Isolation and Identification of the Antibacterial Compounds Produced by Maillard Reaction of Xylose with Phenylalanine or Proline.

Maillard reaction products (MRPs) of xylose with phenylalanine and xylose with proline exhibit high antibacterial activity. However, the active antibacterial compounds in MRPs have not yet been identified or isolated. This study aimed to isolate the active compounds in the two antibacterial MRPs. The organic layer of the MRP solution was separated and purified using silica gel chromatography and high-performance liquid chromatography. The chemical structures of the isolated compounds were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds inhibited the growth of Bacillus cereus and Salmonella Typhimurium at 25 °C for 7 days at a concentration of 0.25 mM. Furthermore, the isolated compounds inhibited the growth of naturally occurring microflora of lettuce and chicken thighs at 25 °C for 2 days at a concentration of 0.5-1.0 mM. The antibacterial compounds found in MRPs demonstrated a wide range of effectiveness and indicated their potential as alternative preservatives.

Isolation and Structure Determination of cis-OPDA-α-Monoglyceride from Arabidopsis thaliana.

cis-12-oxo-Phytodieneoic acid-α-monoglyceride (1) was isolated from Arabidopsis thaliana. The chemical structure of 1 was elucidated based on exhaustive 1D and 2D NMR spectroscopic measurements and supported by FDMS and HRFDMS data. The absolute configuration of the cis-OPDA moiety in 1 was determined by comparison of 1H NMR spectra and ECD measurements. With respect to the absolute configuration of the ß-position of the glycerol backbone, the 2:3 ratio of (S) to (R) was determined by making ester-bonded derivatives with (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride and comparing 1H NMR spectra. Wounding stress did not increase endogenous levels of 1, and it was revealed 1 had an inhibitory effect of A. thaliana post germination growth. Notably, the endogenous amount of 1 was higher than the amounts of (+)-7-iso-jasmonic acid and (+)-cis-OPDA in intact plants. 1 also showed antimicrobial activity against Gram-positive bacteria, but jasmonic acid did not. It was also found that α-linolenic acid-α-monoglyceride was converted into 1 in the A. thaliana plant, which implied α-linolenic acid-α-monoglyceride was a biosynthetic intermediate of 1.

Isolation, structural elucidation, and biological activity of a novel isocoumarin from the dark septate endophytic fungus Phialocephala fortinii.

A novel isocoumarin was isolated from the mycelia of the dark septate endophytic fungus Phialocephala fortinii. The chemical structure was determined to be 8-hydroxy-6-methoxy-3,7-dimethyl-1H-2-benzopyran-1-one based on mass spectrometry, 1H-nuclear magnetic resonance (NMR), and 13C-NMR spectroscopic analyses, including 2D-NMR experiments. The isolated compound inhibited root growth of Arabidopsis thaliana, suggesting its potential as a plant growth regulator.

Investigation Of The Biosynthesis Pathway That Generates cis-Jasmone.

Researchers have established that (+)-7-iso-jasmonic acid ((+)-7-iso-JA) is an intermediate in the production of cis-jasmone (CJ); however, the biosynthetic pathway of CJ has not been fully described. Previous reports stated that CJ, a substructure of pyrethrin II produced by pyrethrum (Tanacetum cinerariifolium), is not biosynthesized through this biosynthetic pathway. To clarify the ambiguity, stable isotope-labelled jasmonates were synthesized, and compounds were applied to apple mint (Mentha suaveolens) via air propagation. The results showed that cis-jasmone is not generated from intermediate (+)-7-iso-JA, and (+)-7-iso-JA is not produced from 3,7-dideydro-JA (3,7-ddh-JA); however, 3,7-didehydro-JA and 4,5-didehydro-7-iso-JA were converted into CJ and JA, respectively.

Pennelliiside D, a New Acyl Glucose from Solanum pennellii and Chemical Synthesis of Pennelliisides.

Acyl glucoses are a group of specialized metabolites produced by Solanaceae. Solanum pennellii, a wild-type tomato plant, produces acyl glucoses in its hair-like epidermal structures known as trichomes. These compounds have been found to be herbicides, microbial growth inhibitors, or allelopathic compounds. However, there are a few reports regarding isolation and investigation of biological activities of acyl glucoses in its pure form due to the difficulty of isolation. Here, we report a new acyl glucose, pennelliiside D, isolated and identified from S. pennellii. Its structure was determined by 1D NMR and 2D NMR, together with FD-MS analysis. To clarify the absolute configuration of the acyl moiety of 2-methylbutyryl in the natural compound, two possible isomers were synthesized starting from ß-D-glucose pentaacetate. By comparing the spectroscopic data of natural and synthesized compounds of isomers, the structure of pennelliiside D was confirmed to be 3,4-O-diisobutyryl-2-O-((S)-2-methylbutyryl)-D-glucose. Pennelliiside D and its constituent fatty acid moiety, (S)-2-methylbutanoic acid, did not show root growth-inhibitory activity. Additionally, in this study, chemical synthesis pathways toward pennelliisides A and B were adapted to give 1,6-O-dibenzylpennelliisides A and B.

A carlactonoic acid methyltransferase that contributes to the inhibition of shoot branching in Arabidopsis.

SignificanceStrigolactones (SLs) are a group of apocarotenoid hormones, which regulates shoot branching and other diverse developmental processes in plants. The major bioactive form(s) of SLs as endogenous hormones has not yet been clarified. Here, we identify an Arabidopsis methyltransferase, CLAMT, responsible for the conversion of an inactive precursor to a biologically active SL that can interact with the SL receptor in vitro. Reverse genetic analysis showed that this enzyme plays an essential role in inhibiting shoot branching. This mutant also contributed to specifying the SL-related metabolites that could move from root to shoot in grafting experiments. Our work has identified a key enzyme necessary for the production of the bioactive form(s) of SLs.

AtGH3.10 is another jasmonic acid-amido synthetase in Arabidopsis thaliana.

Jasmonoyl-isoleucine (JA-Ile) is a key signaling molecule that activates jasmonate-regulated flower development and the wound stress response. For years, JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl-amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine, and the source of persisting JA-Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate through recombinant enzyme assays and loss-of-function mutant analyses that AtGH3.10 functions as a JA-amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA-Ile accumulation in the gh3.10-2 jar1-11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative 12OH-JA-Ile was undetected in the flower buds and unwounded leaves. Residual levels of JA-Ile, JA-Ala, and JA-Val were nonetheless detected in gh3.10-2 jar1-11, suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA-Ile and 12OH-JA-Ile and the expression of JA-responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM-DOMAIN1 observed in WT, gh3.10-1, and jar1-11 leaves were effectively abolished in gh3.10-2 jar1-11. Additionally, an increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10-2 jar1-11. These findings conclusively show that AtGH3.10 contributes to JA-amino acid biosynthesis and functions partially redundantly with AtJAR1 in sustaining flower development and the wound stress response in Arabidopsis.

Potato tuber-inducing activities of jasmonic acid and related-compounds (II).

New information is being accumulated for plant-derived oxylipins, such as jasmonic acid (JA) amino acid conjugates. However, these compounds have not being examined for their activity in promoting potato tuber formation. It was found that (-)-JA had the highest activity followed cis-(-)-OPDA, (+)-4, 5-didehydroJA, cis-(+)-OPDA-l-Ile, and (-)-JA-l-Ile, -Leu, -Phe, -Val, although iso-OPDA and 3,7-didehydroJA did not exhibit activity.

Verification of the versatility of the in vitro enzymatic reaction giving (+)-cis-12-Oxo-phytodienoic acid.

Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic pathway of arabidopsides could occur via MGDG.

A promiscuous fatty acid ω-hydroxylase CYP94A90 is likely to be involved in biosynthesis of a floral nitro compound in loquat (Eriobotrya japonica).

Nitro groups are often associated with synthetically manufactured compounds such as medicines and explosives, and rarely with natural products. Loquat emits a nitro compound, (2-nitroethyl)benzene, as a flower scent. The nitro compound exhibits fungistatic activity and is biosynthesised from l-phenylalanine via (E/Z)-phenylacetaldoxime. Although aldoxime-producing CYP79s have been intensively studied, it is unclear what enzymes form nitro groups from aldoximes either in plants or in other organisms. Here, we report the identification of two cytochrome P450s that are likely to be involved in (2-nitroethyl)benzene biosynthesis in loquat through differential gene expression analysis using RNA-seq and functional identification using yeast and tobacco. CYP79D80 and CYP94A90 catalysed the formation of (E/Z)-phenylacetaldoxime from l-phenylalanine and (2-nitroethyl)benzene from the aldoxime, respectively. Expression profiles of CYP79D80 and CYP94A90 were correlated with the emission of (2-nitroethyl)benzene from loquat flowers. CYP94A90 also functioned as a fatty acid ω-hydroxylase as do other CYP94A fatty acid ω-hydroxylases. The CYP94As tested from other plants were all found to catalyse the formation of (2-nitroethyl)benzene from (E/Z)-phenylacetaldoxime. CYP79D80 and CYP94A90 are likely to operate in concert to biosynthesise (2-nitroethyl)benzene in loquat. CYP94A90 and other CYP94As are 'promiscuous fatty acid ω-hydroxylases', catalysing the formation of nitro groups from aldoximes, and are widely distributed in dicot plants.

Interdependent evolution of biosynthetic gene clusters for momilactone production in rice.

Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other "phytocassane" BGC. This CYP76M8 acts after the CYP99A2/3 from the "momilactone" BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the "momilactone" BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other "phytocassane" BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.

Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

Bioproduction of glucose conjugates of 4-hydroxybenzoic and vanillic acids using bamboo cells transformed to express bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase.

Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.

Gene Variants That Affect Levels of Circulating Tumor Markers Increase Identification of Patients With Pancreatic Cancer.

BACKGROUND & AIMS: Levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and cancer antigen 125 (CA-125) in blood are used as markers to determine the response of patients with cancer to therapy, but are not used to identify patients with pancreatic cancer. METHODS: We obtained blood samples from 504 patients undergoing pancreatic surveillance from 2002 through 2018 who did not develop pancreatic cancer and measured levels of the tumor markers CA19-9, CEA, CA-125, and thrombospondin-2. Single-nucleotide polymorphisms (SNPs) in FUT3, FUT2, ABO, and GAL3ST2 that have been associated with levels of tumor markers were used to establish SNP-defined ranges for each tumor marker. We also tested the association between additional SNPs (in FUT6, MUC16, B3GNT3, FAM3B, and THBS2) with levels of tumor markers. To calculate the diagnostic specificity of each SNP-defined range, we assigned the patients under surveillance into training and validation sets. After determining the SNP-defined ranges, we determined the sensitivity of SNP-adjusted tests for the tumor markers, measuring levels in blood samples from 245 patients who underwent resection for pancreatic ductal adenocarcinoma (PDAC) from 2010 through 2017. RESULTS: A level of CA19-9 that identified patients with PDAC with 99% specificity had 52.7% sensitivity. When we set the cut-off levels of CA19-9 based on each SNP, the test for CA19-9 identified patients with PDAC with 60.8% sensitivity and 98.8% specificity. Among patients with FUT3 alleles that encode a functional protein, levels of CA19-9 greater than the SNP-determined cut-off values identified 66.4% of patients with PDAC, with 99.3% specificity. In the validation set, levels of CEA varied among patients with vs without SNP in FUT2, by blood group, and among smokers vs nonsmokers; levels of CA-125 varied among patients with vs without the SNP in GAL3ST2. The use of the SNPs to define the ranges of CEA and CA-125 did not significantly increase the diagnostic accuracy of the assays for these proteins. Combining data on levels of CA19-9 and CEA, CA19-9 and CA-125, or CA19-9 and thrombospondin-2 increased the sensitivity of detection of PDAC, but slightly reduced specificity. CONCLUSIONS: Including information on SNPs associated with levels of CA19-9, CEA, and CA-125 can improve the diagnostic accuracy of assays for these tumor markers in the identification of patients with PDAC. Clinicaltrials.gov no: NCT02000089.

Cobalt-dependent inhibition of nitrite oxidation in Nitrobacter winogradskyi.

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH3) to nitrate (NO3-) via nitrite (NO2-), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO2- oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l-Histidine effectively restored cell growth and NO2- oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation.

Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the third isozyme with a distinct expression profile.

6-Tuliposide B (PosB), a major secondary metabolite that accumulates in tulip (Tulipa gesneriana), is converted to the antibacterial lactone, tulipalin B (PaB), by PosB-converting enzyme (TCEB). TgTCEB1 and TgTCEB-R, which encode TCEB, are specifically expressed in tulip pollen and roots, respectively, but are hardly expressed in other tissues (e.g. leaves) despite the presence of substantial PosB-converting activity, suggesting the existence of another TCEB isozyme. Here, we describe the identification of TgTCEB-L ("L" for leaf), a paralog of TgTCEB1 and TgTCEB-R, from leaves via native enzyme purification. The enzymatic characters of TgTCEB-L, including catalytic activity and subcellular localization, were substantially the same as those of TgTCEB1 and TgTCEB-R. However, TgTCEB-L did not exhibit tissue-specific expression. Identification of TgTCEB-L explains the PosB-converting activity detected in tissues where TgTCEB1 and TgTCEB-R transcripts could not be detected, indicating that tulip subtilizes the three TgTCEB isozymes depending on the tissue.

Investigating inducible short-chain alcohol dehydrogenases/reductases clarifies rice oryzalexin biosynthesis.

Rice (Oryza sativa) produces a variety of labdane-related diterpenoids as phytoalexins and allelochemicals. The production of these important natural products has been partially elucidated. However, the oxidases responsible for production of the keto groups found in many of these diterpenoids have largely remained unknown. Only one short-chain alcohol dehydrogenase/reductases (SDRs), which has been proposed to catalyze the last step in such a pathway, has been characterized to date. While rice contains >220 SDRs, only the transcription of five has been shown to be induced by the fungal cell wall elicitor chitin. This includes the momilactone A synthase (OsMAS/SDR110C-MS1), with the other four all falling in the same SDR110C family, further suggesting roles in diterpenoid biosynthesis. Here, biochemical characterization with simplified substrate analogs was first used to indicate potential functions, which were then supported by further analyses with key biosynthetic intermediates. Kinetic studies were then employed to further clarify these roles. Surprisingly, OsSDR110C-MS2 more efficiently catalyzes the final oxidation to produce momilactone A that was previously assigned to OsMAS/SDR110C-MS1, and we speculate that this latter SDR may have an alternative function instead. Conversely, two of these SDRs clearly appear to act in oryzalexin biosynthesis, with OsSDR110C-MI3 readily oxidizing the 3α-hydroxyl of oryzalexin D, while OsSDR110C-MS3 can also oxidize the accompanying 7ß-hydroxyl. Together, these SDRs then serve to produce oryzalexins A-C from oryzalexin D, essentially completing elucidation of the biosynthesis of this family of rice phytoalexins.

Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana.

Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways.

Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis.

The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3ß-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2-one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.

The application of synthetic biology to elucidation of plant mono-, sesqui-, and diterpenoid metabolism.

Plants synthesize a huge variety of terpenoid natural products, including photosynthetic pigments, signaling molecules, and defensive substances. These are often produced as complex mixtures, presumably shaped by selective pressure over evolutionary timescales, some of which have been found to have pharmaceutical and other industrial uses. Elucidation of the relevant biosynthetic pathways can provide increased access (e.g., via molecular breeding or metabolic engineering) and enable reverse genetic approaches toward understanding the physiological role of these natural products in plants as well. While such information can be obtained via a variety of approaches, this review describes the emerging use of synthetic biology to recombinantly reconstitute plant terpenoid biosynthetic pathways in heterologous host organisms as a functional discovery tool, with a particular focus on incorporation of the historically problematic cytochrome P450 mono-oxygenases. Also falling under the synthetic biology rubric and discussed here is the nascent application of genome-editing tools to probe physiological function.