Crotonic acid

Surface charge-based rational design of aspartase modifies the optimal pH for efficient beta-aminobutyric acid production.[Pubmed:32888990]

Int J Biol Macromol. 2020 Dec 1;164:4165-4172.

beta-Aminobutyric acid (BABA) can be widely used in the preparation of anti-tumor drugs, AIDS drugs, penicillin antibiotics, and plant initiators. However, the efficient, economical, and environmentally friendly production of BABA still faces challenges. Its important production enzyme, aspartase, catalyzes the substrate Crotonic acid, and depends on harsh conditions, such as high temperatures and the presence of strong alkali. Here, we modified the surface charge of the enzyme to enable it to become more negatively charged (K19E, N87E, N125D, S133D, Q262E, and N451E; from -60 to -80), reducing its optimal pH from 9.0 to 8.0. The M20 enzyme showed improved specific activity (400.21 mU/mg at pH 8.0; 2.47-fold that of aspartase), and at pH 7.0, its activity increased 3-fold. The thermal stability of the enzyme was also improved. For the production of BABA, a 500 g/L whole-cell transformation was obtained with a 1.41-fold increase in yield, and the final production of BABA reached 556.1 g/L within 12 h. Our method provides a new strategy for modifying the characteristics of the enzyme through the modification of its surface charge, which also represents the first modification of the optimal pH for aspartase.

Acidic Versus Alkaline Bacterial Degradation of Lignin Through Engineered Strain E. coli BL21(Lacc): Exploring the Differences in Chemical Structure, Morphology, and Degradation Products.[Pubmed:32714907]

Front Bioeng Biotechnol. 2020 Jun 30;8:671.

There is increasing interest in research on lignin biodegradation compounds as potential building blocks in applications related to renewable products. More attention is necessary to evaluate the effects of the initial pH conditions during the bacterial degradation of lignin. In this study we performed experiments on lignin biodegradation under acidic and mild alkaline conditions. For acidic biodegradation, lignin was chemically pretreated with hydrogen peroxide. Alkaline biodegradation was achieved by developing the bacterial growth on Luria and Bertani medium with alkali lignin as the sole carbon source. The mutant strain Escherichia coli BL21(Lacc) was used to carry out lignin biodegradation over 10 days of incubation. Results demonstrated that under acidic conditions there was a predominance of aliphatic compounds of the C3-C4 type. Alkaline biodegradation was produced in the context of oxidative stress, with a greater abundance of aryl compounds. The final pH values of acidic and alkaline biodegradation of lignin were 2.53 and 7.90, respectively. The results of the gas chromatography mass spectrometry analysis detected compounds such as Crotonic acid, lactic acid and 3-hydroxybutanoic acid for acidic conditions, with potential applications for adhesives and polymer precursors. Under alkaline conditions, detected compounds included 2-phenylethanol and dehydroabietic acid, with potential applications for perfumery and anti tumor/anti-inflammatory medications. Size-exclusion chromatography analysis showed that the weight-average molecular weight of the alkaline biodegraded lignin increased by 6.75-fold compared to the acidic method, resulting in a repolymerization of its molecular structure. Lignin repolymerization coincided with an increase in the relative abundance of dehydroabietic acid and isovanillyl alcohol, from 2.70 and 3.96% on day zero to 13.43 and 10.26% on 10th day. The results of the Fourier-transformed Infrared spectroscopy detected the presence of C = O bond and OH functional group associated with carboxylic acids in the acidic method. In the alkaline method there was a greater preponderance of signals related to skeletal aromatic structures, the amine functional group and the C - O - bond. Lignin biodegradation products from E. coli BL21(Laccase), under different initial pH conditions, demonstrated a promising potential to enlarge the spectrum of renewable products for biorefinery activities.

Synthesis of alpha,beta- and beta-Unsaturated Acids and Hydroxy Acids by Tandem Oxidation, Epoxidation, and Hydrolysis/Hydrogenation of Bioethanol Derivatives.[Pubmed:32052908]

Angew Chem Int Ed Engl. 2020 May 4;59(19):7456-7460.

We report a reaction platform for the synthesis of three different high-value specialty chemical building blocks starting from bio-ethanol, which might have an important impact in the implementation of biorefineries. First, oxidative dehydrogenation of ethanol to acetaldehyde generates an aldehyde-containing stream active for the production of C4 aldehydes via base-catalyzed aldol-condensation. Then, the resulting C4 adduct is selectively converted into Crotonic acid via catalytic aerobic oxidation (62 % yield). Using a sequential epoxidation and hydrogenation of Crotonic acid leads to 29 % yield of beta-hydroxy acid (3-hydroxybutanoic acid). By controlling the pH of the reaction media, it is possible to hydrolyze the oxirane moiety leading to 21 % yield of alpha,beta-dihydroxy acid (2,3-dihydroxybutanoic acid). Crotonic acid, 3-hydroxybutanoic acid, and 2,3-dihydroxybutanoic acid are archetypal specialty chemicals used in the synthesis of polyvinyl-co-unsaturated acids resins, pharmaceutics, and bio-degradable/ -compatible polymers, respectively.

Crotonylation at serine 46 impairs p53 activity.[Pubmed:32035620]

Biochem Biophys Res Commun. 2020 Apr 9;524(3):730-735.

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

Complexes of oxoplatin with rhein and ferulic acid ligands as platinum(iv) prodrugs with high anti-tumor activity.[Pubmed:31942585]

Dalton Trans. 2020 Feb 7;49(5):1613-1619.

We herein designed two new Pt(IV) prodrugs of oxoplatin (cis,cis,cis-[PtCl2(NH3)2(OH)2]), [Pt(IV)Cl2(NH3)2(O2C-FA)2] (Pt-2) and [Pt(IV)Cl2(NH3)2(O2C-RH)2] (Pt-3), by conjugating with ferulic acid (FA-COOH) and rhein (RH-COOH) which have well-known biological activities. Three other Pt(iv) complexes of [Pt(IV)Cl2(NH3)2(O2C-BA)2] (Pt-1), [Pt(IV)Cl2(NH3)2(O2C-CA)2] (Pt-4) and [Pt(IV)Cl2(NH3)2(O2C-TCA)2] (Pt-5) (where BA-COOH = benzoic acid, CA-COOH = Crotonic acid and TCA-COOH = trans-cinnamic acid) were also prepared for the comparative study. Like most Pt(IV) prodrug complexes, the cytotoxicity of Pt-3 containing the biologically active rhein (RH-COOH) ligand against lung carcinoma (A549 and A549/DDP) cells was higher than those of Pt-1, Pt-2, Pt-4, cisplatin and Pt-5. Moreover, the cytotoxicity of Pt-3 in HL-7702 normal cells was lower than those of Pt(IV) derivatives bearing BA-COOH, FA-COOH, TCA-COOH and CA-COOH ligands. The highly efficacious Pt-2 and Pt-3 were found to accumulate strongly in the A549/DDP cells, with the prodrug Pt-3 showing highest levels of penetration into the mitochondria. The prodrug Pt-3 effectively entered the A549/DDP cells and caused mitochondrial damage, significantly greater than Pt-2. In addition, the prodrug Pt-3 exhibited higher antitumor efficacy (inhibition rates (IR) = 67.45%) than Pt-2 (28.12%) and cisplatin (33.05%) in the A549/DDP xenograft mouse model. Thus, the prodrug Pt-3 containing the rhein (RH-COOH) ligand is a promising candidate drug targeting the mitochondria.

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.[Pubmed:31713669]

Appl Microbiol Biotechnol. 2019 Dec;103(23-24):9593-9606.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of Crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

Microwave-assisted synthesis of porphyrin conjugated microporous polymers for microextraction of volatile organic acids in tobaccos.[Pubmed:30799063]

J Chromatogr A. 2019 Jun 7;1594:45-53.

Conjugated microporous polymers (CMPs) with permanent microporosity and extended p-conjugated skeletons have recently shown the fascinating application in separation and enrichment. In this report, porphyrin CMP that possessed microporous structure and nitrogen-rich pyrrole building blocks was successfully synthesized by microwave-assisted method. Then, a novel coating based on porphyrin CMP was fabricated on silica fiber for efficient enrichment volatile organic acids (VOAs). The simulation showed the coating exhibited strong interaction with volatile organic acids based on charge transfer interaction, hydrogen bond and size effect. Hence, we proposed a method for determination of volatile organic acids in tobaccos by headspace solid-phase microextraction (HS-SPME) with porphyrin CMP coating by gas chromatography-mass spectrometry. The results showed that the coating provided high enrichment factors for VOAs ranging from 66,657 to 133,970 and low limits of detection from 4.6 to 22 ng/L. A good linearity was observed for propionic acid and Crotonic acid ranging from 0.050 to 8.0 mug/L, 2-methylheptanoic acid ranging from 0.063 to 1.5 mug/L, others ranging from 0.025 to 3.0 mug/L with the determination coefficient (R(2)) between 0.9900 and 0.9980. The strategy for determination of volatile compounds in complex solid samples was successfully applied to the analysis of volatile organic acids in tobacco leaves. The results showed that the method was accurate and reliable.

Safety assessment of the substance poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) for use in food contact materials.[Pubmed:32626096]

EFSA J. 2019 Jan 25;17(1):e05551.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP Panel) assessed the safety of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) (PHBH), CAS No 147398-31-0 and food contact material (FCM) substance No 1059. This biodegradable copolymer is produced by fermentation of palm oil using a genetically modified microorganism (Cupriavidus necator). Overall migration was up to 5.4 mg/kg. Oligomers are hydroxyl-terminated or with crotyl- and hexenyl end-groups from dehydration of hydroxyl end-groups. In the absence of calibration standards, the total oligomer migration was set at the overall migration values. Other degradation products are Crotonic acid and (E)-2-hexenoic acid. Crotonic acid is authorised for use in FCMs with a specific migration limit (SML) of 0.05 mg/kg food. For (E)-2-hexenoic acid, no indication for genotoxicity was identified by the EFSA CEF Panel in its group evaluation of flavouring substances in FGE.05Rev2 (EFSA CEF Panel, 2010b). The other migrating substances detected, , are from the authorised substance 'palm oil and/or palm fatty acid distillate' (FCM substance No 9) used as a carbon source for the fermentation and do not give rise to safety concern. A PHBH oligomer mixture was synthesized to simulate that migrating. It did not give rise to concern for genotoxicity. From the repeated dose 90-day oral toxicity study in rats, the Panel identified the no-observed-adverse-effect level (NOAEL) at the highest dose tested in males, 1,364 mg/kg body weight (bw) per day. The Panel concluded that the potential for bioaccumulation of oligomers is low. Overall, the CEP Panel concluded that the substance PHBH is not of safety concern for the consumer if it is used alone or blended with other polymers in contact with all kinds of food during more than 6 months at room temperature or below, including hot-fill or a short heating up phase. The specific migration of all oligomers < 1,000 Da should not exceed 5 mg/kg food. The migration of Crotonic acid should not exceed the SML of 0.05 mg/kg food. As the migration of (E)-2-hexenoic acid can be expected to be always lower than that of Crotonic acid, no individual restriction is necessary.

A review on mechanism of action, resistance, synergism, and clinical implications of mupirocin against Staphylococcus aureus.[Pubmed:30551435]

Biomed Pharmacother. 2019 Jan;109:1809-1818.

Mupirocin (MUP), bactroban, or pseudomonic acid is a natural Crotonic acid derivative drug extracted from Pseudomonas fluorescens which is produced by modular polyketide synthases. This antibiotic has a unique chemical structure and mechanism of action. It is a mixture of A-D pseudomonic acids and inhibits protein synthesis through binding to bacterial isoleucyl-tRNA synthetase. MUP is often prescribed to prevent skin and soft tissue infections caused by S. aureus isolates and where the MRSA isolates are epidemic, MUP may be used as a choice drug for nasal decolonization. It is also used for prevention of recurring infections and control the outbreaks. The emergence of MUP resistance has been increasing particularly among methicillin-resistant Staphylococcus aureus (MRSA) isolates in many parts of the world and such resistance is often related with MUP widespread uses. Although both low-level and high-level MUP resistance were reported among MRSA isolates, the rate of resistance is different in various geographic areas. In this review, we will report the global prevalence of MUP resistance, discuss synergism and mechanism of action of MUP, and provide new insights into the clinical use of this antibiotic.

SUPERSEDED: Safety assessment of the substance poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) for use in food contact materials.[Pubmed:32625967]

EFSA J. 2018 Jul 20;16(7):e05326.

This opinion of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) deals with the safety assessment of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate), Chemical Abstracts Service (CAS) No 147398-31-0 and food contact material (FCM) substance No 1059, for contact with dry/solid food. This biodegradable (co)polymer is produced by fermentation of palm oil using a genetically modified microorganism (Cupriavidus necator). No migration of oligomers into food simulant E (10 days at 40 and 60 degrees C) was found at a detection limit per single oligomer of 5 mug/kg food. Migration of the degradation product Crotonic acid was 8 and 25 mug/kg at the two test temperatures, respectively. The other migrating substances detected, , likely originated from or are related to the authorised substance (FCM No. 9) 'palm oil and/or palm fatty acid distillate' used as carbon source for the fermentation. At the migration levels reported, these migrants do not give rise to safety concern. No genotoxicity data are required for poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) because of its high molecular weight. The fraction below 1,000 Da is 0.5%. The major monomeric unit in the copolymer, 3-hydroxybutyric acid, is an intermediate in fatty acid metabolism. The minor monomeric unit, 3-hydroxyhexanoic acid, tested negative for bacterial gene mutations. Degradation products, which may be present in the (co)polymer, are Crotonic acid and (E)-2-hexenoic acid. Crotonic acid is authorised for use in FCM with a specific migration limit (SML) of 0.05 mg/kg food; for (E)-2-hexenoic acid, no indication for genotoxicity was identified by the EFSA CEF Panel in its 2010 group evaluation of flavouring substances in FGE.05Rev2. The CEF Panel concluded that the substance poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) is not of safety concern if used alone or in blends with other polymers for contact with dry/solid food. If the SML of Crotonic acid is met, migration of (E)-2-hexenoic acid will also not exceed 0.05 mg/kg food.