and MUSTER at the backend for further annotation and quality assessment. In addition, KIXBASE provides useful information about critical aspects of KIX domains such as their intrinsic disorder, hydrophobicity profiles, functional classification and annotation based on domain architectures. KIXBASE represents a significant enrichment of the currently annotated KIX dataset, especially in the plant kingdom, thus highlighting potential targets for biochemical characterization. The KIX webserver and database are both freely available to the scientific community, at  Structurally, KIX domains are triple helix bundles, with hydrophobic allosteric sites for various transactivation domains (TADs) of transcription factors like CREB-pKID3, Mixed lineage leukaemia protein (MLL)6, FOXO3a7, breast cancer 1 (BRCA1)8, c-Myb9 and sterol responsive element binding proteins (SREBP)10. The KIX domains not only serve as docking sites for TADs but also assist in proper folding of intrinsically disordered TADs. Studies have shown direct involvement of KIX domains in many biological processes like fatty acid metabolism11, multi-drug resistance2, and long-term memory storage in mammals12. KIX domains provide a paradigm for research on molecular allostery, as well as coupled folding and binding mechanisms with TADs. Interaction of KIX with various proteins is important for normal cell function and thus overall health12. For example, KIX binding with MLL protein is an important step in MLL mediated gene expression and the consequent production of normal blood cells. KIX is the binding partner of MLL-activation domain, that is lost due to chromosomal translocations causing childhood and therapy-induced leukemia13. The importance of KIX domains has been well established and is an active research area not only in mammals but also in yeast and plants14. Although KIX domains are yet to be experimentally characterised in the plant kingdom, the global protein databases like InterPro15 reveal their presence in large numbers in many plant species, including Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Populus trichocarpa, Glycine max, etc. Recent taxon-specific computational studies have also suggested the presence of KIX domains in Arabidopsis and rice homologs of MED15 and CBP related Histone acetyl transferase (HAT) proteins as well as F-box proteins of rice17. Functional inferences have been reported in plants where mutations in KIX expressing genes led to multiple development and phenotypic anomalies18. Expression studies followed by SNP analysis revealed the association of KIX domain with stress response, leaf maturation, floral development and seed development20.Transcription, being one of the most fundamental processes in living organisms, is accomplished through stringently regulated assembly of various protein complexes at transcription start sites. Short conserved regions in these proteins often act as independently folding domains that can mediate recognition and transient binding, providing optimal thermodynamic environments and/or docking sites for other proteins leading to the successful formation of the transcriptional machinery. The kinase-inducible domain interacting (KIX) domain, approximately 90-residues in length, is one such example reported to play an indispensable role in transcription15, Pfam21 or Prosite22, when queried for KIX, return predominantly the first of KIX sub-classes, i.e. KIX domains found in CBP related proteins, and very few MED15 associated KIX domains. Most notably, there is a complete omission of RECQL5 associated KIX family members in the general sequence databases. RECQL5 KIX homologs have been reportedly identified in plants as well14, but no records can be found for this in any public database. For example, human MED15 and human RecQL5 proteins, both of which contain an experimentally characterized KIX domain along with availability of 3D structure (PDBIDs ‘2GUT’ for MED15 and ‘4BK0’ for RECQL5), do not show presence of any KIX domain when queried by InterPro or Pfam. Similarly, in plants, InterPro shows the presence of KIX domains in very few MED15 family members of Arabidopsis thaliana whereas KIX domains have been identified not just in members of Arabidopsis Histone acetyltransferase (HAC) family , but also in CBP/P300 like protein (At1g16705), HAC related protein (At4g32295), RECQL3 protein (AT4G35740) and an uncharacterised protein (At3g24150)16. One possible reason for this omission may be that KIX domains tend to have higher sequence similarity within their own class. Sequence homology is not only difficult to detect between KIX sub-classes but also between taxonomic kingdoms of the same sub-class i.e. between MED15 associated KIX of fungi, metazoans and plants14. Therefore, the conventional methods of local sequence similarity search tend to fail when trying to identify KIX domains among diverse organisms across different kingdoms of life. This lacuna in accurate KIX identification inspired us to design an interactive web platform, KIXBASE, which would provide a platform for detecting the presence of KIX domains as well as a comprehensive online repository of KIX in metazoans, fungi and plants developed through detailed computational meta-analysis of available data in resources like the NCBI RefSeq23, UniProt24 and Phytozome25, followed by HMM based domain identification and prediction along with assessment of features like structural propensities, class-based residue conservation and domain organization. To our knowledge, KIXBASE is the only server that can identify all three classes of KIX domains, including those in CBP, RECQL5 and MED15 containing proteins, whereas no other program can presently do so. The web interface of KIXBASE is user-friendly, providing links to explore multiple features of each KIX entry. All the sequence records are in standard FASTA format and have been further validated through other resources viz. MUSTER26, PsiPred27 and Clustal Omega28. We describe the various capabilities and features of KIXBASE, along with a specific case study. In summary, this work massively expands the known KIX domain repertoire and provides evidence for structural conservation across all three classes of KIX domains despite low conservation in sequence across various life forms. We show that KIX domains maintain conservation of secondary and tertiary structure, as well as very similar spatial locations of intrinsically disordered regions. The database is continually updated and maintained in the laboratory. The KIX domain database has been developed using a combination of public resources  and in-house shell scripts, perl functions, python and R code. KIXBASE presently contains 2526 records of KIX domains representing 581 organisms from metazoans, fungi and plants.Despite the characterization of three major classes of KIX, , there is no web resource that caters to these domains, their identification or comparative analyses. Furthermore, the major global protein domain databases like InterProKIXBASE represents a compilation of KIX domains predicted by a search algorithm based on hidden markov model profiles trained on known KIX domains as described in Methods. The input sequences used for training the profile HMMs can be retrieved from the KIXBASE website. The search algorithm was tested for predictive accuracy in identification of KIX domains in higher organisms, as described in the Benchmarking section below, and was found to be highly accurate. Based on the superiority of our search algorithm as compared to other existing programs, it was developed into a predictive online web server, which was used to scan the full proteomes of all available metazoans, plants and fungi . The resulting 2526 KIX domain records along with the web server were assembled into a single browseable online platform, namely KIXBASE. The following sections describe this database and associated features that enable a through investigation of the largest online KIX domain repertoire available to date for higher organisms. We thus present KIXBASE as the only dedicated database, as well as analysis portal for KIX domains, wherein, apart from identification of new KIX, additional features like secondary structure and disorder prediction, extraction of KIX regions, alignments and scoring etc. are assembled on a single platform.rd helix of KIX domain is partially disordered in unbound form and gains helicity upon binding with TAD and formation of ternary complex, signifying a direct relation of helical propensity with cooperative binding30. These plots provide enhanced visualisation of KIX region in terms of its structural properties. The ‘Get secondary Structure’ option on the navigation bar contains the secondary structure output of every predicted KIX domain. This option provides important information for annotation, as the KIX domain exists as a triple helix interrupted by loops or 3–10 helices. The secondary structure output contains three parameters i.e. Conf (Confidence), Pred (Predicted secondary structure) and AA (Target Sequence), as shown in Fig. E-value gives an idea about the probability of the KIX prediction to be a significant hit , as well as a negative dataset that included KIX structural homologs in order to incorporate an additional level of stringency towards accurate identification. The Jackknife test revealed very good statistical parameters, namely a perfect sensitivity of 100%, accuracy >96%, and a Matthew’s Correlation Coefficient (MCC) of 0.94. Encouraged by such a superior performance, we carried out an objective sequence-by-sequence assessment of performance of KIXBASE with other existing methods available publicly.As described in Methods, the Jackknife test was used to evaluate the performance of KIXBASE, as this test is considered to be the least arbitrary and most objective as compared to other cross-validation methods that evaluate quality of computational models, namely sub-sampling  test, and the independent datasetsArabidopsis thaliana whereas KIX domains have been identified and characterised in several other proteins, all of which are identified and represented in KIXBASE. We ascribe this superiority of domain identification of our platform to the rigorous profile HMMs that have been used for training in KIXBASE.Figure Interestingly, plants have the highest density of KIX domains per genome, as compared to fungi or metazoans. Most notably, as mentioned before, KIXBASE is the only web resource providing annotation for RECQL5 helicase associated KIX domains. This can be seen better in Fig. However, no comparative performance evaluation is complete without a statistical test using a common benchmark protocol. A pre-requisite for such a test would be the original sequences that were used to build the respective profiles, followed by regeneration of the hidden markov model. Pfam, having two distinct KIX domain profiles meets this requirement, and therefore, we were able to perform a Jackknife test on Pfam as described in Methods for KIXBASE benchmarking.hmmbuild program from the HMMER suite.However, InterproScan and Prosite, being consortia of independent domain databases like the CDD, do not have their own KIX profiles. Pfam, on the other hand, has two profiles for KIX, [namely Pfam-KIX and Pfam-KIX-2]. We downloaded the full FASTA formats from multiple sequence alignment (MSA) of both these Pfam entries, and performed a Jackknife re-sampling of the aligned sequences, using Table For a true comparative evaluation, we selected test sequences whose fraction of KIX proteins (positive set) could be treated as similar to the actual frequency of KIX proteins in nature. Accordingly, we tested KIXBASE and both Pfam profiles on subsets of yeast, Arabidopsis and human proteomes, where known KIX-containing proteins were treated as the positive set sequences, while the negative set sequences were proteins known ‘not’ to contain any KIX. Thus, our positive test set contained known Med15, CBP and RECQL proteins having complete/partial KIX domain from uniprotKB. The negative test set contained all reviewed (swissprot) protein sequences with molecular function GO term ‘not’ related to transcription. Results of the stastistical test are shown in Table As can be seen from this table, KIXBASE performs much better than  Pfam in all measures evaluated. As mentioned previously, the two Pfam KIX profiles are very sub-class specific, one for CBP-KIX domains and the other for MED15-KIX of yeast and plants. Consequently, these profiles are unable to identify any KIX domain outside of these classes or taxa. The perfect score of Pfam in case of yeast merely reflects the absence of any experimentally characterized RECQL-5 domains in this organism to date, and consequently RECQL-5 identification was not used for performance evaluation in yeast, providing an example of how difficult it is to reveal the true nature of Pfam deficiency in case of KIX. However, all three classes of KIX are experimentally characterized in humans with structures available in PDB, thereby making it possible to further elaborate the class-wise performance of all three profiles for humans, as shown in Table As can be seen from this table, only KIXBASE can predict all classes of KIX domains whereas both Pfam KIX profiles are only able to identify CBP-KIX. It may be noted here that although Pfam_KIX_2 profile is based on MED15-KIX specific sequences (of yeast and plants), it is still not able to classify Med15 KIX in humans  were added to the training set to improve the accuracy of prediction14. In all, the training set contained 312 KIX domains, all of which have been provided to KIXBASE users on the website under Tutorials/Datasets section. The HMM profile for KIX domains was generated using HMMER v3.1b134. This profile was run on the complete proteomes of 118 plants, 278175 metazoans and 924 fungal species, extracted using NCBI RefSeq23, UniProtKB24 and Phytozome-1025. As shown in the workflow in Fig. 26 in only retain records with highest fold similarity to any of the characterised KIX domain available in the PDB35 library. This incorporation of fold recognition feature makes KIX database highly accurate and reliable.The KIX identification algorithm uses profile Hidden Markov Models. The initial dataset used for generation and training of profile HMMs was compiled by extraction of FASTA sequences of proteins containing KIX domain in various species via a keyword-based search  of various protein family databases, such as Pfam, PROSITE, Superfamily, SMART and Gene3D, which are collectively present in InterPro Consortium. This dataset was filtered for redundancy using the CD-HIT31.Cross-validation is a mode of performance estimation for a predictive model on a dataset, which is not used for generating it. It involves partitioning the data into complementary sub-sets, developing the predictor on one sub-set (the training dataset), and validating the prediction efficacy on the other sub-set . To reduce variability due to sample partition, multiple rounds of cross-validations are performed using different data partitions and results are averaged over all partitions. Three of the most common cross-validation methods used in statistical prediction are independent dataset test, sub-sampling test and jackknife test. Of these, the Jacknife test , has been increasingly and widely used by investigators to examine the quality of various predictors and has also been shown to be a method of choice for evaluation of hidden markov models, since it is considered least arbitrary and can yield a unique result for a given benchmark dataset whereas the other two test methods bear considerable arbitrariness, as elaborated by Chou in a detailed analysis that we strongly recommend to interested readers36 since sequences closely related to KIX would serve as a suitable negative dataset. The final negative test set comprised of 1144 sequences. The training set was based on known KIX sequences which were used to build a multiple sequence alignment (MSA) that was used as input to the HMMER package through hmmbuild option.For evaluating the KIXBASE pipeline, a negative dataset was prepared by collecting TFIIS sequences and its homologs through BLAST. TFIIS was selected on account of its marked structural similarity with RecQL5 KIX. As mentioned previously, RecQL5 KIX mimics TFIIS structure and binds to RNA pol II instead, thus blocking transcription elongation. In addition, we also took KIX structural homologs through DALI serverThe jackknife cross-validation experiment was conducted on KIXBASE to validate the HMM performance for detection of sequences not used in the creation of the profiles (pseudo-novel sequences). In order to follow the jackknife model testing procedure, we consider as “full data set” all members of the HMM seed set plus the 1144 negative examples. In each iteration, one sequence was removed from the MSA of the HMM’s seed set, and a new HMM was constructed from the remaining sequences in the MSA. Then the profile’s ability to correctly classify the removed sequence was measured. To test the negative dataset, this involved x + 1144 iterations, where x is the size of our HMM seed set. For 1144 of these iterations the HMM was identical (since no members of the positive set are being held out), but the point is still to have symmetric treatment of the positive and negative examples in the designated training set, and to test the classification of exactly one data point (the one that is held out) at each iteration. This was followed by assessment of the following statistical parameters for evaluation and performance:In general, sensitivity is the proportion of positive sequences correctly identified as KIX whereas specificity is the proportion of negative sequences correctly identified as non-KIX. Precision is a fraction of retrieved positives that are relevant and accuracy is an overall population of true results in the population. MCC is considered the best measure of the quality of classification when the test datasets are of different sizes during binary classification. MCC ranges from −1 to 1, where values closer to −1, 0 and +1 depicts poor, random and good predictions respectively.In order to support our claim that KIXBASE is superior to global general databases for KIX domain assignment, we have performed Jackknife tests for the two Pfam profiles related to KIX, namely, pfam KIX and pfam KIX-2 where the same negative test set of 1144 sequences was used as in case of KIXBASE above, along with each sequence in the respective MSA as iterative positive test data. Hence our positive set (x) for generating Table 27 was used for prediction of secondary structures of KIX sequences. The UniRef9037 dataset was downloaded in order to run psiBLAST38 search required for PSIPRED runs. A python script was developed to generate 2D plots from result scores of PSIPRED, IUPRED39 and hydrophobicity Kyte-Doolittle index40 for KIX domains. The python script included Numpy41, pylab and Matplotlib42 packages for plotting and graphics environment. Each KIX domain sequence was aligned with a parent set of characterised KIX domains using the Clustal Omega28 program. Domain arrangements plots were generated through a highly efficient pipeline where each sequence was scanned for known domains through ‘hmmscan’ program from HMMER followed by E-value filters and overlap removals. Pfam-A seed alignments were utilized for searching additional annotated domains present in combination with predicted KIX domains on a given sequence. PHP, CSS and HTML programming languages were employed to develop the KIXBASE user interface, with output in the form of tables and coloured plots generated through manual scripts. BLASTP43 service by NCBI is also utilised to search a query across the database. In house perl scripts were developed to color the alignments in accordance with conventional Clustal-O coloring scheme based on physiochemical properties. MSA package from Bioconductor was used to generate conservation-based display of alignments44. A total of 326 metazoans, 170 fungi and 90 plant species are present in the KIXBASE. The complete list of these species along with classification tree can be found in KIXBASE. The training and test sets used for generating and testing profile HMM are also available for download on the website under the Tutorial/Dataset link. The web portal is freely available to the scientific community, at http://www.nipgr.res.in/kixbase/home.php.For the development of the web repository, all KIX domain regions were mapped onto FASTA sequences and highlighted using python code. PSIPRED 3.5


Plant lectins are proteins that reversibly bind carbohydrates and are assumed to play an important role in plant development and resistance. Through the binding of carbohydrate ligands, lectins are involved in the perception of environmental signals and their translation into phenotypical responses. These processes require down-stream signaling cascades, often mediated by interacting proteins. Fusing the respective genes of two interacting proteins can be a way to increase the efficiency of this process. Most recently, proteins containing jacalin-related lectin (JRL) domains became a subject of plant resistance responses research. A meta-data analysis of fusion proteins containing JRL domains across different kingdoms revealed diverse partner domains ranging from kinases to toxins. Among them, proteins containing a JRL domain and a dirigent domain occur exclusively within monocotyledonous plants and show an unexpected high range of family member expansion compared to other JRL-fusion proteins. Rice, wheat, and barley plants overexpressing OsJAC1, a member of this family, are resistant against important fungal pathogens. We discuss the possibility that JRL domains also function as a decoy in fusion proteins and help to alert plants of the presence of attacking pathogens. Arabidopsis thaliana. A prominent example is the brassinosteroid receptor BRI1 (brassinosteroid insensitive 1) that contains an extracellular leucine-rich repeat (LRR) domain .In this review, we will summarize recent advances in our understanding of the role of chimeric lectin-containing proteins in plant defense, with special emphasis on those chimeric proteins containing a JRL domain.The majority of eukaryotic proteins consist of more than one domain, i.e., are chimeric . These mIn general, lectins are sugar-binding proteins with at least one non-catalytic domain enabling them to selectively recognize and reversibly bind to particular carbohydrates . These gGalanthus nivaili agglutinin-related), and C-  types [The majority of known plant lectins consist of one or more lectin domains coupled to another, unrelated domain . The most) types . Beside t) types .Amaranthus was reported to enhance resistance to aphids when ectopically expressed in transgenic tobacco and cotton by affecting the growth and development of the invading pest [Nicotiana tabacum agglutinin, originally discovered in tobacco leaves after jasmonate treatment [O-GlcNAc moieties of core histone proteins. It is hypothesized that the binding of Nictaba to chromatin results in enhanced transcription of defense-related genes [Sambucus (elderberry) a variety of ricin B domain-containing proteins was identified, including SNA-I and SNA-V, and the constitutive expression of the corresponding two genes in transgenic tobacco was shown to enhance resistance to tobacco mosaic virus [Artocarpus integrifolia L.) [Contrary to most lectins, the amaranthin domain itself has no sugar binding site(s); however, a specific head-to-tail arrangement of two amaranthin subunits establishes a T-antigen disaccharide binding site . The nucing pest . Calretiing pest . The faming pest . Nictabareatment . Expressed genes . The riced genes . Unlike ed genes . Within ic virus ,23. Seveic virus . jacalinolia L.) . JRLs arolia L.) . Althougolia L.) .Arabidopsis thaliana [Fusarium graminearum and Blumeria graminis [Many JRLs have been shown to be associated with disease resistance, abiotic stress signaling, wounding, and insect damage . For exathaliana . Moreovegraminis . OsJAC1,graminis .Most recently, Krattinger et al.  identifiProteins containing JRL domains are not exclusive to plants, but rather are present in various species of different taxonomic groups. We identified 3000 JRL domain-containing proteins in a Uniprot data base search using the Pfam domain PF01419 as query . Out of Phytophthora protein 1 (NPP1) domain, are specific for Oomycetes and 4.7% of JRL proteins in Oomycetes comprise an NPP1 domain (In bacteria the majority of JRL proteins (69.6%) are fusion proteins with one or more exonuclease-endonuclease-phosphatase (EEP) domain . The lar1 domain . ProteinDrosophila kelch open reading frame (ORF) 1 protein [In plants, single-domain JRL proteins and multi-JRL domain proteins account for the majority of proteins with a JRL domain; however, a variety of chimeric JRL proteins containing unrelated domains can be found . In eudi protein ,40. The  protein . The F-b protein .Members of the Liliopsida show the highest diversification of chimeric JRL proteins compared to plants of other classes . FrequenProteins containing a dirigent, Pkc, or RX-CC domain are discussed regarding their involvement in pathogen resistance. Proteins of the RX-CC domain superfamily are similar to the potato resistance protein Rx. Rx has a modular structure consisting of a coiled-coil (CC) domain, a nucleotide-binding (NB), and a leucine-rich repeat (LRR) domain, and mediates resistance to potato virus X . The P-lSo far, we have focused on the question of which unrelated domain superfamilies occur as partners in JRL fusion proteins and how frequently they are found in different taxonomic groups. Next, we were interested in the question of whether there are specific domain compositions within the plethora of different chimeric JRL-proteins that appear noticeably more often than others, or are specific for different taxonomic groups. In Fusarium head blight [By correlating these data, we show that while single- and multi-domain JRL proteins with up to three JRL domains are found in prokaryota and eukaryota, proteins with more than three JRL domains are only present in eukaryota . It is pd blight .Domains of plant-specific chimeric JRLs include kelch, RX-CC, ricin, PKc, or dirigent domains. Interestingly, JRL proteins with three carboxy-terminal kelch domains or an amino-terminal F-box domain are specific for eudicotyledonous plants and, to our knowledge, cannot be found in the group of monocotyledons (Liliopsida) . In contDirigent proteins are generally assumed to play an important role in the biosynthesis of lignins and lignans . In bothPoaceae-specific monocot chimeric jacalins [Magnaporthe oryzae, Rhizoctonia solanii, or Xanthomonas oryzae [Recently, we reported our novel findings regarding the dirigent domain-containing chimeric JRL protein OsJAC1 of rice . OsJAC1 jacalins . When ovs oryzae . Overexps oryzae . The arts oryzae .The Rosetta stone theory hypothesizes that fusion proteins are an indication for the cooperative activity of proteins with the respective single domains . In thisBoth aspects—the identification of interacting pairs of dirigent and JRL proteins in dicots, as well as the characterization of the still poorly-described mode of action of monocot chimeric jacalins—will be important steps in advancing our understanding of plant resistance.


Hybrid fungal CDP synthetases are constructed from three different origins to produce highly active cyclodepsipeptides up to g L–1 scale. in trans expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their in vivo biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using Aspergillus niger as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L–1) and octa-beauvericin (10.8 mg L–1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L–1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against Leishmania donovani and Trypanosoma cruzi compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines.Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation,  N-methylated cyclodepsipeptides (CDPs) of different ring sizes. Hexa-CDPs such as enniatin B (Fusarium ssp.) and beauvericin (Beauveria bassiana) as well as octa-CDPs like PF1022A (Rosellinia sp.) and bassianolide (B. bassiana) , transfer (T) and condensation (C) of the substrate, as well as a terminal T2b–C3 bidomain (1) activates d-α-hydroxycarboxylic acids (d-HA) and has been shown to be promiscuous concerning aliphatic and aromatic d-HAs to varying extents.4 The d-HA is subsequently coupled to an l-α-amino acid (l-AA), which is activated and N-methylated by the second module (M2) containing an auxiliary N-methyltransferase (Mt) domain. Module 2 has also been reported to process different N-Me-l-amino acid (N-Me-l-AA) building blocks to a certain degree.6 Concerning substrate elongation, it has been proposed that the growing depsipeptide chain is elongated either in dipeptidol units 8 or in a stepwise fashion using single HA or AA building blocks  domains in fungal linear NRPSs.11 Still, various biosynthetic features of CDP synthetases are only partially understood, including mechanistic insights into how hexa- and octa-CDPs are assembled.Nonribosomal peptide synthetases (NRPSs) assemble a great variety of linear and cyclic peptides, which display a diverse spectrum of valuable pharmacologically useful bioactivities. Amongst those biosynthetic machineries, iterative-type NRPSs from filamentous fungi are a remarkable subgroup, which produce assiana)  show varbidomain . The fir12 substitution,13 extension14 and deletion15 of NRPS segments. Initial attempts in fungal NRPS-engineering have shown encouraging results in vivo, leading to novel nonribosomal peptides (NRPs) including derivatives of enniatin and beauvericin with altered d-HA side chains.18 Since the chemical synthesis of CDPs has some drawbacks and limitations, biotechnological production in heterologous microbial cell factories is a promising alternative for sustainable CDP production.19 However, as long as our mechanistic understanding of fungal NRPS assembly remains limited, an effective engineering of fungal iterative NRPSs is unfeasible. We thus focused on the functional analysis of various catalytic steps of CDP synthetases, whilst concomitantly establishing an in vivo expression system for new-to-nature CDP derivatives with altered bioactivities. While we were preparing this manuscript, Yu et al. proposed a linear CDP biosynthesis model, which complements our findings.10Due to their modular architecture and high homology, CDP synthetases appear well suited to study domain-specific contributions by combinatorial exchanges, while generating novel peptide structures. Consequently, fungal NRPSs offer an excellent opportunity for engineering approaches. Inspiration comes from various engineering attempts in bacterial systems, including module fusion,5C6 bidomain of tyrocidine synthetase from Brevibacillus brevis .20 Using the TycC5–6 T–C bidomain as a template, structural models of EnSYN-, BeSYN- and BaSYN-T1–C2 and T2b–C3 bidomains were generated (SWISS-MODEL21). The homology models were aligned to TycC5–6  to identify promising swapping sites in the linker regions. The structurally identified swapping regions were annotated in a multiple sequence alignment with the synthetases PF-, En-, Be- and BaSYN in order to define the distinct T1–C2 and T2b–C3 swapping sites  of surfactin A-synthetase from Bacillus subtilis .23 The identified truncation region was annotated in a multiple sequence alignment with PF-, En-, Be- and BaSYN to define the distinct C1 truncation site .24 Structural information about A domains was obtained from the crystal structure of GrsA 24 to identify secondary structure elements flanking the loop region in which Mt domains are commonly embedded. The loop region was annotated in a multiple sequence alignment of the PF-, En-, Be- and BaSYN-A2 domains to identify the distinct truncation sites  and aligned (PyMOL) to confirm the construction of a loop in A2 similar to A1 , harboring the fungal phosphopantetheinyl transferase (PPTase) npgA derived from A. nidulans DM3365 on a second plasmid. The co-expressed NpgA displayed a 14-fold enhanced production of the fungal CDP products in comparison to the endogenous PPTase and 4.8-fold increase when compared to Sfp  were successfully produced in E. coli co-expressing NpgA  and monitored in vivo production of enniatins with and without co-expression of the excised EnSYN-C3 domain. EnSYNΔC3 was unable to produce enniatins, however, CDP production could be restored in trans by co-expression with the free-standing EnSYN-C3 domain C3 with a heterologous C3 domain derived from the octa-CDP-producing BaSYN even enabled the production of new-to-nature octa-enniatin B , while for BaSYN, no natural non-methylated species have been reported so far. Finally, we challenged the linear CDP elongation model similar to a recent BeSYN-based approach10 by constructing versions of EnSYN with mutations at the conserved serines bearing the phosphopantetheine groups Ppant2a (S2538A) or Ppant2b (S2632A). Like the BeSYN-variants, both mutants  were still able to produce their wild-type CDP enniatin in vivo in E. coli C3 and BeSYN–Ba(T)C3 still produced trace amounts of the natural smaller ring size C3 and BaSYN–Be(T)C3 produced the respective hybrid CDP hexa-bassianolide  and fungal Cterm domains,28 the C3 domain consists of an N- and C-terminal subdomain , which together form a V-shaped pseudo-dimer with the active site buried in the cleft between the two subdomains  synthetase that is closed at its acceptor site,22 the C3 domain of fungal CDP synthetases also catalyzes condensation reactions.10 Hence, the C3 domain features a functional acceptor site competent to catalyze ester bond formation. In order to gain insights into the molecular determinants of macrocyclization and ring size control by the C3 domain, we started with a sequence-based shuffling approach. An alignment of the C3 domains revealed ten residues potentially involved in ring size determination . Exchanging these five amino acids did not hamper CDP production, but did not lead to an altered ring size either , the region which crosses over to C3NTD and forms a lid above the cyclization pocket -mediated transformation of two A. niger strains differing in their protease profiles (strains AB1.13 and MA169.4) with the hybrid synthetase genes of EnSYN–BaTC3, BeSYN–BaTC3, BaSYN–EnTC3 and BaSYN–BeTC3. The genes were integrated into the fungal genome by homologous recombination . Since both wild-type BaSYN and EnSYN naturally produce minor amounts of hexa-bassianolide, the building blocks d-Hiv and l-Leu represent cognate substrates for both NRPS systems, thus ensuring efficient processing. To date, hexa-bassianolide could only be obtained in low amounts either from chemical synthesis,29 by precursor-directed biosynthesis (2.2 mg L–1)30 or from extraction of cultures of Verticillium hemipterigenum .27 In a very recent report on heterologous CDP production in S. cerevisiae, no yields were mentioned.10All hybrid CDPs were purified by extraction with ethyl acetate, flash chromatography and reversed phase HPLC. Purification of the hybrid octa-CDPs from inations , which w32 octa-enniatin B, octa-beauvericin and hexa-bassianolide were tested for antiparasitic, antibacterial and antifungal activity along with cytotoxicity measurements  than the medical reference drug benznidazole (6.53 μM). Furthermore, they are superior to their corresponding natural hexa-CDPs enniatin B (2.31 μM) and beauvericin (0.76 μM), respectively. Against L. donovani, octa-enniatin B and octa-beauvericin showed up to >8-fold lower IC50  compared to the medically used reference drug miltefosine (0.58 μM). In comparison to enniatin B and beauvericin , both compounds were also significantly more active and showed a similar cytotoxicity against rat-derived L6 cells  were capable of producing the predicted hexa- or octa-CDPs in E. coli co-expressing NpgA  remained in the same range as that of wild-type EnSYN (0.15 mg L–1) and BaSYN (0.06 mg L–1), with the exception of BaSYN–EnC3 (24 mg L–1). The fact that BaSYN–EnC3 showed such a superior production in comparison to BaSYN–EnTC3 (0.88 mg L–1) highlights the influence of artificial T–C domain pairings in hybrid synthetases, which have a crucial role, i.e. in ester bond formation and macrocyclization. Surprisingly, the hybrid synthetases containing multiple swaps  showed an enhanced production up to a factor of 520 (55–78 mg L–1) compared to the wild-type EnSYN (0.15 mg L–1), despite having more heterologous interfaces for domain–domain interactions. This is in stark contrast to virtually all previous studies on module and domain exchanges in bacterial NRPS systems suffering from a significant drop in product titers.32 Notably, the relatively high titer was achieved in the bacterial heterologous host E. coli, which previously rendered low production yields.17 Fungal NRPS systems might thus represent a viable alternative as an engineering platform with promising potential. Furthermore, the results suggest that the efficiency of the biosynthesis is influenced by the N-terminal part of the synthetase. This is supported by the fact that the hybrid EnSYN–BaM2 contains a homologous module 1 and T2bC3, thus pointing to highly important inter-modular interactions like the proposed C3–T1 interaction during ester bond formation.10 However, the highest production was achieved with a heterologous C3–T1 interface (BeM1–BaM2–EnTC3). The assumption of a readily exchangeable “bio-brick” system thus appears somewhat simplistic for a sustainable engineering approach.Since bacterial NRPS-systems do not tolerate this type of swapping well,3 domain performs macrocyclization and determines the ring size independently from C1. In contrast to recent findings,10 truncated synthetases lacking the C1 domain are indeed functional in vivo. However, since the absence of C1 significantly affects the production yields of CDPs (drop of synthesis to 1–55%), a stabilizing and/or supportive role of C1 in the catalytic cycle is apparent. In accordance with a recent crystal structure comprising the C–A–T–Te domains of the bacterial NRPS AB3403,28 the C1 domain may serve as a resting site during precursor adenylation in the A1 domain. This stabilization may also apply to the A1 domain itself, since the C domains can affect the tertiary structure33 and substrate specificities of their downstream A domain partners.34Fungal CDP synthetases produce six- and eight-membered CDPs with a broad range of pharmacologically relevant bioactivities. Based on their modular structure, high homology and manageable size, these nonribosomal assembly lines bear good prospects to be an effective engineering platform for the production of novel CDP compounds. Still, a tailored engineering of CDP synthetases is somewhat limited by our poor understanding of the biosynthesis mechanisms. The systematic investigation of the individual contributions of domains to the catalytic activity and synthesis products in this study allow us to confirm the following model: the C-terminal C3 domain demonstrated that the C3 domain tolerates aliphatic as well as aromatic side chains . Apart from esterification, C3 also inherits the role of a gauge, measuring product chain length. In contrast to recent statements,10 this gauge also allows for cyclization of premature depsipeptide chains. Deletion of the Mt domain, affording the corresponding non-methylated peptides, was able to demonstrate that the absence of structure-modulating N-methylations is tolerated. As previous Mt domain deletions reportedly only affected β-methylation of Glu in daptomycin,13 our findings represent a rare example of complete in vivo NRP backbone engineering. The construction of a number of artificial CDP synthetases by means of domain swapping yielded hybrid hexa- and octa-CDPs under in vivo conditions. This way, we were able to produce the hybrid CDP hexa-bassianolide at very high titer of 1.3 g L–1 as well as new-to-nature octa-enniatin B (4 mg L–1) and octa-beauvericin (10.8 mg L–1) by using A. niger as a robust and cognate heterologous host. While previous approaches with the daptomycin synthetase have shown that multiple domain swaps lead to reduced product yields,13 we could significantly enhance CDP production in vivo by constructing functional hybrid synthetases from three different NRPSs. The results highlight the potential of fungal NRPS systems as engineering platforms as well as the crucial role of domain–domain and module–module interfaces in NRPS efficiency.With regard to substrate specificity, various functional hybrid synthetases with a heterologous C3 subdomain uncovered functional aspects of macrocyclization and ring size control. According to the bacterial Te mechanism, macrocyclization by the fungal C3 domain is performed when the free-standing hydroxy group at the tail of the linear depsipeptidyl chain is in close proximity to Hiscat of C3 and the substrate thioester. In consequence, CDP ring size probably depends on the size and composition of the cyclization pocket formed by C3NTD and C3CTD, along with a competitive process between the elongation and cyclization reaction. This assumption is supported by our observations that, unlike the octa-CDP-producing synthetases, all hexa-CDP-generating hybrids almost completely lost the capability of producing the cognate eight-membered ring. One reason might be the steric demand of a linear octa-depsipeptide, which may not fit well into the cyclization pocket of a hexa-CDP-producing synthetase. In addition to steric restrictions imposed by the (heterologous) cyclization pocket, ring size may also depend on intrinsic conformational properties of the growing depsipeptide chain. The nature of side chains, and in particular of backbone N-methylation, dictates the available conformational space of the depsipeptide backbone and thus affects cyclization efficiencies. For example, Ramachandran plots for peptides comprising N-Me-AAs demonstrate the restricted conformational freedom compared to their non-methylated analogs.35 However, the distinct mechanism of CDP ring size determination remains to be elucidated. By confirming recent findings of CDP production in synthetase variants lacking Ppant2a/b, our data also give strong support to the so-called “linear” CDP biosynthesis model. From a formal aspect, employing an alternating upstream and downstream elongation, this NRPS type might be reclassified as belonging to the “complex-type”32 rather than simply linear or iterative. We therefore suggest the term “looping mode”, implicating the substrate shuttling between two C domains in a consecutive manner. Still, since the role of the T2b domain remains unclear and odd-numbered T domain-bound intermediates are yet to be observed,8 neither of the two models can be completely ruled out. Hence, further information must be gathered with synthetase mutants, e.g. stalled at certain biosynthesis steps, as it has been applied to the bacterial balhimycin synthetase36 and rifamycin polyketide synthase.37Swapping of parts of the CT. cruzi (up to 12-fold) and L. donovani (up to 8.3-fold) compared to the reference drugs benznidazole and miltefosine, respectively, accompanied by similar cytotoxicity against L6 cells. Our findings give distinct clues for the active and applicable reprogramming of both iterative and linear NRPS assembly lines in further protein engineering approaches. In the future, we aim to expand the existing range of CDP ring sizes beyond the natural hexa- and octa-CDPs, giving rise to a whole class of new-to-nature cyclic fungal products with superior pharmacological activities and efficiencies.Finally, our experiments show that the design of bio-combinatorial swaps in NRPSs can serve well for the synthesis of compounds with enhanced bioactivities. Accordingly, the bioactivity profiling of octa-enniatin B and octa-beauvericin rendered a significantly stronger antiparasitic activity against There are no conflicts of interest to declare.


Phyllanthus niruri L., a well-known medicinal plant, has been used as a folk antitumor remedy in the worldwide scale. However, the antitumor components in P. niruri have not been reported. In order to verify the antitumor components of P. niruri and the plants which have the high content of these components, we isolated the antitumor components with bioguided fraction and isolation, by different chromatographic methods from the ethyl acetate fraction of P. niruri., and identified them as ethyl brevifolincarboxylate and corilagin by 1H-NMR, 13C-NMR, 2D-NMR, and mass spectrometric analyses. Cell cytotoxicity assays showed that corilagin has broad-spectrum antitumor activity, a better antitumor potential, and lower toxicity in normal cells. Besides, the coefficient of drug interaction (CDI) of 10 μM corilagin and 20 μM cDDP reached up to 0.77, which means corilagin can promote the antitumor activity of cDDP. Furthermore, by the extensive screening among 10 species of plants reported to contain corilagin, we found that Dimocarpus longan Lour. has the maximum content of corilagin. In conclusion, corilagin is the major active antitumor composition in P. niruri. L. on HCC cells and has high content in D. longan. Phyllanthus niruri L. (P. niruri) is a member of the Euphorbiaceae family that consists of over 600 species of shrubs, trees, and annual or biennial herbs [ P. niruri is used as medicated food to nurse people's health, especial hair [ P. niruri has been claimed to present beneficial therapeutic effects in treating kidney and gallbladder stones, liver related diseases such as jaundice and liver cancer, viral infections such as hepatitis and tuberculosis, malaria, diabetes, and fever [al herbs . It usuaial hair , intestiial hair , liver [ial hair , and kidial hair  problemsnd fever . P. niruri have been reported gradually. P. niruri has been found to exhibit antitumor [ P. niruri extract shows potential in reducing chemically induced skin papillomas by enhancing antioxidant defense systems [ P. niruri exhibited a 59.5% inhibition of rat aortic vascular growth and showed a significant decrease of 37.9% in the tube formation assay involving human umbilical vein endothelial cells (HUVECs) on Matrigel [In recent years, systematic studies on the constituents and medicinal effects ofntitumor , antivirntitumor , antioxintitumor , anti-inntitumor , and antntitumor  activitintitumor . Particu systems  and showMatrigel . P. niruri is a candidate folk medicine [ in vitro/vivo just focused on the crude extracts from P. niruri [ P. niruri have antitumor activity. Fujiki indicated that, to investigate P. niruri, a useful and practical method for screening preventive cancer agents is needed [Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world and was ranked the second cause of cancer death . The maimedicine , 18, 19.. niruri , 20. Thes needed . P. niruri and determine their biological activity on HCC.Thus, due to the presence of various medicinal properties, the present study was designed to isolate, screen, and identify the structure of really active components from P. niruri plants were collected from Gulangyu Islet, Fujian province, China, in October 2009 and identified by Professor Yong-Tian Zhang, Fujian Province Institute of Subtropical Botany, China. A voucher specimen (YZY20091026) was deposited at Xiamen Overseas Chinese Subtropical Plant Introduction Garden, China.WholeAll solvents and chemicals used for extraction in this study were of analytical grade and obtained from Tianjin Reagent Company . Materials for column chromatography included polyamide resin  and Sephadex LH-20 . The SMMC7721, Bel7402, MHCC97-H, HepG2, OC316, SGC7901, QBC939, and Chang-liver normal liver cell line were obtained from the Cell Bank of the Chinese Academy of Sciences . RPMI 1640 medium and Dulbecco's Modified Eagle Media (DMEM) were obtained from Gibco . The 3--2,5-diphenyltetrazolium bromide (MTT) reagent was obtained from Sigma . Trypsin, Leibovitz's L-15 medium, fetal bovine serum (FBS), and penicillin/streptomycin solution (200x) were obtained from Mediatech Inc. .μm); DAD detector detection wavelength: 280 nm; column temperature: 30°C; mobile phase: Acetonitrile (A) 0.1% KH2PO4 (B); flow speed: 1 mL/min; injection volume: 20 μL. Elution procedures are as follows: 0–10 min, V(A) : V(B) = 15 : 85; 10–15 min, V(A) : V(B) = 20 : 80; 15–20 min, V(A) : V(B) = 25 : 75.The chromate column was Waters XBridge™ Shield RP18  and concentration (X).We weighed 2.5 mg corilagin precisely, dissolved it in a 10 mL measuring flask with 50% methanol, and prepared a standard solution whose concentration is 0.25 mg/mL; diluted corilagin standard solution into 0.025, 0.050, 0.075, 0.100, and 0.125 mg/mL; injected the diluted solution into HPLC and then determined absorption spectrums. We could get the linear relation from peak area . The solvent was evaporated in vacuo, and the dried ethanol extract was dissolved in 50% methanol (100 mL) and then we filtered it with 0.45  1H-, 13C-, and 2D-NMR spectra were recorded on Bruker AV-400 spectrometers at room temperature . Mass spectrometry was carried out on a VG AutoSpec-3000 spectrometer or a Finnigan MAT 90 instrument. EI-MS measurements were carried out on an LCQ-Finnigan instrument at room temperature.Compounds were purified on polyamide column, D101 MCI gel and Sephadex LH-20 column, analyzed qualitatively by TLC and quantitatively on ODS-C18 column (150 × 4.6 mm) with a UV detector and MeOH-water (7 : 3) as mobile phase, and determined by HPLC. IR spectra were obtained with a Nicolet AVAIAR 360 FT-IR spectrometer with KBr pellets. The2SO4 in ethanol.Column chromatography was performed with polyamide resin (100–200 mesh) and Sephadex LH-20. Fractions were monitored using TLC, and spots were visualized by heating silica gel plates or spraying with 5% H P. niruri (3 kg) were extracted with 75% EtOH (3 × 5 L). The 75% EtOH extract was combined and evaporated under reduced pressure to yield a residue. Then, it was suspended in water and partitioned with petroleum ether, CHCl3, EtOAc, and n-BuOH , resulting in five fractions: petroleum ether fraction , CHCl3 fraction , EtOAc fraction , n-BuOH fraction , and water fraction . After that, the cytotoxicity of five fractions on HCC cell lines was determined by MTT assay. Fraction showing the highest cytotoxicity was further fractionated by column chromatography each time.The dried and powdered whole2O-EtOH  and then 3.5% NH3-H2O to yield seven fractions (Fr.3.1–Fr.3.7). For Fr.3.2, the solvent was allowed to slowly evaporate, causing the compound to precipitate, which was then collected by filtration and recrystallized to yield yellow, diamond, flake crystallization . The filtrate was then separated by chromatography on a Sephadex LH-20 column and eluted with MeOH to obtain compound 2 (43.0 mg) (Fr.3 was separated by polyamide column chromatography using a gradient of H43.0 mg) .μg/mL) in a humidified incubator aerated with 5% CO2 and 95% air at 37°C.Cells were cultured in RPMI 1640  or DMEM  supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin  for 48 h. Then, 20 μL of MTT (5 mg/mL) was added to each well, and the plates were incubated for additional 4 h at 37°C. The medium was replaced with DMSO to dissolve the formazan produced from MTT by viable cells. Absorbance at 492 nm is proportional to the live cell count, and cell survival was expressed as the absorbance of MTT-treated cells relative that of DMSO-treated controls.The effect of constituents on the growth of SMMC7721, Bel7402, and Chang-liver cells was determined using the MTT assay. Briefly, 5000 cells per well in 96-well microtiter plates were treated with corilagin at 0, 10, 20, 40, 80, 120, and 160 μM) and cis-platinum  alone or together with SMMC7721 for 48 h. The coefficient of drug interaction (CDI) was calculated as follows: CDI = AB/(A × B). According to the absorbance of each group, AB is the absorbance of the combination group; A or B is the absorbance of the single drug group. Thus, CDI value <1, =1, or >1 indicates that the drugs are synergistic, additive, or antagonistic, respectively. CDI value <0.7 indicates that the drugs are significantly synergistic. Thus, CDI was used to analyze effects of drug combinations [As cell cytotoxicity assays, SMMC7721 was treated with corilagin , EtOAc fraction (Fr.3), n-BuOH fraction (Fr.4), and water fraction (Fr.5), were extracted from 75% EtOH extract of P. niruri. We estimated the antitumor activity of five fractions by MTT assay and found that the IC50 values of the Fr.3 and Fr.4 were lower than others , CHCln others . So Fr.32O-EtOH  and 3.5% NH3·H2O, respectively, to yield seven fractions (Fr.3.1–Fr.3.7). MTT assay showed that the IC50 value of Fr.3.2 was the lowest one  and corilagin (2).Chromatographic purification ofEthyl brevifolincarboxylate (1). 1H-NMR  δ: 1.19 ; 4.05 ; 2.44 ; 2.98 ; 4.40 ; 7.29 . 13C-NMR : δC 145.77 (C=2), 138.49 (C=3), 114.93 (C=3a), 143.63 (C=140.20), 149.60 (C=6), 108.02 (C=7), 112.98 (C=7a), 160.11 (C=8), 40.69 (C=9), 37.01 (C=10), 193.02 (C=11), 171.99 (C=O), 60.56 (C=12), and 13.86 (C=13).Corilagin (2). 1H-NMR : δH 7.01 , 6.55 , 6.49 , 6.20 , 4.58 , 4.35 , 4.22 , 4.21 , 3.94 , and 3.87 ; 13C-NMR : δC 167.2 and 167.5 (C=O), 145.6 (C-3′′′), 145.3 (C-3′′), 144.4 (C-5′′′), 144.8 (C-5′′), 135.9 (C-4′′′), 136.0 (C-4′′), 116.0 (C-1′′′), 116.3 (C-1′′), 123.5 (C-2′′′), 124.4 (C-2′′), 106.6 (C-6′′′), and 107.4 (C-6′′); galloyl δC 165.3 (C=O), 146.0 (C-3′ and C-5′), 139.5 (C-4′), 119.2 (C-1′), and 109.5 (C-2′ and C-6′); glucose δC 92.7 (C-1), 78.0 (C-3), 76.8 (C-5), 72.1 (C-2), 64.4 (C-6), and 62.6 (C-4).μM) for 48 h, and the cell viability was detected by MTT assay. 50  147.16 ± 18.9 μM). To evaluate the cytotoxicity of corilagin, Chang-liver, SMMC7721, Bel7402, HepG2, MHCC97-H, OC316, SGC7901, and QBC-939 cells were treated with increasing concentrations of corilagin . This result suggests that corilagin can promote the antitumor activity of cDDP.Subsequently, we analyzed the nature of the interaction between corilagin and the positive control cDDP using CDI, which quantitatively measures the interaction of two drugs. As shown in Y) and concentration (X), we could get the equation of the standard curve: Y = 1.03333 + 24742.3X (R2 = 0.9998). The linear range was 0–125 μg/mL. So we get the data of different corilagin content of plants ( Dimocarpus longan Lour. seed contents the maximum corilagin, up to 643 μg/g (According to the linear relation from peak area (f plants . Results643 μg/g . P. niruri has gained a reputation in folk and traditional medicine, for its multiple healing properties. Previous study tried to induce liver cancer in mice that had been pretreated with a water extract of P. niruri. The results indicated the P. niruri extract dose dependently lowered tumor incidence, levels of carcinogen-metabolizing enzymes, levels of liver cancer markers, and liver injury markers [Over the last few years, the markers . So the 50 value on HCC cell lines, which indicates that corilagin is the antitumor compound from P. niruri [ P. niruri. can be easier to be separated.In this study, with bioguided fraction and isolation, EtOAc fraction significantly inhibits the growth of HCC cell lines. Two compounds were obtained and identified as ethyl brevifolincarboxylate and corilagin. Between them, corilagin had a lower IC. niruri . So biog in vitro, which is consistent with the most recent report that corilagin could retard the in vivo growth of xenografted Hep3B HCC cells [Corilagin, a tannin, was mostly focused on the antioxidant activity , 26, wheCC cells  and induCC cells . But the Dimocarpus longan Lour. among 10 species of plants reported to contain corilagin, up to 643 μg/g., which means that corilagin has rich sources and is easy to be acquired.Besides the favorable antitumor activity, corilagin also has the maximum content in P. niruri. L. has great inhibiting effect on HCC cells. Corilagin is the major antitumor component that can significantly inhibit the growth of HCC cells and that shows low toxicity to normal cells. So corilagin is the major active antitumor composition in P. niruri. L. on HCC cells with growth inhibition.With bioguided fraction and isolation, we found that


Protein content was also correlated with dough stability (r = 0.86 **), extensibility (r = 0.8 **), and negatively correlated with dough weakness (r = −0.69 **). Bread firmness and dough weakness were positively correlated (r = 0.66 **). Sensory analysis indicated Doumah-2 was the best appreciated whilst Doumah 40765 and 46055 were the least appreciated which may suggest their suitability for biscuit preparation rather than bread making.The relationships between breadmaking quality, kernel properties , and dough rheology were investigated using flours from six genotypes of Syrian wheat lines, comprising both commercially grown cultivars and advanced breeding lines. Genotypes were grown in 2008/2009 season in irrigated plots in the Eastern part of Syria. Grain samples were evaluated for vitreousness, test weight, 1000-kernel weight and then milled and tested for protein content, ash, and water content. Dough rheology of the samples was studied by the determination of the mixing time, stability, weakness, resistance and the extensibility of the dough. Loaf baking quality was evaluated by the measurement of the specific weight, resilience and firmness in addition to the sensory analysis. A comparative study between the six Syrian wheat genotypes and two English flour samples was conducted. Significant differences were observed among Syrian genotypes in vitreousness (69.3%–95.0%), 1000-kernel weight (35.2–46.9 g) and the test weight (82.2–88.0 kg/hL). All samples exhibited high falling numbers (346 to 417 s for the Syrian samples and 285 and 305 s for the English flours). A significant positive correlation was exhibited between the protein content of the flour and its absorption of water ( Bread wheat is an important crop worldwide especially in the Mediterranean basin. During the last ten years Syrian bread wheat production constituted more than half of Syria’s total wheat production . This leThe baking potential of wheat flours is influenced by many factors, most notably protein content ,6,7,8. Pet al. [R2 = 0.70 in terms of equation to product quality. Dowell et al. [R2 of 0.78–0.93. The three previous works cited indicate the difficulty in developing models to predict wheat baking performance, and confirms it is still necessary to perform baking tests in order to achieve a reliable evaluation.In recent years, various researchers have attempted to avoid the baking tests by predicting bread quality through prediction models in which combination of measurements made from grain, flour and dough were used. For instance when trying to predict loaf volume, Miller  includedet al.  includedl et al.  combinedThe purpose of the present study was to investigate the major physical, chemical and rheological characteristics of some widely cultivated varieties in addition to some of those in the process of accreditation. The impact of these characteristics on the quality of the produced bread was also studied. For reliable evaluation of the ability of the Syrian genotypes for Western style bread preparation, a comparative study with some commonly used English flour was conducted. Consumer judgment is the most valuable tool in food quality assessment; therefore, a sensory analysis for bread quality evaluation was performed using a panel of assessors who regularly consume the product. Six Syrian bread wheat genotypes, Sham-6, Sham-8, Bouhouth-8, Doumah-3, Doumah-40765 and Doumah-46055 were selected. These genotypes are recommended for sowing in irrigated areas. Doumah 40765 and Doumah 46055 genotypes are awaiting certification. Samples were provided from the Ministry of Agriculture, the General Commission for Scientific Agricultural Research, Deir Ezzor Research Station, which is located in the east of Syria. Genotypes were grown at the same location and under the same agro ecological conditions in the crop year 2009. The English flour samples were obtained from the commonly used flour in the market .Test weight was determined using the approved method of the American Association of Cereal Chemists 55-10 , and theN × 5.7 [Water content of the kernels and the flour was determined by the approved AACC method 44-15 . ProteinN × 5.7 . FallingN × 5.7 . Ash conN × 5.7 . ResultsL *, a * and b *. The L *, a * and b * readings were treated using the software Colortools V3.1. The L * value gives a measure of the lightness of the product colour from 100 for perfect white to zero for black as the eye would evaluate it. The redness+/greenness− and the yellowness+/blueness− are denoted by the a * and b * values respectively. The colour of each sample was measured three times.The colour of the flour and the breads was assessed using a Datacolour Spectra . In the Datacolour spectrophotometer SF600 Plus, the colour of a sample is denoted by the three dimensions, Flour production was achieved by cleaning the samples using sieves. Samples were tempered to 14% water content overnight . Moistened samples were milled into flour using a Brabender Quadrumat Junior Experimental Mill . Flour extraction was expressed on a total product basis; its rate was 72%.Bread preparation was conducted using a recipie and a protocol supplied by an industrial partner (not identified by request). The recipe of dough preparation was as shown in The Extensibility of dough was studied using a texture analyzer  calibrated for a load cell of 30 kg. The Extensibility of dough was determined by the tensile test using the Kieffer rig . More than 15 strips of dough were tested from each batch. Water absorption of flour was evaluated using the farinograph apparatus. Rheological properties of dough  were determined according to the approved AACC method 54-21.01 [Bread firmness was determined using a texture analyzer , calibrated for a load cell of 30 kg. Bread loaves were sliced mechanically into 12.5 mm slice thickness using Greaf bread slicing machine . Two slices were stacked together for each test, discarding two end slices of the loaf. Bread firmness was measured with a probe 25 mm in diameter and at 40% compression. Firmness was defined as the maximum force obtained during compression. Bread firmness and resilience (ability to recover from compression) were measured on three successive days using the method described as AACC Standard Method 74-09 . Breads Loaf volume was measured by small seeds displacement method . A contaSensory evaluation for bread was carried out under white light at room temperature in individual sensory booths at the Food Technology Laboratory, Hollings Faculty, MMU, Manchester, UK. The sensory laboratory conforms to ISO standard guidelines for the design of test rooms  and ASTM’s physical requirements guidelines for sensory evaluation laboratories . Bread was evaluated by generic descriptive analysis in accordance to Lawless and Heymann . Ten expp < 0.05. Correlation coefficients were run between the different variables using Microsoft Excel at the significance level of p < 0.05, 0.01, 0.005. A total of 10 loaves of bread were made. All the experiments were replicated in triplicate unless otherwise stated. The coefficient of variability of all the tests was lower than 10%. Analysis of variance (ANOVA) and the linear regressions followed by Tukey’s test in addition to the correlations between factors were performed using SPSS 16.0 . All the calculations were done at the significance level of Physical characteristics such as vitreousness, kernel weight and test weight of the Syrian genotypes were determined to assess their contribution to the quality of the prepared bread. Vitreousness is an important factor in the determination of the quality of the wheat type because it reflects the texture of the endosperm and consequently the end use of the wheat ,13,25. Rr = −0.79 ** and r = −0.35 respectively. There was a significant positive correlation between the kernel weight and the test weight (r = 0.62 **) and a negative effect of the water content on the test weight of all the genotypes (r = −51 *)  .A range of chemical properties of the Syrian and the English flour samples are presented in r = 0.54 *), while a negative significant correlation between the protein content and the kernel weight was noticed (r = −0.8 **)  . Negative correlations were observed between the flour colour L * and the ash and protein with correlation values r of −0.62 ** and −0.78 ** respectively. This observation can be explained by the negative effect of the increase of both the protein and ash on the flour brightness. This conclusion can be enhanced by the negative correlation between the flour colour value L * and the water absorption. The redness values of the flour samples a * did not show significant difference, while the yellowness values b * varied from 8.57 for the weak English flour to 11.55 for Sham-8 which is due to more likely to the difference of pigments among samples, the negative significant correlation between b * values and ash values enhance the previous conclusion (r = −0.49 *).Protein content and vitreousness were correlated positively (−0.8 **) . A previ0.84 **) . Concern0.84 **) . The colr = −0.69), while a positive correlation existed between the dough stability and the protein content of the samples (r = 0.86 **) and also with water absorption of the flour (r = 64 **) (r = −69 **), in addition to a logic negative correlation between dough weakness and its stability (r = −0.8). Dough extensibility was positively correlated with protein (r = 0.8 **) and also with the water absorption of the flour and the mixing time and resistance with r values of 0.69 **, 0.83 ** and 0.48 ** respectively. On the other hand, dough extensibility was negatively correlated with weakness (r = −0.78)  . A negat= −0.78) . Other rThe samples did not show significant differences in the specific volume of bread loaf which varied from 3.48 for Sham-6 to 3.73 for Bouhouth-8. All Syrian genotypes showed a similar ability to produce bread when compared to the English. The resilience 0 values did not show significant differences between samples except Doumah-40567 and the weak English flour sample which had low values compared to the others . The resr) between firmness 1 and 2 was 0.67 **, and 0.81 ** with firmness 3, while the correlation value between firmness2 and 3 was 0.5 * . The differences in physical, chemical and rheological characteristics between samples did not give significant variations in specific loaf volume, but those differences affected the quality of the loaf. Bread sensory analysis clearly demonstrated the effect of the kernel physical characteristics, flour chemical traits and the dough rheology on bread quality. Colour, appearance and texture were the major factor in bread evaluation. Results proved conclusively the validity of the Syrian genotypes for Western style bread making which is not one of the mostly commonly produced bread on the Syrian market. The Syrian variety Doumah-2 was the most appreciated among all samples, while Doumah 40765 and 46055 were the least appreciated by the assessors which may suggest their suitability for biscuit preparation rather than bread making. Correlation analyses confirmed the importance of the vitreousness, protein content and the rheological traits of dough on the quality of bread especially the resilience and firmness. It is common in breeding programs to assess the suitability of grain solely on the properties of the raw material. In this study, sensory analysis was prioritized to assess bread quality rather than relying on judging the quality of the final product merely through quantitative evaluation of kernel and flour characteristics.