Carob is widely cultivated Mediterranean plant, but its flour is scarcely used in bread making. Previous studies investigated the quality of wheat bread with added carob flour showing discrepant results. This study aims to investigate the fermentation performance, antioxidant activity, rheological behaviour and baking application of carob sourdough.Lactobacillus brevis or Lactobacillus fermentum combined with Saccharomyces cerevisiae for 24 h at 30 °C. At the end of sourdough fermentation, number of viable lactic acid bacteria and yeast cells, total titratable acidity, pH value, antioxidant activity, phenolics and sugar content were determined. Carob flour (12% flour mass fraction) or sourdough equivalent (22.5% dough mass fraction) was applied in making composite partially baked frozen bread. Dough rheology was monitored using a farinograph. Nutritive value, physical properties and sensory attributes of the rebaked bread samples were evaluated using a hedonic test.Carob sourdough was fermented with By the end of fermentation, carob sourdough reached pH=4.2-4.5 and total acidity 9.9-12.3 mL of 0.1 M NaOH, sugar content on dry mass basis was reduced by 8 g/100 g, while total phenolics and antioxidant activity were increased up to 21%, depending on the starter culture. Addition of carob flour or sourdough to wheat dough resulted in firmer consistency, longer development time, and lower quality number. Regardless, bread with carob flour had significantly improved specific volume. Compared with common wheat bread, carob bread had increased dietary fibre content (46%), total phenolics (140-200%) and antioxidant activity (240-300%), higher shape, larger volume, reduced crumbliness, unchanged firmness and darker crumb colour. Consumer preference and overall acceptability scores of carob sour bread were comparable to those of wheat bread, falling into the category of ’liking very much’.L. brevis together with S. cerevisiae was better adapted to the substrate than L. fermentum. The carob sourdough could be used as a natural ingredient for improvement of nutritive value and reduction of crumbliness of partially baked frozen bread.To our knowledge, this is the first study that proved the feasibility of carob sourdough fermentation using mixed starter cultures, where  Bread was partially baked and frozen as is today’s common practice in bakery industry, offering consumers a wide range of freshly baked bakery products throughout the whole day.This study aims to investigate the possibility of sourdough fermentation of wholemeal carob flour (containing pulp and seeds) with typical heterofermentative sourdough cultures Commercial white wheat flour  contained 12% moisture, 12.5% proteins, 33% wet gluten and 0.5% ash. It showed 63.5% of farinograph water absorption and amylograph maximum viscosity 2490 BU according to ICC standards 115/1  contained 8.9% moisture, 5.2% proteins, 2.9% ash and 0.6% fat. After determination of sugar and fibre content, starch was calculated by subtracting the content of moisture, proteins, ash, fat, dietary fibre and sugar from 100%. Particle size distribution of carob flour was determined according to Benković Lactobacillus fermentum DSM 20052 or L. brevis DSM 20054  and incubated for 48 h at 37 °C. Malt extract broth  was inoculated with an overnight culture of Saccharomyces cerevisiae and incubated for 48 h at 30 °C. After centrifugation  at 2000×g for 10 min, microbial cells were used as inoculum.MRS broth  was inoculated with the overnight culture of L. fermentum or L. brevis (approx. 6 log colony forming units (CFU)/g) each combined with yeast S. cerevisiae (approx. 4 log CFU/g). Dough yield of 400 was necessary to obtain adequate consistency and sugar concentration. Sourdough was fermented (in duplicate) for 24 h at 30 °C with constant stirring, after which it was stored at 4 °C and used for baking within 2 h or lyophilized for analyses.Carob flour (150 g) was mixed with water  and the inoculum of Viable lactic acid bacteria (LAB) and yeast cells were enumerated in the inoculum and in sourdough under anaerobic conditions at 30 °C following the ISO 15214:1998 (Lactobacillus culture). The recipe (expressed as flour mass) for the control bread consisted of wheat flour (100%), farinograph water (63%), compressed yeast (2.5%), salt (1.5%), and sugar (1.9%) according to ICC standard 131 (3/m3). Partially baked bread, previously cooled at ambient conditions to 20 °C, was frozen in a blast freezer  and packaged in polyethylene bags. After storage at -18 °C for 30 days, and defrosting for 20 min at ambient conditions, the bread was rebaked for 11 min at 220 °C with 50 mL of steam.Four bread formulations were tested: control wheat and three types of carob bread  and pH value of sourdough or bread were determined on 10 g of a sample homogenized with 90 mL of distilled water. TTA is expressed as the volume of 0.1 M sodium hydroxide solution necessary to adjust the pH of 10 g of sample in 90 mL of distilled water to 8.5  in a test tube. Tubes were vortexed for 3 min, shaken in a water bath  for 1 h at room temperature and centrifuged  at 2500×g for 10 min to recover the supernatant. Vortexing, shaking and centrifugation were repeated with another 20 mL of acetone/water (φ=0.7) added to the residue. Combined methanolic and acetonic extracts were centrifuged at 3500×g for 15 min to obtain the supernatant for the determination of TPC and antioxidant activity. TPC were determined with Folin–Ciocalteu assay according to Durazzo et al. (m/V). After 2 h of reaction at room temperature, the absorbance was measured at 760 nm against a blank. Gallic acid was used as the standard and results are expressed on dry mass basis as gallic acid equivalents (GAE) per 100 g.Total phenolic compounds (TPC) and antioxidant activity were measured spectrophotometrically  after extraction. Sample (1 g) was mixed with 25 mL of acidic methanol/water =0.5 . After 4 min of incubation at 37 °C, the absorbance was measured at 595 nm. A calibration curve was made with known concentrations of Trolox and results are expressed on dry mass basis as Trolox equivalents (TE) per 100 g.Antioxidant activity assessment was based on the reduction of iron(III)-TPTZ  complex to iron(II)-TPTZ at acidic pH by means of ferric reducing antioxidant power (FRAP) as described earlier by Čukelj A farinograph  was used to determine the water absorption, development time and stability of the dough systems  were evaluated with a colorimeter  in the CIELab system , the employees of the Faculty of Food Technology and Biotechnology, Zagreb, Croatia, who were familiar with the main bread characteristics. For the 9-point hedonic test the sensory attributes selected were: appearance, smell, taste, texture and overall perception  to identify statistically significant differences (p≤0.05) between bread samples using Statistica v. 12  μm, 50th percentile d[50]=(187±7) μm, and 10th percentile d[10]=(38±2) μm. Tsatsaragkou Lactobacillus brevis or L. fermentum were used together with yeast Saccharomyces cerevisiae to inoculate the sourdough. Sourdough was evaluated by the measurement of microflora CFU, pH and acidity, which are the main criteria for effective fermentation. Generally, a sourdough contains a variable number of LAB and yeasts, ranging from 107 to 109 and 105 to 107 CFU/g, respectively, with a ratio of about 100:1  log CFU/g on average. Both LAB had a lower cell yield and growth rate in carob than in other legumes  lower pH and higher TTA than L. fermentum sourdough, which is consistent with the cell numbers. The difference between carob flour and sourdough in pH and TTA was reflected in the resulting bread samples; likewise, bread with carob flour was more acidic than the control wheat bread , but much higher TTA values, between 20 and 27 mL 0.1 M NaOH, as well as higher cell number of LAB (9.8-10.2 log CFU/g). This just confirms that sourdough acidity and microbial ecology depend on numerous endogenous and exogenous factors  after sourdough fermentation with L. brevis (21%) and L. fermentum (16%). In accordance with our results, Curiel et al.  or L. fermentum (21%). Curiel et al. , but higher with L. brevis sourdough (1.8%). Purhagen et al.   decreased after adding carob flour (26-fold), and even more after adding ecrease) . ContrarL* and yellow pigment b*, but higher redness value a* of the crumb. Bread with carob sourdough had higher intensities of a* and b* than the bread containing carob flour. This could be related to the higher content of phenolic compounds in the sourdough bread. Consistent with our results, lower L* values have been reported after using carob flour for partial substitution of rice in gluten-free bread  lower et al. . The only significant difference (p=0.02) between sensory characteristics of bread samples containing carob and the control bread was in their appearance , probablL. fermentum sourdough bread was most preferred with the highest number of times having the sum of ranks 37, but its average rank (2.34±1.08) and the sum of ranks were similar to those for L. brevis sourdough bread (2.28±1.00) (The Saccharomyces cerevisiae. Lactobacillus brevis associated with S. cerevisiae is better adapted to carob substrate than L. fermentum, which seems to be sensitive to the high amount of phenolic compounds. Sourdough fermentation of carob flour can be recommended for further enhancement of its phenolic content and antioxidant activity, the reduction of sugar content, as well for shortening the dough mixing time. Despite some differences in dough rheology, the choice between starter cultures L. brevis or L. fermentum does not make major differences in bread quality. Carob flour or sourdough can be used as a natural improver of partially baked and then frozen bread to reduce crumbliness. Volume, crumb texture and consumers acceptability of carob breads is comparable to common white wheat bread. Because of its nutritional benefits, consumer liking, technological suitability and acceptable pricing, fermented carob flour is a promising ingredient for health-promoting bakery foods. Future studies should investigate the influence of different parameters of carob sourdough fermentation and its application in gluten-free bakery products.In this study we investigated the possibility of using carob flour or its sourdough for enrichment of wheat bread with dietary fibre and phenolic antioxidants. Sourdough fermentation of carob flour is feasible with mixed starters of lactobacilli and yeast


The presence of pathogen-specific antibodies in an individual’s blood-sample is used as an indication of previous exposure and infection to that specific pathogen . Measurement of the diagnostic antibodies is routinely achieved using solid phase immuno-assays such as ELISA tests and western blots. Here, we describe a sero-diagnostic approach based on phage-display of epitope arrays we term “Domain-Scan”. We harness Next-generation sequencing (NGS) to measure the serum binding to dozens of epitopes derived from HIV-1 and HCV simultaneously. The distinction of healthy individuals from those infected with either HIV-1 or HCV, is modeled as a machine-learning classification problem, in which each determinant (“domain”) is considered as a feature, and its NGS read-out provides values that correspond to the level of determinant-specific antibodies in the sample. We show that following training of a machine-learning model on labeled examples, we can very accurately classify unlabeled samples and pinpoint the domains that contribute most to the classification. Our experimental/computational Domain-Scan approach is general and can be adapted to other pathogens as long as sufficient training samples are provided. B-cells respond to infection by producing pathogen-specific antibodies that bind and neutralize infectious agents , 2. In abona fide infected person. Unfortunately, specificity and sensitivity are never perfect. In order to increase accuracy, several repeats of a test are performed, often using more than one methodology. For example, Enzyme-Linked Immunosorbent Assay (ELISA) tests are routinely run in duplicates and triplicates. However, further confirmation of positive ELISA test results is possible through western blot analysis , “Domains”. In order to test multiple serum samples against multiple pathogen peptide arrays simultaneously in a single sample, NGS data have been analyzed The fth1 filamentous bacteriophage vector  was modiTCTAGAGCCGACCG-3’Sense oligonucleotide: 5’-TAGGGGATCCAGGNNNNNNNNNNNNBglII-digested fth1 vector, using a Gibson assembly reaction (via PCR amplification). The cloned vectors were transformed into E. coli MC1061 by electroporation. The transformed cells were cultured in 500 ml of LB medium with tetracycline (20 μg/ml). The culture was grown overnight at 37°C with shaking at 225 rpm. The culture was then centrifuged at 8,000 rpm, for 20 min. The supernatant was discarded, and the pellet was used for plasmid DNA extraction .The anti-sense oligonucleotide (5’- CGGTCGGCTCTAGA-3’) was complementary to the 14 bases at the 3’ end of the sense oligonucleotide (underlined). The two oligonucleotides were annealed and filled in with Klenow DNA polymerase large fragment . The insert containing the 12 random-bases was cloned into reaction   starting at the first codon of each gene and shifting by 15 or 30 bases (5 aa or 10 aa) towards the 3’ end of the gene. The size of each domain in every library was validated in agarose gel, and each insert was cloned into the digested and purified “fth1-BC” vector. The cloned vectors were used to transform DH5alphaF- competent bacteria. Clones were isolated and sequenced by standard Sanger sequencing in order to confirm the correctness of each cloned domain-peptide sequence, and to determine its corresponding barcode.A total of six Domain-Scan libraries representing antigens derived from HCV and HIV-1 were cloned and expressed in fth1 Protein III and Protein VIII  such that, upon annealing, 3’-overhangs corresponding to the overhangs generated after cleavage of the 5’ and 3’ SfiI-digested “fth1-BC” vector via a Gibson assembly. The same general scheme was used for the selected E2 and NS3 peptides , and the 5’ ssDNA aspects of the constructs were filled in with Klenow DNA polymerase. The products were ligated into ides see Table S2protein III gene sequence flanking either side of the BstXI sites of the “fth1-BC” vector. Each domain was cloned, individually, into BstXI-digested “fth1-BC” vector using a Gibson assembly reaction. Each clone was sequenced to validate the sequence for correctness and to identify its corresponding 12-bp domain-specific barcode. Similarly, two selected peptides of 68 aa and 76 aa derived from the NS3 antigen were designed and cloned into the BstXI sites using the same general procedure . The template used for the production of the HIV-1 gp160-ConS peptides were kindly provided by Dr. Barton Haynes . Oligonudure see Table S2Once the DNA clones, corresponding to the peptides derived from the six antigens used in this study were confirmed and their barcodes determined, they were consolidated respectively, to produce six individual DNA mixtures . For this, 150 ng DNA aliquots from each domain were combined to produce the six separate DNA mixtures. Next, the three DNA mixtures of Protein VIII-expression libraries were combined into one library (consolidated Protein VIII Library). Similarly, the DNA mixtures of the three Protein III-expression libraries were combined as well (consolidated Protein III Library). The consolidated Protein VIII and Protein III DNA mixtures were used separately to transform competent DH5alphaF-. Cultures were grown, and supernatants were collected. Bacteriophages were precipitated with polyethylene glycol (PEG-NaCl) overnight at 4°C, centrifuged at 8,000 rpm to harvest the bacteriophages, and re-suspended in Tris-buffered saline (TBS).Polyclonal serum-samples were collected from three sources: The Israeli National Blood Bank , Chaim Sheba Medical Center and Altai State University. A total of 85 different samples were analyzed in this study, representing three biological conditions: 15 HIV-1 positive, 40 HCV positive, and 30 healthy individuals. All sera were collected under informed consent and approved by the Institutional Review Boards. All samples were stored at -20°C.10 phages) of the Protein VIII or Protein III Domain-Scan libraries were mixed with 1 µl of serum sample and 1x1010wt-fth1 phages , in TBS containing 3% BSA (completed to 100 µl) and incubated for 1 h on a rotating mixer at room temperature. Next, 50 µl of Protein-G coated magnetic beads  were added, and the mix was incubated for an additional 30 min on a rotating mixer at room temperature. The vials were then placed on a magnetic stand  for 2 min to collect the beads, and the supernatants were discarded. Subsequently, the beads were washed three times with 200 µl ice-cold Tris-buffered saline containing 0.5% Tween-20 detergent (TBST), re-suspended in 100 µl TBST, and transferred to a new vial. The vials were then placed on the magnetic stand, and the supernatants were discarded. Bound phages were eluted with 105 μl of elution buffer  for 10 min at room temperature. The eluate was collected and neutralized with 19 μl of neutralizing buffer (1 M Tris-HCl pH 9.1). Each serum tested was used to screen the Domain-Scan libraries in triplicate, thus generating three independent samples sent to NGS. In order to multiplex dozens of samples together on a single NGS chip, an 8-bp sample-indexing barcode was introduced by PCR during sample preparation (see below). Hence, each serum sample was analyzed in triplicate, thus corresponding to three sample-indexing barcodes.For biopanning, 10 µl .“Reverse”5’-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTATCGCGGTCGGCTCTAGA-3’.TM SuperFi™ PCR Master Mix), 10 pmol of “Forward” and “Reverse” primers, and ultra-pure H2O to a total reaction volume of 25 µl. The thermal profile was:For each sample, 1 µl of the eluted phages, was mixed with 12.5 µl high-fidelity DNA polymerase  and measured for concentration using a Qubit fluorometer. The samples were then diluted to 10 nM, combined and sent for Illumina NGS sequencing. Hence, three “samples”, each tagged with an 8-bp indexing-barcode, were produced for every serum that was analyzed. NGS output DNA reads contained a sample-indexing barcode followed by a Domain-defining barcode, a total of 54 bp fragment, flanked by the Illumina Adaptor-sequences .NGS was performed at the Weizmann Crown Institute for Genomics. 11 pM of samples were used for clustering PCR followed by sequencing of 1x75 bp using an Illumina NextSeq500. The lane was spiked with 20% PhiX DNA. Approximately 100 samples were run on a single lane and a total of six runs were conducted in this study. The 85 serum samples (see above) were screened against the six Domain-Scan libraries representing 339 domains Table 1.The input to the machine-learning classifier was a list of sequence counts for each domain in each sample  relative to “noise”. For example, when the sample is from an HIV-1 positive sample, the normalized ratio for an HIV-1 domain should be high. In contrast, for an HIV-1 negative sample, the ratio should be substantially lower.An average over the normalized counts of the triplicates of each domain, per individual, was computed to form a single sample per individual, i.e., after these steps, we obtained 85 samples, corresponding to the number of individuals. The data for each domain before and after processing are given in We considered the problem of identifying whether a patient is infected with HIV-1 or not as a machine-learning binary classification problem (and similarly for HCV). For each sample, the input to the machine-learning algorithm was sequence counts of each domain (HIV-1 and HCV domain names are given in The following classifiers were tested: Naïve Bayes (NB), Support Vector Machine (SVM), Logistic regression (LR), Linear-Discriminant Analysis (LDA), Random Forest (RF), and k-Nearest Neighbors (KNN). We used the Python implementation of these algorithms, available in the “Scikit-learn” package .For some domains, the median value of the healthy individuals was found higher than the median of infected individuals, indicating that these domains bind serum non-specifically. Thus, based on the training set, these domains were excluded from both the training and the testing data. To test the accuracy of each classifier on the training data, we used a repeated five-fold cross-validation procedure. Specifically, we divided the training data to five disjoint folds, each comprising 20% of the training data. Here too, we applied stratified sampling to form the folds. Each classifier was trained on four folds (comprising 80% of the training data) and its performance was evaluated on the remaining fifth fold using the AUC (area under the ROC curve) score. This process was repeated five times, each time with a different fold used for performance evaluation. This process of dividing to five random folds was repeated 50 times and the average AUC was measured over the 250 folds used for validation.The above repeated cross-validation procedure on the training data was used both for parameter tuning and for additional feature selection, performed for each classifier. Parameter tuning was used both to improve the classifier performance and to avoid overfitting, i.e., accurate classification of the training data and poor classification of the validation data. Model regularization was introduced by limiting the number of trees, the tree depth, and the number of samples in the nodes of the tree in the Random Forest classifier, and by introducing the Lasso and Ridge Regression in the logistic regression classifier. The following feature-selection methods were considered: recursive feature elimination and select from model, as implemented in Scikit-learn . The setTo examine a potential effect of sequencing noise produced by the NGS on our analysis we applied two complementary approaches: (i) We tested how stable our results are. To do so, instead of averaging the triplicates we randomly selected a single sample from each triplicate. We then repeated the entire machine-learning pipeline as described above, this time using the single samples instead of the average over triplicates; (ii) We tried to test how sequencing coverage affects the obtained results. To do so, we followed a down-sampling approach, in which we randomly sampled a subset of the reads. Specifically, we sampled 50%, 60%, 70%, 80%, and 90% of the reads. We repeated the entire machine-learning pipeline for each such sampling.Combinatorial diagnostics is based on individually measuring the binding of serum-antibodies to members of a panel of pathogen-defining markers. In this study, we applied Deep-Panning analysis of pathogen-defining Domain-Scan peptides, displayed on filamentous bacteriophages in order to develop a high throughput and multiplex serum diagnostic platform.Initially, we used Protein VIII to express 105 short peptides derived from the CORE, E2 and NS3 antigens of HCV and 41 peptides representing p24 of HIV-1. Subsequently, in order to expand the range of diagnostic antigens and potentially include conformational targets, we added 205 “domains” as Protein III fusions representing HCV NS3 and NS5 as well as HIV-1 gp160. Ultimately, a total of 339 peptides, representing “domains” derived from six different antigens of HIV-1 and HCV, were analyzed.Materials and Methods and panels A and B in The Domain-Scan libraries were prepared using a novel fth1-BC vector system. In order to obtain an optimized and uniform read-out using NGS, we modified the fth1 vector by inserting a barcode-library of 12 random bases into an intergenic region of the vector. Each cloned domain was thus associated with a unique 12-bp barcode  vector was sequenced to confirm the expected high variability of the barcode region (protein III gene (using the BstXI sites) or protein VIII gene (using the SfiI sites). As a result, each cloned peptide could be associated with a unique barcode. The sample preparation for Illumina NGS was performed by PCR, employing sense and anti-sense primers containing the Adaptor A and B sequences, respectively . Moreover, a sample indexing-barcode was introduced just prior to the domain-defining barcode. The PCR product (152-bp) was “ready to use”, generating reads of uniform size containing first the sample index barcode (8-bp) followed by the domain barcode (12-bp), a total uniform read length of 54-bp irrespective of the size of the domain or whether it was displayed on Protein III or Protein VIII , and as a result, the PCR primers used for NGS sample preparation did not generate signals from the fth1 vector DNA. Thus, the dilution of the Domain-Scan phage-display libraries with carrier phages, led to decreased background noise, without introducing reads from fth1 carrier phages.SfiI cloning sites of the recombinant protein VIII gene of the fth1-BC vector; (i) 41 overlapping domains of the p24 antigen of HIV-1, (ii) 27 domains from the N-terminal aspect of HCV-CORE antigen (residues 1-135), and (iii) 75 domains representing HCV-E2 antigen. In addition, phages expressing selected non-overlapping peptides derived from the HCV-NS3 antigen were also prepared . The serum samples were screened in triplicate against each of the two phage-display Domain-Scan libraries . For each serum sample, an additional “sample-defining” 8-bp DNA barcode was introduced during the sample preparation for NGS  and (ii) are described in detail in the Materials and Methods. Step (iii) is the calculation of an “enrichment score” for each domain and determination of the utility of a domain as a discriminating diagnostic marker. This was conducted in a number of steps as described herewith: For each serum sample, a set of disease-related domains were identified and compared against “irrelevant domains”. As a case in point, the Protein VIII domains derived from the CORE, E2 and NS3 antigens are regarded as “disease-related domains” when analyzing HCV. Consequently, the p24 HIV-1 derived domains were taken as the “irrelevant domains” for comparison. For this, the reads of the p24 HIV-1 domains were summed and used as a reference “normalization-constant”. Then, the observed reads for each HCV-domain were divided by the normalization-constant, resulting in a “domain-enrichment score”. For the naïve healthy sera, enrichment scores were calculated in the same manner . Next, the irrelevant domains were removed from the analysis Table S4First, the data were randomly divided into two distinct sets: 80% of the data were used for training and 20% of the data were left aside in order to be used as a test set to assess the ability of the platform to diagnose the clinical status of un-seen sera. After training the classifiers on the training data as described in the methods, the classifier was fitted to the training set and the AUC was calculated both for the training data and for the test data. In total, six classifiers were evaluated, as described in the methods.We first analyzed only the data obtained from the Protein VIII Domain-Scan library containing; CORE (26 domains), E2 (75 domains), and NS3 (17 domains) for HCV, and p24 (41 domains) for HIV-1. A total of 69 domains remained after discarding the irrelevant domains: 22, 43, 3, and 1 for HCV-CORE, HCV-E2, HCV-NS3, and HIV-1-p24, respectively.Regarding the HCV classification, the Random Forest classifier outperformed all other classifiers with a perfect classification (AUC of 1.0) both on the training set and on the test data. In addition to AUC, the results of the classification can also be described in a confusion matrix, which provides the frequencies of true positives , false positives , true negatives , and false negatives . The HCV training set included 32 infected and 24 healthy serum samples, while the test set included eight HCV-infected and six non-infected serum samples and the accuracy of the HCV classification using Random Forest was 100% both on the training and the test sets.Materials and Methods). This domain had a median of 0 both in the healthy and infected individuals and consequently, no further analysis could be done on the data. It suggests that p24 domains alone provide only poor sero-diagnostic power for HIV-1.Regarding HIV-1 classification (based on the Protein VIII Domain-Scan library), only one domain remained after discarding the domains for which the median of the healthy individuals was higher than that of infected individuals  we might gain conformational epitopes as well. We added Domain-Scans for both HIV-1 and HCV, anticipating that HIV-1 classification will now become feasible. We expected the already perfect HCV classification to remain the same. The domains added in the Protein III Domain-Scan consolidated library included NS3 and NS5A for the HCV  and gp160 (79 domains) for HIV-1. A total of 71 domains remained in this library after discarding the irrelevant domains .Regarding HCV classification, Random Forest showed the best performance with an AUC of 0.999 on the training and 0.875 on the test. Combining the domains of both the Protein VIII and Protein III libraries resulted with a perfect AUC of 1.0 both on the training and the test sets (with Random Forest as the best classifier). Thus, the combined analysis maintained the perfect AUC score achieved using the Protein VIII library domains alone, both for the training and the test datasets. The accuracy remained perfect as well.The inclusion of 50-residue gp160 domains enabled accurate HIV-1 classification. The best performing classifier was Random Forest, and the resulting AUC was perfect (AUC of 1.0) both for the training and test sets. The corresponding accuracy was also perfect.The complete training and test results of all the classifiers, for the Protein VIII Domain-Scan library, for the Protein III Domain-Scan library, and for the combined analyses, are available in During the classification process, a feature-selection procedure was conducted in order to achieve better accuracy for the classification. Features (domains) that were identified as the most meaningful from our data were kept while the rest were discarded. The feature-selection procedure also provided a feature importance score for each feature, reflecting its contribution to the classification accuracy, the higher the score, the more important is the feature for classification. The most important features were CORE_26-40 for HCV and gp160_291-340 for HIV-1. Furthermore, the feature selection process selected domains from all the antigens  against serum samples taken from HIV-1 positive individuals, HCV positive individuals and compared against otherwise healthy individuals. The objective of these analyses was to test the diagnostic performance of our multiplex platform and to identify specific peptide domains that best represent the humoral response raised against either HIV-1 or HCV infection. These domains were used collectively as bait in mixed reactions to measure the presence of specific and corresponding antibodies, independent of one another. The rationale underlying this combinatorial approach  is that Our results illustrate that for the separate viral antigens we detected several specific domains that were found to be effective diagnostic markers, listed in Further improvement of combinatorial diagnostics is gained through the implementation of the supervised machine-learning approach illustrated in this study. According to this approach, the weights of the different domains were computed in the training phase of the algorithm, from known cases . The benefit of the machine-learning approach is that it can improve when more data are analyzed: As the number of known cases increases, it is expected that better weighting of the different domains is achieved, leading to better classification accuracy. Another advantage of the machine-learning approach developed here is that the classifier can learn inter-relationships between the separate domains. For example, if each of five separate domains is marginally enriched and not in itself statistically significant, the trained classifier can learn that such a combined signal is enough to classify an unknown sample as positive. The trained classifier can also learn that an enrichment of five other domains may not be enough to classify an unknown sample as positive. Thus, our trained classifier does not merely count the number of statistically significant domains, but rather, specifically considers which domains are enriched, and to which extent. Similarly, the classifier can detect “sticky” domains, which are highly enriched, yet are non-informative for classification. Moreover, the machine-learning classifier does not only provide a binary decision regarding whether a sample is positive or not for a specific pathogen, but rather, it provides a score reflecting how confident a classification is. This allows pointing to samples for which a repeated test should be performed or should be tested by complementary methods.The application of disease-defining phage-displayed diagnostic markers could be especially important in screening blood donations for multiple pathogen contaminations and thus improve transfusion safety. It is anticipated that by using the Domain-Scan approach and supervised combinatorial diagnostics it should be possible to construct effective epitope arrays for dozens to even hundreds of specific pathogens of concern that could be screened in a single multiplex assay to secure safe blood donations and disqualify those suspected of pathogen contamination.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.SH-N and YW-O - design and construction of libraries and execution of experiments. NW, OA, and TP - computational pipeline design and execution. NW, OA, HA, and TP - data preparation. YM - HCV biology and epidemiology. ES - HCV biology. DS - HIV serology. SH-N, YWO, NW, TP, and JMG - wrote the primary draft. SH-N completed the manuscript writing with the contributions from all authors. TP and JMG supervised the study. SH-N, YW-O, and NW equally contributed. All authors contributed to the article and approved the submitted version.This research was supported by two Grants of the Israel Science Foundation to JG. NW, OA, and HA were supported in part by a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel Aviv University. NW was supported in part by the Manna Center Program for Food Safety and Security at Tel Aviv University. OA was supported in part by the Dalia and Eli Hurvitz Foundation. HA was supported in part by Humboldt Research Fellowship for Postdoctoral Researchers from the Alexander von Humboldt Foundation. YM and ES were supported in part by a grant from the Dr. Sima Lior research. SH-N and YW-O were recipients of Jakov, Miriana, and Jorge Saia Fellowships.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our in vitro study clearly shows that the ASC PYD domain of corresponding amino acid 1–96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1–83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1–83 and 1–96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.The formation of inflammasome complexes contributes inactivation of inflammatory caspases  NLR andin vitro. The Ni-affinity chromatography and size exclusion chromatography (SEC) was used for protein purification. The main peak of the PYD domain of POP3 in SEC was eluted at 16 mL, which suggests the formation of a dimer in solution, and was found homogenous as was judged by the SDS-PAGE, which was subsequently characterized by a MALS. Furthermore, our study shows that in extremely low pH (pH5.0) conditions and high salt conditions (500 mM NaCl) influence the protein behavior and aggregation. The Native PAGE shows that the ASC PYD domain's self-oligomerization inhibited the formed complex with PYD domain of pyrin only protein 3 in vitro consistent with a study done by Narayanan KB et al, shows that ASC PYD easily self-oligomerized to prevent the formation of a complex with NALP3 PYD in vitro. Finally, the purity and homogeneity of the PYD domain of POP3 and ASC PYD, together with complex, were investigated by SDS-PAGE.The PYD-only proteins (POPs) form the inflammasome regulator family . POPs ar22.1g, the bacteria expressing each protein were pelleted, resuspended, and then sonicated in 40 mL of buffer (lysis) with added to phenyl methane sulfonyl fluoride (PMSF). Subsequently, centrifugation was carried out of lysate. After the addition of a 70 mL buffer (Wash), the unbound proteins were eliminated. Finally, the buffer (elution) was used to elute the target proteins. Total of 2 mL protein samples being collected with equally divided in 4* 0.5 mL eluted fractions. The SD-PAGE was used to check the purity of the target protein. The protein fractions containing more than 90% homogenous PYD domain of Pyrin only protein 3 were combined. The sample was loaded to the column superdex 200 at the final purification stage, which was pre-equilibrated with a 20 mM Tris-HCl solution at pH8.0 and 150mMNaCl. For biochemical characterization, the SEC PYD domain protein fractions were pooled & then stored at 4 °C. SDS-PAGE was used to judge the homogeneity of the PYD domain of Pyrin only protein- 3.Cloning, protein expression, and purification were conducted as described previously (The cDNA of Human full-length PYRIN Domain-Only Protein 3 and ASCPYD domain were used to perform the polymerase chain reaction (PCR). Afterward, PCR products were digested using restriction enzymes. The digestion of the plasmid pET24a was carried. The PYD domain (1–83) of Pyrin only protein 3 and ASC PYD (1–96) were sub-cloned in a C-terminally His –tag (His6) plasmid (pET24a) (Novagen). Following, the transformation of the plasmids into the BL21 cell (DE3) competent cells. The agar plates possessing Luria Bertani (LB) comprising the appropriate antibiotics (50ul/mL) were used to gently spread each clone on it, following the incubation of agar plates for 16hr at 37 °C. A micro-loop was used to inoculate single colonies from each construct into a 5 mL LB medium, incubated overnight in a 37 °C shaking incubator. The next day, 5 mL of overnight pre-inoculated culture was inoculated into 2000 mL of the flask, which contained 100 mL LB media, and was incubated for 4 hrs at 37 °C with shaking. Also, the routinely tested optical density (O.D) until it reached between 0.6 and 0.7 nm, by 0.25 mM IPTG treatment at 20 °C overnight following protein overexpression. By centrifugation at 10,000eviously .2.2g) was followed by removing the precipitate at 4 °C for 10 min before the loading on column HR 10/30, pre-balanced with a 20 mM Tris-HCl at pH8.0 & 150 mM NaCl. Furthermore, three-angle refractive index detectors for light dispersion were connected to the system and analyzed by the ASTRA software after every 0.5 s, providing a molar mass of each sample.To determine the absolute molecular mass, the PYD domain of Pyrin only protein 3 (1–83 aa) was used by a technique known as MALS. The protein of interest was purified by two rapid steps, Nickel-AC and SEC. Only protein 3 corresponding amino acids 1–83 were obtained from the key peak fractions. The centrifugation  and ASCPYD (1–96 aa), as well as the complex formation. Protein samples purified by chromatography were loaded on the gel, followed by bright blue coomassie stain.2.4Purified protein samples of POP3 PYD domain of corresponding amino acid 1–83 and ASC PYD domain of corresponding amino acid 1–96 aa were mixed in a molar ratio of approximately 1:1 and subjected to pre-incubation for 60 min at 4 °C. The protein mixture was further purified via superdex 200 gel filtration column HR 10/30 equilibrated by 20 mM Tris-HCl at pH 8.0 & 150mMNaCl.3PYRIN Domain-Only Protein 3 (POP3) consists of 113 amino acids with PYD domain A, which in vitro biochemical characterization, we produced the overexpression construct of each protein viz, POP3PYD (1–83 aa) and ASCPYD (1–96 aa). Each protein was over-expressed in bacterial cell BL21 and purified by Nickel chromatography and size exclusion chromatography (SEC). The SEC of, the POP3PYD domain with corresponding amino acid 1–83 aa, was eluted at approximately 16 mL in Tris and NaCl buffer conditions  and ASCPYD domain (1–96 aa) using structural and biochemical assays in vitro because of oligomerization of PYD domain inhibited assembly by self. Further research to understand the molecular function in vivo would give more insight into the PYD domain of POP3 and ASC PYD biology.We performed size exclusion chromatography to confirm our results. The mixtures of PYD DOMAIN of POP3 (1–83 aa) did not produce complex peaks . This re4in vitro analysis explaining the complex formation by PYD-PYD interaction.Large molecular complexes are the formation of inflammasomes triggered by pathogens that guide to caspase-1 activation and are involved in the both inflammatory and adaptive immune response. The key components of inflammasome NALP3 are ASC, NALP3, and caspase-1, coupled with NOD-like receptors (NLRs). Each protein comprises the superfamily of the DD, caspase-1 CARD, ASC, and NALP3 PYD to permit protein module interaction. ASC constituted of both CARD and PYD domain at N-and C terminal, respectively. Interaction of ASCPYD and POP3 PYD is critical for inflammasome recruitment of other essential proteins for its activation. The structure of ASC PYD showed a bundle of 6– helix of the DD superfamily. However, no structure of the POP3PYD domain is reported to date, and also no in vitro complex assembly formation. Both proteins were purified through -affinity chromatography and size exclusion chromatography (SEC). The PYD domain of POP3 forms the dimer in solution based on our size exclusion chromatography results. The homogeneity of protein was detected on SDS PAGE gel. To confirm the stoichiometry, we performed the MALS of the PYD domain of POP3 protein. In addition, protein behavior was influenced by extremely low pH (pH5.0) conditions and high salt conditions (500 mM NaCl) and aggregated as detected on SDS PAGE gel. Based on our results, our in vitro study confirmed that self-oligomerization of ASC PYD domain inhibited assembly of the complex with PYD domain of pyrin only proteins 3 consistent with a previous study done by Narayanan KB et al. The homogeneity of this complex assembly was confirmed by SDS-PAGE.To understand the biochemical insight on a molecular basis, the Human PYD domain of Pyrin only protein 3 and ASC PYD domain was used to elucidate its biochemical characterization together with in vitro, and a particular binding region of POP3 PYD was analyzed. The PYD domain of ASC contributed to self-oligomerization, which inhibited the binding to the PYD domain of POP3 protein. This strategy may be necessary for the ASC PYD domain's inhibitory activity to the PYD domain of POP3 . Furthermore, we showed dimer formation of POP3 PYD in solution. Moreover, we find unstable POP3 PYD protein behavior in extremely low pH (pH5.0) conditions, and the proteins get aggregated at high salt conditions (500 mM NaCl). This can act as a template for further study to understand the molecular mechanisms, which will widen the field of pyrin-only protein biology.All authors declare that there are no conflicts of interest.


Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma. Non-ribosomal peptide synthases are multimodular enzymes comprised of adenylation (A), condensation (C) and thiolation domains. Here, the authors show that non-ribosomal peptides can be generated solely by A domain substitutions, providing evidence that the postulated substrate specifying role of C-domains may be rare in nature. Although modified lipopeptides were detected using mass spectrometry, in each case the yield of modified lipopeptide was substantially diminished, to only trace levels2. Evidence that C domains exhibit stringent specificity toward the acceptor substrate activated by their cognate (downstream) A domains offered an explanation for the reduced yields4. This evidence was based on NRPS enzymes artificially loaded with amino-acyl CoA or aminoacyl-N-acetylcysteamine thioesters, which mimic an amino acid attached to a T domain. Soon after, a substrate binding pocket of the C domain was suggested to play a role in controlling the direction of biosynthesis5. Subsequent efforts at substituting cognate C-A domains together enjoyed modest success, reinforcing the belief that this was necessary to bypass C domain substrate specificity7. Since then the most successful engineering attempts have focused on substituting C-A domains together9 or A-T-C domains with the condition of not disrupting C domain acceptor site specificity12. It is now widely accepted in the field that successful domain substitution requires working within the constraints imposed by C domain specificity13.The earliest reported attempts to create artificial non-ribosomal peptide synthetase (NRPS) enzymes were substitutions of A-T domains into the second and seventh modules of the NRPSs involved in the biosynthesis of the lipopeptide surfactinPseudomonas aeruginosa PAO1 is a UV-fluorescent siderophore formed from an 11-membered peptide, encoded by NRPS modules that will here be referred to as Pa1-Pa11 14 yielded detectable pyoverdine products, versus zero of nine substitutions of A domains specifying alternative residues15. In contrast, non-synonymous C-A domain substitutions generated modified pyoverdines in three out of ten cases, suggesting that a C domain with compatible acceptor site specificity was required for functionality16.Until now, our own work using pyoverdine as a model system has been consistent with this dogma. Pyoverdine from a11 Fig. . We obse3.We now show that our previous failure to generate modified pyoverdines by A domain substitution is surmountable by use of more effective recombination boundaries, and that the C domains in the pyoverdine NRPS system do not impose stringent proof-reading constraints. We perform comprehensive evolutionary analyses across three different bacterial genera that are consistent with this conclusion, indicating that non-ribosomal peptide divergence is primarily driven by recombination of A domains or sub-domains, independent of partner C domains. Tellingly, we show that we can also incorporate leucine as the acceptor residue in the tyrocidine PheATE/ProCAT dimodular NRPS model Fig. , which w19. We instead adopted a semi-rational shuffling strategy, by combining regions of DNA encoding C domains that incorporate different amino acid substrates, then seeking to retrospectively identify important substrate-defining residues. For this we selected the Lys-specific module Pa8 and the Thr-specific module Pa11, as we had previously found a pyoverdine with Lys at position 11 could be generated by substituting the C-A domains (but not the A domain) from Pa8 into Pa11 of PvdD9. At the time, we interpreted this result as showing that the Pa8 A domain can function within the PvdD environment, but only when paired with a Lys-specifying C domain. An added attraction of the model is that the Pa8 and Pa11 C domains appear to be paralogs, as they are nearly identical apart from three stretches of low sequence identity  suggested the three regions could be shuffled effectively, with only a minimal number of amino acid perturbations introduced  and Pa11 (presumed T-specific) C domains were shuffled to yield all eight possible combinations . Identification of a more permissive recombination boundary that did not exchange any plausible proof-reading residues21 was inconsistent with the hypothesis that C domain acceptor site specificity had caused our previous A domain substitutions to be non-functional15.Region 3 contains 38 non-identical residues between the Pa8 and Pa11 C domains . As adenylation is independent of any catalytic role of the C domain, the inconsistency has been attributed to indirect conformational effects30, which in turn suggests that isolated A domains may appear in vitro to be more promiscuous than they actually are in vivo. Thus, while it is possible that diversification via A domain substitution in the microcystin, aeruginosin, hormaomycin and lipo-octapeptide pathways has been enabled by unusually relaxed-specificity C domains, our experimental success in generating modified pyoverdines led us to consider that these examples might not be so unusual, and that A domain substitution might be a more global phenomenon driving NRPS diversification.Our success in generating modified pyoverdines via A domain substitution led us to consider whether this approach might be applicable to other pathways. Compared to natural evolution, the number of substitutions that can be created in the lab is infinitesimal, and we reasoned that we could gain valuable insight by investigating whether A domain substitution has occurred frequently during natural NRPS diversification. The tight acceptor site specificity originally proposed by Belshaw et al.P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas syringae). This revealed strong clustering of A domains by substrate specificity, providing evidence that C and A domains had evolved independently  described the formation of a modified peptide, the rate of formation of which was greatly reduced in vitro and not tested further in vivo35. We tested partial A domain substitution in PvdD using boundaries suggested by our recombination analysis, but did not achieve visible production of pyoverdine in any instance  experimental attempts at partial A domain substitution were generally unsuccessful, or at best highly inefficient35. In contrast, because natural recombination processes favour short sequences38, this appears to be a preferred region of recombination during natural NRPS evolution, with the low success rate presumably offset by the high frequency of recombination events. Irrespective, our diverse analyses provide unanimous support for partial or complete A domain substitution being a primary mechanism for NRPS diversification.Partial A domain substitution has previously been attempted in two key laboratory studiesles Fig. . Whereasles Fig. , S12. We39. However, the possibility remained that the Pa8 and Pa11 C domains are more relaxed than C domains such as that in module 2 of tyrocidine biosynthesis—the system that Belshaw et al.3 used to develop the original hypothesis of C domains exhibiting strong acceptor substrate specificity. We therefore considered it important to test whether we could create novel dipeptides by performing A domain substitution within the same system.Our high success rates in modifying pyoverdine via A domain substitution and our bioinformatics analyses suggested that C domain acceptor substrate specificity is less of a barrier to NRPS engineering than previously proposed by several groups, including usd-Phe) and module two  could be artificially loaded with different amino acids in vitro, and the resulting dipeptide purified and analysed3. Artificial loading with l-Pro resulted in the expected product, but no product resulted when ProCAT was loaded with l-Leu, suggesting stringent substrate specificity during condensation. If this hypothesis is true, the C-domain of ProCAT will not accept l-Leu. We therefore considered that efficient production of d-Phe-l-Leu dipeptides via A domain substitution in the ProCAT module would disprove the C domain substrate specificity hypothesis in this foundational model. To avoid potential for in vitro inconsistencies and facilitate construct generation and analysis we used a similar two-plasmid in vivo system to previous researchers41, with the replacement of the T domain from ProCAT with the T-Te domains from SrfC, to enable release of linear d-Phe-l-Leu dipeptides43. The genetic regions encoding four different Leu-specifying A domains were selected for substitution into the ProCATTe plasmid and compared to an unsubstituted control  substitution. This observation is inconsistent with widespread acceptor site specificity existing as a barrier to successful A domain substitution. Further work will be needed to understand how broadly these findings can be applied, but we consider they hold considerable potential for efficient rational and combinatorial improvement of medically and industrially relevant peptides.While natural evolution has given rise to a large diversity of non-ribosomal peptides, effective re-engineering of NRPS templates in the laboratory has proven difficultAll plasmids, primers and sequences used in this study are provided in the Supplementary file Supplementary Data pvdD, the PBAD promoter was excised from pSW196 using the restriction sites NsiI and SacI and ligated into pUCP22 using the restriction sites PstI and SacI. The resulting plasmid was named pUCBAD. Next, the pvdD gene lacking the C-A domains from the second module (module Pa11) was excised from the plasmid pSMC9 using NheI and SacI restriction sites and annealed into the pUCBAD vector using the restriction sites NheI and SacI to create the vector pUCBAD-SMC. The Thr-specific A domain from the second module of PvdD and the Lys-specific A domain from the first module of PvdJ (module Pa8) were PCR amplified and separately ligated into the pUCBAD-SMC vector. This resulted in the creation of the vectors pDEC-Thr and pDEC-Lys, which contained a Thr-specific A domain and a Lys- specific A domain variant of the pvdD gene, respectively. For modifying the third variable region of the C domain from pvdD, the first two variable regions of the pvdD C domain from module Pa11 were PCR amplified. The resulting fragment was ligated into the pDEC-Thr vector using a 5′ SpeI/XbaI and a 3′ SalI/SalI ligation to create the plasmid pTRN. The destruction of the SpeI restriction site within the vector by SpeI/XbaI ligation meant the introduced SpeI site downstream to region 2 of the C domain was unique within the plasmid, allowing insertion of region 3 using SpeI and SalI restriction sites.To create vectors for substituting domains into Pseudomonas aeruginosa PA01; Ps5-Ser indicates the fifth serine-incorporating pyoverdine NRPS module from Pseudomonas syringae 1448a; Pf6-Orn indicates the sixth ornithine-incorporating pyoverdine NRPS module from Pseudomonas fluorescens SBW25; Supplementary Fig. C domains were created by overlap PCR or synthesis . C domain sequences were amplified using the appropriate forward and reverse primers specific to the C-domain from modules Pa8-Lys, Pa11-Thr, Ps5-Ser or Pf6-Orn  following codon optimisation for 62, and using the Swiss-Model server (http://swissmodel.expasy.org/)63 and Modeller 9.1164. Models created from each method were submitted to the QMEAN server to obtain QMEAN6 and QMEANclust scores66. The model RaptorXmsa was selected to work with as it scored best overall considering both measurements. The model RaptorXmsa aligned well to the C domain structure from TycC21 with a root mean-square deviation for the backbone α carbons of 0.381 Å.Structural models of the C domain from the second module of PvdD were created by submitting the C domain sequence to multiple automated servers22 was queried for all NRPS biosynthetic gene clusters from the genera Pseudomonas, Bacillus and Streptomyces. The genbank files for each cluster were downloaded, and the python script extractCATdomains_consensus.py  used to find, extract and save DNA encoding LCL-A-T tridomains into a separate FASTA file for each genera. The criteria for extracting tridomains were that the A domain had an antiSMASH consensus substrate specificity prediction, and that C-A-T domains were located on the same protein in the correct order, with fewer than 250 amino acid residues between domains. Each extracted DNA sequence was annotated with the consensus substrate specificity. Sequences were dereplicated using USEARCH 10.0.24067, and clustered at 95% identity  calculated the average number of clashes using SCHEMA for each recombination point between Pa11 and the modules used as a source of A-domains.The structure 2VSQ was used as it was identified as the top template for modelling the CAT-domains from Pa11 using the Swiss-Model serverl-arabinose and 4 g/L succinate (pH 7.0). Cultures were grown for 37 °C for 24 h, centrifuged to pellet bacteria, and then 100 μL of supernatant transferred to a fresh 96 well plate and diluted twofold in fresh M9 media to give a total volume of 200 μL. Absorbance (400 nm) was measured using an EnSpire 2300 Multilabel Reader . For mass spectrometry analysis, 1 μL of supernatant was mixed with 20 μL of matrix 70. Aliquots of 0.5 μL were spotted in triplicate onto an Opti-TOF® 384 well MALDI plate  and allowed to dry at room temperature. Spots were analysed using a MALDI TOF/TOF 5800 mass spectrometer (Applied Biosystems) in positive ion mode. Peaks were externally calibrated using cal2 calibration mixture (Applied Biosystems). Peaks in spectra were labelled in Data Explorer (Applied Biosystems).Each strain to be analysed was first used to inoculate 200 μL of low salt LB in a 96 well plate. After 24 h growth at 37 °C, 10 μL of each starter culture was used to inoculate 190 μL of M9 media amended with 0.1 % (w/v) E. coli strain BAP144. A 100 µL aliquot from an overnight culture was used to inoculate 10 mL M9 media amended with 0.1% (w/v) casamino acids and 0.4% (w/v) d-glucose. The culture was grown at 30 °C for 5 h and then incubated on ice for 20 min. IPTG was added to a final concentration of 1 mM, and the culture grown for a further 16 h at 18 °C. The cells were pelleted by centrifugation at 2000 g for 20 min. The supernatant was transferred to a fresh tube and immediately extracted with 4:1 butanol–chloroform (vol/vol). The organic layer was washed with 1 volume of 0.1 M NaCl and the aqueous phase removed. Solvent was removed under vacuum and residue dissolved in 150 µL of 10% CH3CN. HPLC separation was performed on an Agilent 1200 LC system using a C18 reverse-phase column . The column oven was set at 40 °C and injection volume at 30 µL. Elution was conducted with a mobile phase consisting of (A) water + 0.1% formic acid, and (B) CH3CN + 0.1% formic acid. Following 1 min at 10% B, a gradient up to 25% B was performed in 17 min. The flow rate was set at 1 mL/min. Concentrations were determined by comparison to chemically synthesised standards with 98% purity by LifeTein . For HPLC-MS, separation was performed on an Agilent 6530 Accurate-Mass Q-TOF LC/MS system equipped with an Agilent 1260 HPLC system . The same HPLC conditions as previously were used except the column oven was set at 45 °C and injection volume at 1 µL.The plasmids containing PheATE and ProCATTe were transformed into the Further information on research design is available in the Supplementary InformationPeer Review FileDescriptions of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The surface morphologies of residual impressions with various holding times were investigated. It was indicated that generation of secondary cracks and propagation of both main and secondary cracks were the dominating mechanism for time-dependent plastic deformation at the initial holding stage, and the density and length of cracks were invariable at the steady-state holding stage, which suggested a nonlocalized plastic deformation beneath the indenter. It could be concluded that time-dependent plastic deformation of brittle ceramic under nanoindentation is composed of instant cracking as the continuation of loading sequence and homogeneous creep flow by high shear-compression stress at room temperature.Recently, instrumental nanoindentation has been widely applied to detect time-dependent plastic deformation or creep behavior in numerous materials, particularly thin films and heterogeneous materials. However, deformation mechanism at nanoindentation holding stage has not been well revealed hitherto. In the current work, nanoindentation holding tests with high loads were performed on a brittle LiTaO Creep is classically defined as a time-dependent plastic deformation, which is occurred during elastic holding and facilitated by high temperature . Creep rTo weaken thermal drift influence on the mechanical response, nanoindentation load-holding tests were commonly conducted at room temperature. At the onset of the nanoindentation holding stage, plastic deformation was generally occurred particularly by adopting Berkovich indenter. In this scenario, the mechanism of time-dependent plastic deformation under nanoindentation could be much different to that of the thermally activated creep flow by conventional method. To the authors’ best knowledge, the nanoindentation creep mechanism was not well revealed and merely qualitatively discussed from the viewpoint of high-temperature creep . It is m3) has been extensively used in the functional devices like laser, communication, acoustic fields as an excellent piezoelectric material [3 wafer are required. Due to its soft-brittle nature, catastrophic fracture of LiTaO3 is always occurred during precise machining [3 wafer suffers long-term point-contact deformation during grinding and polishing, nanoindentation exploration has attracted lots of attentions on revealing the mechanical properties and deformation features of LiTaO3 at the microscale [Lithium tantalite  and polyurethane polish pad were adopted on the Nanopoli-100. After 45-min polishing, LiTaO3 samples were carefully cleaned in the anhydrous alcohol by ultrasonic cleaning [2 as exhibited in Ra could be measured.A LiTaOcleaning . Top vien, generally within the range from −0.05 and +0.05) correction which was calibrated at 10% of the maximum load during the unloading process was strictly performed for each test. The reduced nanoindentation displacement could be obtained by h-t × n. The morphologies of all the residual impressions were observed by SEM (sigma hv-01-43). All the sixteen residual impressions at each holding condition were carefully detected, and the count and size of cracks were recorded.Nanoindentation load-holding tests were performed on Agilent Nano Indenter G200 at ambient temperature of 23 °C by air condition. A spherical tip with nominal radius of 5 μm was adopted to study the crack evolution during nanoindentation holding. The effective spherical tip radius was obtained as 2.95 μm by calibrating on the standard fused silica. The loading rate and peak load were fixed to 2 mN/s and 150 mN, respectively. Under the current testing conditions, both the residual impression and crack feature at the beginning of duration could be clearly captured by scanning electron microscope (SEM). Four individual holding segments at peak load were adopted to investigate the time-dependent plastic deformation under nanoindentation, which were ranged from 0 to 1500 s . It should be pointed that nanoindentation holding test was performed individually, rather than cyclic loading at the same location. Sixteen independent measurements  were conducted for each case. All the holding tests were launched until thermal drift reduced to 0.03 nm/s. Meanwhile, thermal drift (P-h) curves for the holding tests with different durations. Clearly, the permanent deformation continuously appeared at the holding stage, and the time-dependent plastic deformation was more pronounced with increasing holding time. The P-h curves exhibited a well repeatability for each holding time. 2 > 0.99) by an empirical law [h0, t0 are the displacement and time at the beginning of holding stage, a, b, k are the fitting constants. Based on the creep fitting lines, the nanoindentation strain rates of time-dependent plastic deformation under spherical tip were estimated by A is the contact area, equal to 2πRhc. R is the tip radius, the contact depth hc could be deduced as hc = h − ε × P/S, where h is the recorded indenter displacement, ε = 0.75 for a spherical tip, S is the contact stiffness [−3 s−1  and then decreased to abut 1 × 10−4 s−1 as holding time increased to about 50~100 s. At the ending of each holding stage, the average strain rates were about 1.8 × 10−5, 1.45 × 10−5 and 1.15 × 10−5 s−1 for the 250, 500 and 1500 s holdings, respectively. This means the time-dependent plastic deformation turned into the steady state from the 250 holding as the strain rate was very slowly changed. The strain rate at the onset of holding stage could be obtained by H is hardness) [P was the peak load 150 mN. Thus the strain rate of initial time-dependent plastic deformation was comparable to that (0.0067 s−1) of loading sequence.ical law :h(t) = htiffness . It shouardness) , where Ph at the end of holding stage were recorded for each measurement, the mean values were 20, 33.5 and 56.6 nm for the 250, 500 and 1500 s holdings, respectively. The mean contact radii at the peak load could be obtained by P is the peak load and S is the contact stiffness. As shown in The increased displacements Δ3 under spherical nanoindentation. The radii of residual impressions were carefully measured for all the tests as also exhibited in 3 on the three holding stages could be qualitatively unfolded, relying on the length evolution of pre-existing main cracks and generation of new secondary cracks.From the SEM images, it could be seen that symmetric cracks of several micrometers in length appeared along four directions with an angle of 90 degrees, as indicated by white arrows in For the initial holding stage (0~250 s), the total count of secondary cracks quickly increased from 27 to 40 in a total of sixteen tests. Meanwhile, the mean lengths of both main and secondary cracks were enlarged ~100 nm simultaneously, which held at approximately 3.15 and 1.25 μm, respectively. We can conclude that the initial time-dependent plastic deformation was induced by both generation of secondary cracks and elongation of main and secondary cracks. Consequently, deformation strain rate at this stage was evidently higher than the subsequent stage and comparable to the strain rate at instant deformation at the same load. The deformation manner and strain rate at initial holding stage could even be regarded as the continuation of loading sequence by stress overshoot .−5 to 1 × 10−5 s−1, as indicated in As related to deformation behavior on the second holding stage, i.e., 250~500 s, the displacement was almost linearly increased with holding time, which represented a typical steady-state creep flow. In this holding stage, the strain rate was gradually decreased from about 3 × 10−5 s−1, and the slow increase of residual radius also confirmed the sluggish plastic deformation in this stage. Obviously, the count and length of main and secondary cracks were invariable on the whole stage. In this scene, the time-dependent plastic deformation could be drove by homogeneous creep flow, notwithstanding its mechanism should be distinct to high-temperature creep deformation. The nanoindentaiton strain rate could be transformed to uniaxial strain rate empirically by −6 s−1 order. From the perspective of deformation kinetics, plastic deformation under such low strain rate was already beyond the scope of quasistatic deformation, and entirely met the requirement of creep deformation. In comparison, the herein local creep strain rate under high shear stress were within the strain rate ranges by macroscopical creep tests, i.e., bending, compressure and tension at high temperature and low stress. In Duong’s report, the steady-state creep strain rate of a single crystal calcium oxide was in the range approximately from 10−7 to 10−5 s−1 at 1350 °C under low stress (5~15 MPa) [−6 to 10−4 s−1 by enhancing the applied stress from 20 to 100 MPa [−8 to 10−5 s−1 for SiC/SiC composites by increasing holding stress [3, of which melting point was higher than 1600 °C, its room-temperature creep deformation or homogeneous plastic flow was rarely studied, as well as its high-temperature creep behavior. The occurrence of creep deformation and its mechanism should be discussed.For the time-dependent deformation on third holding stage (500~1500 s), its strain rate was slowly reduced to less than 2 × 10u=0.09εI . Thus th~15 MPa) . Lee stu 100 MPa . In Jingg stress . For the3 single crystal [3 even though the deformation strain exceeded its elastic limit by nanoindentation. Therefore, the present main cracks along symmetrical directions were induced by high-stress squeezing and the disordered secondary cracks were formed from the twinning deformation at those “fertile” places with defects [a/2) the indenter, the time-dependent plastic deformation at the steady-state stage was expected to occur beneath indenter. Thus the little change of cracks morphology on the surface as holding time increased from 250 to 1500 s could be understandable.At the onset of the holding stage, the peak load (150 mN) had already exceeded the nanoindentation yielding point of LiTaO crystal . The pla crystal . Accordi defects . In comp3 single crystal, it was rarely studied and its mechanism was unknown under both uniaxial and nanoindentation holdings. In comparison to metals and alloys, it requires much more experiments and simulations in future to reveal the creep behavior and mechanism of LiTaO3, and we simply discussed the room-temperature creep deformation in LiTaO3 from the view of creep mechanisms of alloys. In the current work, we reported that the transient stage of time-dependent plastic deformation was crack-dominated rather than homogeneous creep flow in brittle ceramic, for the first time. It is promising that localized plastic deformation could play an important role at the nanoindentation holding stage for other materials provided holding time is limited. Importantly, it suggests that the determinations of creep behavior including creep strain and strain rate sensitivity by nanoindentation could be misleading at short duration.In brief, the deformation mechanisms herein during the holding stage could be generally determined as crack evolution at transient stage and homogeneous creep flow at the steady-state stage, respectively. In the interim, segment between the two stages, crack extension and creep flow could occur simultaneously. It should be noted that the crack-dominated holding stage was transient and could be expected approximate 0~100 s, based on the change of strain rate at holding stage. Besides, the critical point for the pure creep deformation could be much earlier than 500 s. According to the changes of strain rate and deformation morphology, the mechanisms of time-dependent plastic deformation could be qualitatively discerned, while the specific transition time for different deformation manners was difficult to trace. Basically, the transition of deformation mode would largely rely on the testing conditions, e.g., crack-dominated deformation segment could be increased by increasing the applied stress or strain and strain rate of the loading sequence. For the homogeneous creep flow in brittle LiTaO3 single crystal was investigated relying on a nanoindentation constant load-holding method. The permanent plastic deformation clearly occurred whilst its strain rate was precipitously reduced with increasing holding time. The density and length of cracks around the residual impression were evidently increased after 250 s holding. By further extending holding time, the morphology of surface cracks was rarely changed. Two distinct deformation mechanisms were informed for the time-dependent plastic deformation in this brittle ceramic under high shear-compression stress. At the beginning of the holding stage, generation of secondary cracks and propagation of both main and secondary cracks were the dominating deformation manners, which was consistent to high strain rate of this stage. At the steady-state segment of holding stage, of which strain rate was approximately lower than 10−5 s−1 and very slowly changed with holding time, the time-dependent plastic deformation could be ascribed to homogeneous creep flow which occurred in the severely plastic region beneath indenter. Furthermore, there existed an interim stage between the local cracking and homogeneous creep deformations, where there was deformation by cracking extension and creep flow simultaneously.In summary, the time-dependent plastic deformation of a LiTaO