Non-ribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI.

Nocardicin A is a monocyclic ß-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of gram-negative bacteria. The monobactams are of renewed interest due to emerging gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-L-phenylglycine (L-pHPG) into positions 1, 3, and 5 and L-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is L-pHPG-L-Arg-D-pHPG-L-Ser-L-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the L-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis.

FEMA GRAS assessment of natural flavor complexes: Sage oil, Orris Root Extract and Tagetes Oil and related flavoring ingredients.

In recent years, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) has conducted a program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavor ingredients. This publication, twelfth in the series, details the re-evaluation of NFCs whose constituent profiles are characterized by alicyclic or linear ketones. In its re-evaluation, the Expert Panel applies a scientific constituent-based procedure for the safety evaluation of NFCs in commerce using a congeneric group approach. Estimated intakes of each congeneric group of the NFC are evaluated using the well-established and conservative Threshold of Toxicological Concern (TTC) approach. In addition, studies on the toxicity and genotoxicity of members of the congeneric groups and the NFCs under evaluation are reviewed. The scope of the safety evaluation of the NFCs contained herein does not include added use in dietary supplements or any products other than food. Thirteen (13) NFCs derived from the Boronia, Cinnamomum, Thuja, Ruta, Salvia, Tagetes, Hyssopus, Iris, Perilla and Artemisia genera are affirmed as generally recognized as safe (GRAS) under conditions of their intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of derivatives of basil, nutmeg, parsley, tarragon and related allylalkoxybenzene-containing natural flavor complexes.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural alerts for genotoxicity and when exceeded, a margin of exposure (MOE) is calculated. BMDL10 values are derived from benchmark dose analyses using Bayesian model averaging for safrole, estragole and methyl eugenol using EPA's BMDS software version 3.2. BMDL10 values for myristicin, elemicin and parsley apiole were estimated by read-across using relative potency factors. Margins of safety for each constituent congeneric group and MOEs for each allylalkoxybenzene constituent for each NFC were determined that indicate no safety concern. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food. Ten NFCs, derived from basil, estragon (tarragon), mace, nutmeg, parsley and Canadian snakeroot were determined or affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Allspice, anise, fennel-derived and related flavoring ingredients.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.

FEMA GRAS assessment of natural flavor complexes: Asafetida oil, garlic oil and onion oil.

The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) applies its procedure for the safety evaluation of natural flavor complexes (NFCs) to re-evaluate the safety of Asafetida Oil (Ferula assa-foetida L.) FEMA 2108, Garlic Oil (Allium sativum L.) FEMA 2503 and Onion Oil (Allium cepa L.) FEMA 2817 for use as flavoring in food. This safety evaluation is part of a series of evaluations of NFCs for use as flavoring ingredients conducted by the Expert Panel that applies a scientific procedure published in 2005 and updated in 2018. Using a group approach that relies on a complete chemical characterization of the NFC intended for commerce, the constituents of each NFC are organized into well-defined congeneric groups and the estimated intake of each constituent congeneric group is evaluated using the conservative threshold of toxicological concern (TTC) concept. Data on the metabolism, genotoxic potential and toxicology for each constituent congeneric group are reviewed as well as studies on each NFC. Based on the safety evaluation, Asafetida Oil (Ferula assa-foetida L.), Garlic Oil (Allium sativum L.) and Onion Oil (Allium cepa L.) were affirmed as generally recognized as safe (GRASa) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Lemongrass oil, chamomile oils, citronella oil and related flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, eleventh in the series, evaluates the safety of NFCs characterized by primary alcohol, aldehyde, carboxylic acid, ester and lactone constituents derived from terpenoid biosynthetic pathways and/or lipid metabolism. The scientific-based evaluation procedure published in 2005 and updated in 2018 that relies on a complete constituent characterization of the NFC and organization of the constituents into congeneric groups. The safety of the NFCs is evaluated using the threshold of toxicological concern (TTC) concept in addition to data on estimated intake, metabolism and toxicology of members of the congeneric groups and for the NFC under evaluation. The scope of the safety evaluation does not include added use in dietary supplements or any products other than food. Twenty-three NFCs, derived from the Hibiscus, Melissa, Ricinus, Anthemis, Matricaria, Cymbopogon, Saussurea, Spartium, Pelargonium, Levisticum, Rosa, Santalum, Viola, Cryptocarya and Litsea genera were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Eucalyptus oil and other cyclic ether-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, the sixth in the series, will summarize the re-evaluation of eight NFCs whose constituent profiles are characterized by significant amounts of eucalyptol and/or other cyclic ethers. This re-evaluation was based on a procedure first published in 2005 and subsequently updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and the organization of its chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of the constituents of the congeneric groups and the NFC under evaluation. Eight NFCs derived from the Eucalyptus, Melaleuca, Origanum, Laurus, Rosmarinus and Salvia genera were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Origanum oil, thyme oil and related phenol derivative-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients, mostly consisting of a variety of essential oils and botanical extracts. This publication, seventh in the series, re-evaluates NFCs with constituent profiles dominated by phenolic derivatives including carvacrol, thymol and related compounds using a constituent-based procedure first published in 2005 and updated in 2018. The procedure is based on the chemical characterization of each NFC as intended for commerce and the estimated intake of the constituent congeneric groups. The procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology of the constituent congeneric groups and the NFC under evaluation. Herein, the FEMA Expert Panel affirmed the generally recognized as safe (GRAS) status of seven phenolic derivative-based NFCs, Origanum Oil (Extractive) (FEMA 2828), Savory Summer Oil (FEMA 3013), Savory Summer Oleoresin (FEMA 3014), Savory Winter Oil (FEMA 3016), Savory Winter Oleoresin (FEMA 3017), Thyme Oil (FEMA 3064) and Thyme White Oil (FEMA 3065) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Cinnamomum and Myroxylon-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, third in the series, considers NFCs composed primarily of constituents with the 3-phenyl-2-propenyl or a cinnamyl functional group, using the procedure outlined in 2005 and updated in 2018 to evaluate the safety of naturally-occurring mixtures for their intended use as flavor ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of each NFC's chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of members of the congeneric groups and the NFC under evaluation. Six NFCs from the Myroxylon and Cinnamomum genera, Balsam Oil, Peru (FEMA 2117), Tolu Balsam Extract (FEMA 3069), Cassia Bark Extract (FEMA 2257), Cassia Bark Oil (FEMA 2258), Cinnamon Bark Extract (FEMA 2290) and Cinnamon Bark Oil (FEMA 2291) were evaluated and affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Mint, buchu, dill and caraway derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. NFC flavor materials include a variety of essential oils and botanical extracts. The re-evaluation of NFCs is conducted based on a constituent-based procedure outlined in 2005 and updated in 2018 that evaluates the safety of NFCs for their intended use as flavor ingredients. This procedure is applied in the re-evaluation of the generally recognized as safe (GRAS) status of NFCs with constituent profiles that are dominated by alicyclic ketones such as menthone and carvone, secondary alcohols such as menthol and carveol, and related compounds. The FEMA Expert Panel affirmed the GRAS status of Peppermint Oil (FEMA 2848), Spearmint Oil (FEMA 3032), Spearmint Extract (FEMA 3031), Cornmint Oil (FEMA 4219), Erospicata Oil (FEMA 4777), Curly Mint Oil (FEMA 4778), Pennyroyal Oil (FEMA 2839), Buchu Leaves Oil (FEMA 2169), Caraway Oil (FEMA 2238) and Dill Oil (FEMA 2383) and determined FEMA GRAS status for Buchu Leaves Extract (FEMA 4923), Peppermint Oil, Terpeneless (FEMA 4924) and Spearmint Oil, Terpeneless (FEMA 4925).

FEMA GRAS assessment of natural flavor complexes: Clove, cinnamon leaf and West Indian bay leaf-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as "generally recognized as safe (GRAS)" under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 µg/person per day for compounds with structural alerts for genotoxicity.

FEMA GRAS assessment of natural flavor complexes: Lavender, Guaiac Coriander-derived and related flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, fifth in the series, evaluates the safety of NFCs containing linalool and/or other characteristic mono- and sesquiterpenoid tertiary alcohols and esters using the safety evaluation procedure published by the FEMA Expert Panel in 2005 and updated in 2018. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of the chemical constituents of each NFC into well-defined congeneric groups. The safety of each NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of both the constituent congeneric groups and the NFCs. Sixteen NFCs, derived from the Lavandula, Aniba, Elettaria, Daucus, Salvia, Coriandrum, Ribes, Guaiacum/Bulnesia, Citrus, Pogostemon, Melaleuca and Michelia genera, were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

Identification and characterization of NocR as a positive transcriptional regulator of the beta-lactam nocardicin A in Nocardia uniformis.

Nocardicin A is a monocyclic beta-lactam isolated from the actinomycete Nocardia uniformis, which shows moderate activity against a broad spectrum of gram-negative bacteria. Within the biosynthetic gene cluster of nocardicin A, nocR encodes a 583-amino-acid protein with high similarity to a class of transcriptional regulators known as streptomyces antibiotic regulatory proteins. Insertional inactivation of this gene resulted in a mutant showing morphology and growth characteristics similar to the wild type, but one that did not produce detectable levels of nocardicin A or the early precursor p-hydroxybenzoyl formate. Similar disruptions of nocD, nocE, and nocO yielded mutants that maintained production of nocardicin A at levels similar to the wild-type strain. In trans complementation of the nocR::apr mutant partially restored the wild-type phenotype. Transcriptional analysis of the nocR::apr mutant using reverse transcription-PCR found an absence of mRNA transcripts for the early-stage nocardicin A biosynthetic genes. In addition, transcription of the genes responsible for the biosynthesis of the nonproteinogenic p-hydroxyphenylglycine (pHPG) precursor was attenuated on the nocR disruption mutant. NocR was heterologously expressed and purified from Escherichia coli as an N-terminal maltose binding protein-tagged fusion protein. DNA binding assays demonstrated that NocR is a DNA binding protein, targeting the 126-bp intergenic region between nocF and nocA. Within this intergenic region is the likely binding motif, a direct hexameric repeat, TGATAA, with a 5-bp spacer. These experiments establish NocR as a positive transcriptional regulator of the nocardicin A biosynthetic pathway, coordinating the initial steps of nocardicin A biosynthesis to the production of its pHPG precursor.

FEMA GRAS assessment of natural flavor complexes: Citrus-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients. This publication is the first in a series and summarizes the evaluation of 54 Citrus-derived NFCs using the procedure outlined in Smith et al. (2005) and updated in Cohen et al. (2018) to evaluate the safety of naturally-occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of each NFC intended for commerce and organization of each NFC's chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of members of the congeneric groups and the NFC under evaluation. As a result of the application of the procedure, 54 natural flavor complexes derived from botanicals of the Citrus genus were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavoring ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

Updated procedure for the safety evaluation of natural flavor complexes used as ingredients in food.

An effective and thorough approach for the safety evaluation of natural flavor complexes (NFCs) was published in 2005 by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). An updated procedure is provided here, which maintains the essential concepts of the use of the congeneric group approach and the reliance on the threshold of toxicological concern (TTC) concept. The updated procedure emphasizes more rigorous considerations of unidentified constituents and the genotoxic potential of constituents. The update of the previously established procedure is the first step in a multi-year project to conduct safety re-evaluations for more than 250 NFCs that have uses that are currently considered Generally Recognized as Safe (GRAS) by the FEMA Expert Panel. In addition, this procedure can be more generally employed in the safety evaluation of NFCs.

In vivo characterization of nonribosomal peptide synthetases NocA and NocB in the biosynthesis of nocardicin A.

Two nonribosomal peptide synthetases (NRPS), NocA and NocB, together comprising five modules, are essential for the biosynthesis of the D,L,D configured tripeptide backbone of the monocyclic ß-lactam nocardicin A. We report a double replacement gene strategy in which point mutations were engineered in the two encoding NRPS genes without disruption of the nocABC operon by placing selective markers in adjacent genes. A series of mutants was constructed to inactivate the thiolation (T) domain of each module and to evaluate an HHxxxDR catalytic motif in NocA and an atypical extended histidine motif in NocB. The loss of nocardicin A production in each of the T domain mutants indicates that all five modules are essential for its biosynthesis. Conversely, production of nocardicin A was not affected by mutation of the NocB histidine motif or the R828G mutation in NocA.