Search

Caldimonas thermodepolymerans, a Gram-negative, moderately thermophilic bacterium, exhibits a remarkable biotechnological potential. Given the presence of genes in its genome dedicated to the metabolization of ferulic acid (FA), this study aimed to explore the bacterium's capability for biotransforming FA into high-value metabolites. The results unequivocally demonstrate the bacterium's proficiency in the efficient and rapid conversion of FA into vanillyl alcohol (VOH) and vanillic acid (VA). By manipulating key cultivation parameters, such as adjusting initial FA doses and varying cultivation periods, the product profile can be tailored. Higher initial doses and shorter cultivation periods favor the production of VOH, while lower FA doses and extended cultivation periods lead to the predominant formation of VA. Furthermore, the process can be operated in a repeated-batch scenario. This underscores the potential of C. thermodepolymerans for industrial biotransformation of FA, presenting a promising avenue for leveraging its capabilities in practical applications., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Substituted benzaldehydes are the most commonly used natural-occurring flavours in the world. The consumer's preference for 'natural or organic' aromas has increased the request for flavours possessing the 'natural' status. The resulting shortage of aromatic aldehydes of extractive origin, such as vanillin, veratraldehyde and piperonal, can be offset by developing a new biotechnological synthesis method. Here, we report a study on the microbiological reduction of five natural benzoic acid derivatives, namely p-anisic, vanillic, veratric, piperonylic and eudesmic acids, to produce the corresponding fragrant aldehydes. We found that different Basidiomycota strains can efficiently perform this transformation, with good chemical selectivity and tolerance to the toxicity of substrates and products. Besides confirming the carboxylic acid reductase activity of the already studied fungi Pycnoporus cinnabarinus, we discovered that other species such as Pleurotus eryngii, Pleurotus sapidus and Laetiporus sulphureus as well as the non-ligninolytic fungi Lepista nuda are valuable microorganisms for the synthesis of anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde from the corresponding acids. According to our findings, we propose a reliable process for the preparation of the above-mentioned aldehydes, in natural form. KEY POINTS: • Fragrant benzaldehydes were obtained by biotransformation. • Basidiomycota strains reduced substituted benzoic acid to the corresponding aldehydes. • Anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde were prepared in natural form., (© 2024. The Author(s).)

The process of preprocessing techniques such as acid and alkali pretreatment in lignocellulosic industry generates substantial solid residues and lignocellulosic pretreatment wastewater (LPW) containing glucose, xylose and toxic byproducts. In this study, furfural and vanillin were selected as model toxic byproducts. Kurthia huakuii as potential strain could tolerate to high concentrations of inhibitors. The results indicated that vanillin exhibited a higher inhibitory effect on K. huakuii (3.95 % inhibition rate at 1 g/L than furfural (0.45 %). However, 0.5 g/L vanillin promoted the bacterial growth (-2.35 % inhibition rate). Interestingly, the combination of furfural and vanillin exhibited antagonistic effects on bacterial growth (Q<0.85). Furfural and vanillin could be bio-transformed into less toxic molecules (furfuryl alcohol, furoic acid, vanillyl alcohol, and vanillic acid) by K. huakuii, and inhibitor degradation rate could be promoted by expression of antioxidant enzymes. This study provides important insights into how bacteria detoxify inhibitors in LPW, potentially enhancing resource utilization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. We declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The nosocomial bacterium Acinetobacter baumannii is protected from antibiotic treatment by acquiring antibiotic resistances and by forming biofilms. Cell attachment, one of the first steps in biofilm formation, is normally induced by environmental metabolites. We hypothesized that vanillic acid (VA), the oxidized form of vanillin and a widely available metabolite, may play a role in A. baumannii cell attachment. We first discovered that A. baumannii actively breaks down VA through the evolutionarily conserved vanABKP genes. These genes are under the control of the repressor VanR, which we show binds directly to VanR binding sites within the vanABKP genes bidirectional promoter. VA in turn counteracts VanR inhibition. We identified a VanR binding site and searched for it throughout the genome, especially in pili encoding promoter genes. We found a VanR binding site in the pilus encoding csu operon promoter and showed that VanR binds specifically to it. As expected, a strain lacking VanR overproduces Csu pili and makes robust biofilms. Our study uncovers the role that VA plays in facilitating the attachment of A. baumannii cells to surfaces, a crucial step in biofilm formation. These findings provide valuable insights into a previously obscure catabolic pathway with significant clinical implications., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Maize bran, an agro-processing waste residue, is a good source of ferulic acid that can be further valorized for vanillin production. However, extraction of ferulic acid from natural sources has been challenging due to low concentrations and intensive extraction procedures. In the present work, ferulic acid streams (purities ranging from 5% to 75%) extracted from maize bran using thermochemical methods were evaluated for biotransformation to vanillin, employing Amycolatopsis sp. as a whole-cell biocatalyst. Initial adaptation studies were critical in improving ferulic acid assimilation and its conversion to vanillin by 65% and 56%, respectively by the fourth adaptation cycle. The effect of cell's physiological states and vanillic acid supplementation on vanillin production was studied using standard ferulic acid as a substrate in an effort to achieve further improvement in vanillin yield. In the presence of vanillic acid, 18 h cultured cells using 2 g/L of standard and isolated ferulic acid produced vanillin concentrations of up to 0.71 and 0.48 g/L, respectively. Furthermore, intermediates involved in the ferulic acid catabolic pathway and their interrelations were studied using GC-MS analysis. Results indicated that two different routes were involved in the catabolism of standard ferulic acid, and similar metabolic routes were observed for an isolated ferulic acid stream. These findings effectively evaluated isolated ferulic acid for sustainable vanillin production while reducing agro-industrial waste pollution., (© 2024 American Institute of Chemical Engineers.)

Vanillic acid (VA), as a plant-derived phenolic acid compound, has widespread applications and good market prospects. However, the traditional production process cannot meet market demand. In this study, Pseudomonas putida KT2440 was used for de novo biosynthesis of VA. Multiple metabolic engineering strategies were applied to construct these P. putida -based cell factories, including the introduction of a Hs-OMT opt , engineering the cofactor S -adenosylmethionine supply pathway through the overexpression of metX and metH , reforming solubility of Hs-OMT opt , increasing a second copy of Hs-OMT opt , and the optimization of the fermentation medium. The resulting strain, XCS17, de novo biosynthesized 5.4 g/L VA from glucose in a fed-batch fermentation system; this is the highest VA production titer reported up to recently. This study showed that P. putida KT2440 is a robust platform for achieving the effective production of phenolic acids.

Tyrosinase is a key enzyme in melanogenesis and its inhibitors have become increasingly because of their potential activity as hypopigmenting agents which have less side effects. Nipa palm vinegar is an aqueous product that is normally used as a food supplement. The aim of this study was to study the determination of antioxidant activity and tyrosinase inhibitory activities of aqueous extract of original nipa palm vinegar (AE O-NPV), nipa palm vinegar powder (NPV-P) and aqueous extract of nipa palm vinegar powder (AE NPV-P) were examined. Nipa palm vinegars were evaluated the phenolic and flavonoid content, and the active compounds which were submitted to molecular docking and molecular dynamic simulation, chemoinformatics, rule of five, skin absorption and toxicity. The highest phenolic and flavonoid contents in the AE O-NPV were 2.36 ± 0.23 mg gallic acid equivalents/g extract and 5.11 ± 0.59 mg quercetin equivalents/g, and the highest ABTS radical cation scavenging activity was also found. The AE O-NPV, NPV-P and AE NPV-P showed anti-mushroom tyrosinase activity. The HPLC analysis showed that there were vanillic acid and three flavonoids (catechin, rutin and quercetin). The molecular docking study revealed that the binding of the vanillic acid and three flavonoids occurred in the active site residues (histidine and other amino acids). Moreover, the number of hydrogen bond acceptors/donors, solubility, polar surface area and bioavailability score of the vanillic acid and three flavonoids were acceptable compared to Lipinski's Rule of Five. The molecular dynamic simulation showed that vanillic acid interacts with HIS284 through π - π stacking hydrophobic interactions and forms a metal-acceptor interaction with the copper molecule at the tyrosinase active site. All compounds revealed good skin permeability and nontoxicity. Nipa palm vinegar could be a promising source of a new ingredient for tyrosinase inhibition for cosmetics or pharmaceutical products., Competing Interests: The authors declare there are no competing interests., (©2023 Chatatikun et al.)

Intermolecular interactions play a significant role on the physicochemical properties and digestibility of starchy foods. This study investigated the covalent and non-covalent interactions between vanillic acid (VA) and porous starch (PS) as well as their effects on digestibility using solid-state NMR. VA-PS conjugates and mixtures were synthesized and characterized using 1 H NMR, FT-IR, SEM and XRD. 13 C NMR peaks at 163 ppm and FT-IR signals at 1737 cm -1 indicated the formation of ester bond in VA-PS conjugates. While differences between covalent and non-covalent interactions were also probed by solid-state NMR. The specific binding sites between VA and PS were subsequently identified by 1 H 13 C HETCOR spectra before assessing the impact of covalent and non-covalent interactions on digestibility through an in vitro digestion test. The results revealed 13 C chemical shifts of about 2.0 ppm, indicating stronger intermolecular interactions, and reduced mobility of the VA-PS conjugate due to its covalent bonding. Overall, the results showed that the VA-PS conjugate, characterized by stronger covalent interactions, exhibited superior effects in inhibiting starch digestibility compared with non-covalent interactions., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Lignin-derived compounds (LDCs) bioconversion into lipids is a promising yet challenging task. This study focuses on the isolation of the ligninolytic bacterium Citricoccus sp. P2 and investigates its mechanism for producing lipids from LDCs. Although strain P2 exhibits a relatively low lignin degradation rate of 44.63%, it efficiently degrades various concentrations of LDCs. The highest degradation rate is observed when incubated with 0.6 g/L vanillic acid, 0.6 g/L syringic acid, 0.8 g/L p-coumaric acid, and 0.4 g/L phenol, resulting in respective lipid yields of 0.16 g/L, 0.13 g/L, 0.24 g/L, and 0.13 g/L. The genome of strain P2 provides insights into LDCs bioconversion into lipids and stress tolerance. Moreover, Citricoccus sp. P2 has been successfully developed a non-sterilized lipid production using its native alkali-halophilic characteristics, which significantly enhances the lipid yield. This study presents a promising platform for lipids production from LDCs and has potential to promote valorization of lignin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aging is the root cause of several pathologies like neurological and cardiovascular diseases. Identifying compounds that improve health span and extend life span, called geroprotectors, could be crucial to preventing or at least delaying the onset of age-related diseases. In this regard, the nematode Caenorhabditis elegans (C. elegans) is emerging as an easy, efficient, low-cost model system to screen natural products and identify novel geroprotectors. Phenolic acids can be found in a wide range of natural products that are part of the human diet. Vanillic acid (VA) is a phenolic acid that has previously been attributed with antioxidant, anti-inflammatory, and neuroprotective features. To determine whether these beneficial health effects amount to an extension of health span and life span, in this work, we thoroughly explore the effect of VA on C. elegans stress resistance and life span. We found that VA increases thermotolerance (19.4%), reduces protein aggregation (between 30% and 40%), improves motility, and extends life span by almost 50%, an extent hardly ever achieved with a natural compound. The increased thermotolerance induced by VA is independent of the insulin/insulin-like growth factor-1 signaling pathway but requires heat shock factor-1 and is associated with increased heat shock protein-4 (HSP-4) and hsp-16.2 expression. These results provide new insight into understanding the therapeutical properties of VA and warrant further investigation of VA as a novel geroprotector., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The host stimulator of interferon genes (STING) signaling pathway is a major innate immune sensing pathway, and the stimulation of this pathway within antigen-presenting cells shows promise in targeting immune-suppressed tumors. Macrophages resident in tumors exhibit anti-inflammatory properties and enhance tumor growth and development. Polarizing such macrophages towards a pro-inflammatory phenotype is an effective strategy for tumor suppression. In the present study, we observed that the STING pathway was inactivated in breast and lung carcinomas, and a positive correlation existed between STING and macrophage markers in these tumors. We found that vanillic acid (VA) could stimulate the STING/TBK1/IRF3 pathway. VA mediated the production of type I IFN and promoted macrophage polarization into the M1 phenotype; this activity was dependent on STING activation. A direct-contact co-culture model and a transwell co-culture model revealed that macrophages with VA-induced STING activation exhibited anti-proliferative effects on SKBR3 and H1299 cells, although a STING antagonist and M2 macrophage-related cytokines alleviated this anti-proliferative effect. Further investigation indicated that phagocytosis and apoptosis-inducing effects were the major mediators of the anti-tumor effect of VA-treated macrophages. Mechanistically, VA promoted the polarization of macrophages to a M1 phenotype via IL-6R/JAK signaling, resulting in enhanced phagocytosis and apoptosis-induction effects. Additionally, STING activation-induced IFNβ production also participated in the apoptosis mediated by VA-treated macrophage in SKBR3 and H1299 cells. Mouse models with 4 T1 tumors confirmed the anti-tumor properties of VA in vivo and revealed the infiltration of VA-induced cytotoxic T cells into the tumors. These data suggest that VA is an effective agonist of STING and provides a new perspective for cancer immunotherapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Background: Methamphetamine is widely abused in all parts of the world. It has been reported that short-term and long-term methamphetamine exposure could damage the dopaminergic system and induce cardiomyopathy and cardiotoxicity via mitochondrial dysfunction and oxidative stress. Vanillic acid (VA), a phenolic acid compound derived from plants, is known for its antioxidant and mitochondrial protection properties., Methods: In the current study we used VA for attenuating of Methamphetamine-induced mitochondrial toxicity in cardiac mitochondria. Isolated mitochondria obtained from rat heart were grouped as: control, methamphetamine (250 µM), VA (10, 50 and 100 µM) was cotreated with methamphetamine (250 µM) and VA (100 µM) alone. After 60 min, mitochondrial fraction including: succinate dehydrogenases (SDH) activity, mitochondrial membrane potential (MMP), mitochondrial swelling, mitochondrial glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation (LPO) were evaluated., Results: Methamphetamine exposure significantly disrupted mitochondrial function and induced ROS formation, lipid peroxidation, GSH depletion, MMP collapse and mitochondrial swelling, while VA significantly increased SDH activity as indicator of mitochondrial toxicity and dysfunction. VA also significantly decreased ROS formation, lipid peroxidation, mitochondrial swelling, MMP collapse and depletion of GSH in cardiac mitochondria in the presence of methamphetamine., Conclusion: These findings suggested that VA is able to reduce methamphetamine-induced mitochondrial dysfunction and oxidative stress. Our results demonstrate that VA could potentially serve as a promising and accessible cardioprotective agent against methamphetamine-induced cardiotoxicity, via antioxidant and mitochondrial protection properties., (© 2023. The Author(s).)

Microbial tolerance to lignocellulose-derived inhibitors, such as aromatic acids, is critical for the economical production of biofuels and biochemicals. Here, adaptive laboratory evolution was applied to improve the tolerance of Yarrowia lipolytica to a representative aromatic acid inhibitor vanillic acid. The transcriptome profiling of evolved strain suggested that the tolerance could be related to the up-regulation of RNA processing and multidrug transporting pathways. Further analysis by reverse engineering confirmed that the amplification of YALI0&#95;F13475g coding for transcriptional coactivator and YALI0&#95;E25201g coding for multidrug transporter conferred tolerance not only to vanillic acid but also towards ferulic acid, p-coumaric acid, p-hydroxybenzoic acid and syringic acid. These findings suggested that regulation of RNA processing and multidrug transporting pathways may be important for enhanced aromatic acid tolerance in Y. lipolytica. This study provides valuable genetic information for robust strain construction for lignocellulosic biorefinery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Diabetes is the most common component of metabolic syndrome, including abdominal obesity, insulin resistance, hypertension, and dyslipoproteinemia., Objective: This study aims to determine whether vanillic acid has antihyperlipidemic properties in diabetic hypertensive rats., Methods: For this study healthy male albino Wister rats (180-220 gm) were selected. A 20-week highfat diet (HFD) was given to produce diabetic hypertension in Wister rats. Control and diabetic hypertensive rats were treated with vanillic acid. Vanillic acid effects on lipid profiles (cholesterol, triglycerides, phospholipids, free fatty acids, high-density lipoproteins (HDL)) and lipid metabolizing enzymes LPL, LCAT, and HMG CoA reductase studied by a conventional method. To understand the effect of vanillic acid control, experimental rat lipid and metabolic enzymes were studied and treated and controlled animal liver tissues were observed using the different histology staining agents., Results: Vanillic acid caused considerable lipid profile reductions except for HDL and increased plasma HDL levels. After eight weeks of vanillic acid administration also boosts lipid marker enzyme activity (HMG CoA reductase, LPL, and LCAT). In addition, vanillic acid reduces the accumulation of collagen in liver tissues., Conclusion: These research studies suggest that vanillic acid has antihyperlipidemic effects in diabetic hypertensive rats fed an HFD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Numerous formulations have been utilized in the cosmetic and pharmaceutical industries to effectively deliver bioactive ingredients., Methods: We selected a well-known liposomal formulation of bilayer lipid vesicles composed of ceramide NP. Ethosomes contain hydrophilic vanillic acid or lipophilic α-bisabolol, and their physicochemical properties were evaluated. Vanillic acid is encapsulated in the aqueous core while α-bisabolol is engaged with the lipid phase. The formulation was prepared by the high-pressure homogenization method at 800 bar for 5 min. The particle size, polydispersity index and zeta potential of the ethosome dispersion were analyzed by dynamic light scattering. In order to measure the skin absorption efficiency from artificial skin, an in vitro assay was performed using the Franz diffusion cell method for 24 hours. In addition, ultracentrifuges for encapsulation efficiency, dialysis membranes for active ingredient release, and low-temperature transmission electron microscopy (TEM) to evaluate the morphology of vesicles were utilized., Results: The particle size of the ethosome containing ceramide NP and vanillic acid was in the range of 80 ~ 130 nm, whereas the particle size of the ethosome containing ceramide NP and α-bisabolol was 150 ~ 170 nm. In the vanillic acid-containing ethosome, increasing the amount of ceramide NP decreased the particle size, whereas the size of the α-bisabolol ethosome did not change. The stability of the prepared ethosome did not change significantly for 4 weeks at 25°C, 4°C, and 45°C. The skin absorption efficiency of ceramide NP and vanillic acid-containing ethosome was increased by about 15% compared to the control group, whereas the ethosome containing α-bisabolol and ceramide NP showed slightly higher skin absorption efficiency than the control group. In addition, encapsulation efficiency evaluation, active ingredient release measurement and cryo-TEM were taken., Conclusion and Perspective: Based on the results of these studies, we suggest that ethosome formulations containing ceramide NP can be widely used in the cosmetic industry together with other cosmetic formulations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In the current study, the hepatoprotective activity of vanillic acid, silymarin, and vanillic acid-loaded silver nanoparticles (AgNPs) against CCl 4 -induced hepatotoxicity was tested in male rats for four weeks. Thirty male rats were divided into five groups ( n = 6). The 1st group was a negative control, the 2nd group was a positive control, the 3rd group was treated with 100 mg/kg b.w. of vanillic acid, the 4th group was treated with 100 mg/kg b.w. of vanillic acid-AgNPs, and the 5th group was treated with 50 mg/kg b.w. of silymarin. The CCl 4 -induced hepatic toxicity in the 2nd group was revealed by the liver function and all other biochemical tests. Liver enzymes, bilirubin, lipid peroxidation, lactate dehydrogenase, and interleukin-6 were elevated, whereas, total protein, antioxidant enzymes, and irisin were decreased compared to the negative control. The hepatic tissues were also injured as a result of the CCl 4 -induced hepatotoxicity. Treating the hepatotoxic rats with vanillic acid moderately protected the rats of the 3rd group, whereas treatment with vanillic AgNPs and silymarin in G4 and G5, respectively, greatly protected the rats against the CCl 4 hepatotoxicity, approaching the normal biochemical levels and liver tissue appearance. The biochemical tests were confirmed by the histological investigations of liver tissue.

Vanillic acid has always been in high-demand in pharmaceutical, cosmetic, food, flavor, alcohol and polymer industries. Present study achieved highly pure synthesis of vanillic acid from vanillin using whole cells of Ochrobactrum anthropi strain T5&#95;1. The complete biotransformation of vanillin (2 g/L) in to vanillic acid (2.2 g/L) with 95 % yield was achieved in single step in 7 h, whereas 5 g/L vanillin was converted to vanillic acid in 31 h. The vanillic acid thus produced was validated using LC-MS, GC-MS, FTIR and NMR. Further, vanillic acid was evaluated for in vitro anti-tyrosinase and cytotoxic properties on B16F1 skin cell line in dose dependent manner with IC 50 values of 15.84 mM and 9.24 mM respectively. The in silico Swiss target study predicted carbonic acid anhydrase IX and XII as key targets of vanillic acid inside the B16F1 skin cell line and revealed the possible mechanism underlying cell toxicity. Molecular docking indicated a strong linkage between vanillic acid and tyrosinase through four hydrogen and several hydrophobic bonds, with ΔG of -3.36 kJ/mol and Ki of 3.46 mM. The bioavailability of vanillic acid was confirmed by the Swiss ADME study with no violation of Lipinski's five rules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Growth and maintenance of skeletal muscle is essential for athletic performance and a healthy life. Stimulating the proliferation and differentiation of muscle cells may help prevent loss of muscle mass. To discover effective natural substances enabling to mitigate muscle loss without side effects, we evaluated muscle growth with several compounds extracted from Catalpa bignonioides Walt. Among these compounds, pinoresinol and vanillic acid increased C2C12, a mouse myoblast cell line, proliferation being the most without cytotoxicity. These substances activated the Akt/mammalian target of the rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, the results of in silico molecular docking study showed that they may bind to the active site of insulin-like growth factor 1 receptor (IGF-1R), which is an upstream of the Akt/mTOR pathway, indicating that both pinoresinol and vanillic acid stimulate myoblast proliferation through direct interaction with IGF-1R. These results suggest that pinoresinol and vanillic acid may be a natural supplement to improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus strengthen muscles.

Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here, the acvABCDEF genes were identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S carboxylated the reaction products generated from acetovanillone/acetosyringone by AcvAB and AcvF into vanilloyl acetic acid/3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid. To demonstrate the feasibility of producing cis , cis -muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis , cis -muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximately equimolar cis , cis -muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling. IMPORTANCE In the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p -hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p -hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4'-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This study elucidated the acetovanillone/acetosyringone catabolic system and demonstrates the potential of using these genes for the production of value-added chemicals from these compounds.

Diabetes is the most prevalent disorder in the world characterized by uncontrolled high blood glucose levels and nephropathy is one of the chief complications allied with hyperglycemia. Vanillic acid; the main bioactive compound derived from natural sources such as vegetables, fruits and plants possesses various pharmacological activities such as antioxidant, anti-inflammatory and anti-proliferative. The current study was designed to investigate the antidiabetic and renoprotective effects of vanillic acid by its various pharmacological activities. Streptozotocin (50 mg/kg)/nicotinamide (110 mg/kg) was used to induce diabetes in rats. Oral administration of vanillic acid once daily for 6 weeks (25, 50 and 100 mg/kg) significantly reduced the hyperglycemia, increased liver enzymes and normalized lipid profile that was altered in diabetic rats. Moreover, vanillic acid attenuated the impaired renal function as evidenced by a reduction in serum creatinine, urea, uric acid and urinary microproteinuria levels with a concomitant increase in urinary creatinine clearance in the nephropathic rats. Diabetic rats showed a marked increase in thiobarbituric acid reactive substances (TBARS) and superoxide anion generation (SAG) along with decreased reduced glutathione (GSH) in the renal tissue which was ameliorated in the vanillic acid-treated rats. Histopathologically, vanillic acid treatment was associated with reduced damage with normalized structural changes in renal tissue. Furthermore, treatment groups showed the suppression of upregulation of nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, cyclo-oxygenase (COX)-2 and up-regulation of Nuclear factor-erythroid 2-related factor 2 (Nrf-2) in the renal tissue. In conclusion, vanillic acid's ameliorative impact on diabetic nephropathic rats may be attributed to its powerful free radical scavenging property, down-regulation of NF-κB, TNF-α, COX-2 and up-regulation of Nrf-2 proteins in renal tissue., (© 2022 John Wiley & Sons Ltd.)

A feasible approach against the low yield of microalgae biomass involves the use of a stimulator for microalgal growth. In this research, vanillic acid present in the hydrolysate of agricultural waste, was applied to the cultivation of unicellular microalga Euglena gracilis. At the optimal dosage of 800 mg L -1 vanillic acid, biomass yield at treatment increased 2.08-fold. Correspondingly, the content of chlorophyll a and carotenoids was 3.48 and 2.69 fold than of the control ground, respectively. Increased in cell aspect ratio demonstrated that the alga was more active after vanillic acid treatment. Furthermore, relative lipid and carbohydrate content were analyzed using Fourier transform infrared spectroscopy, the result showed that vanillic acid increased the lipid content in algal cells without sacrificing biomass, which would be a promising way for future biofuel production., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Two of the most important Mexican plant-foods are tomato (Solanum lycopersicum L.) and husk tomato (Physalis ixocarpa Brot.). In this study three objectives were followed: i) to evaluate the bioaccessible phenolic compounds (PC) in T and HT during upper gastrointestinal digestion, ii) to in vitro ferment the indigestible fractions of the samples to evaluate the short-chain fatty acids (SCFA) production, iii) the microbial metabolites, bioconverted PC and volatile organic compounds (VOCs) generated during the fermentation. Vanillic acid was the most bioaccessible PC and after 48 h, 3-hydroxyphenylacetic acid was the most abundant microbial metabolite identified in both samples. The identification of VOCs belonging to terpenes (and derivatives) group in T and HT can be product of the microbial metabolism of carotenoids. The study shows new knowledge of the in vitro intestinal digestion and fermentation of T and HT final compounds with biological potential which should be evaluated in further studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase. Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of CO from OCH-vanillate. The CO evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NHNO) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation. A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed. [ABSTRACT FROM AUTHOR]

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released CO from COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi. [ABSTRACT FROM AUTHOR]

The developmental period is critical in delineating plastic response to internal and external events. However, neurobehavioural effects of global cerebral ischemia (GCI) in the maturing brain remain largely unknown. This study characterised the effects of GCI experienced at puberty on adulthood (1) hippocampus CA1 neuronal damage, (2) cognitive and emotional impairments, and (3) glucocorticoid receptor (GR) expression. Effects of adolescent exposure to the phenol vanillic acid (VA) on post-ischemic outcomes were also determined. Male Long Evans rats (n = 35) were supplemented for 21 consecutive days (postnatal days 33-53) with VA (91 mg/kg) or nut paste vehicle (control) prior to a 10-min GCI or sham surgery. As adults, rats were tested in the Open Field Test (OFT), Elevated-Plus Maze (EPM), and Barnes Maze (BM). GR expression was determined in the basolateral amygdala (BLA), CA1, and paraventricular nucleus (PVN), and brain injury assessed via CA1 neuronal density. Adolescent GCI exposure induced extensive hippocampal CA1 injury, which was not prevented by VA supplementation. Behaviourally, GCI increased EPM exploration while having no impact on spatial memory. VA intake increased OFT peripheral exploration. Notably, while no delayed changes in CA1 and PVN GR immunoreactivity were noted, both treatments separately increased BLA GR expression when compared with sham-nut paste rats. Age at GCI occurrence plays a critical role on post-ischemic impairments. The observation of minimal functional impairments despite important CA1 neuronal damage supports use of compensatory mechanisms. Our findings also show daily VA supplementation during adolescence to have no protective effects on post-ischemic outcomes, contrasting adult intake., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved., Results: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds., Conclusions: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories., (© 2021. The Author(s).)

A novel gene cluster involved in the degradation of lignin-derived monoaromatics such as p -hydroxybenzoate, vanillate, and ferulate has been identified in the thermophilic nitrate reducer Thermus oshimai JL-2. Based on conserved domain analyses and metabolic pathway mapping, the cluster was classified into upper- and peripheral-pathway operons. The upper-pathway genes, responsible for the degradation of p -hydroxybenzoate and vanillate, are located on a 0.27-Mb plasmid, whereas the peripheral-pathway genes, responsible for the transformation of ferulate, are spread throughout the plasmid and the chromosome. In addition, a lower-pathway operon was also identified in the plasmid that corresponds to the meta- cleavage pathway of catechol. Spectrophotometric and gene induction data suggest that the upper and lower operons are induced by p -hydroxybenzoate, which the strain can degrade completely within 4 days of incubation, whereas the peripheral genes are expressed constitutively. The upper degradation pathway follows a less common route, proceeding via the decarboxylation of protocatechuate to form catechol, and involves a novel thermostable γ-carboxymuconolactone decarboxylase homolog, identified as protocatechuate decarboxylase based on gene deletion experiments. This gene cluster is conserved in only a few members of the Thermales and shows traces of vertical expansion of catabolic pathways in these organisms toward lignoaromatics. IMPORTANCE High-temperature steam treatment of lignocellulosic biomass during the extraction of cellulose and hemicellulose fractions leads to the release of a wide array of lignin-derived aromatics into the natural ecosystem, some of which can have detrimental effects on the environment. Not only will identifying organisms capable of using such aromatics aid in environmental cleanup, but thermostable enzymes, if characterized, can also be used for efficient lignin valorization. However, no thermophilic lignin degraders have been reported thus far. The present study reports T. oshimai JL-2 as a thermophilic bacterium with the potential to use lignin-derived aromatics. The identification of a novel thermostable protocatechuate decarboxylase gene in the strain further adds to its significance, as such an enzyme can be efficiently used in the biosynthesis of cis , cis -muconate, an important intermediate in the commercial production of plastics., (Copyright © 2021 American Society for Microbiology.)

Streptomyces tunisiensis DSM 42037 exhibited growth capacity on a minimum medium containing 1% barley bran. This peculiar strain released 83.5% of total ferulic acid present in barley bran after 5 days of incubation and the highest amount of released ferulic acid (19 mg/L) was observed on the 3rd day of incubation. The concentrated supernatant of S. tunisiensis also released ferulic acid from the parietal arabinoxylan complex of barley bran. This strain was able to convert the free ferulic acid into 4-vinyl guaiacol (14 mg/L) and acetovanillone (12 mg/L) at molar yield of 97% and 83% respectively. The biotransformation products were successively purified by preparative thin layer and silica gel column chromatography followed by HPLC and identified by 1 H nuclear magnetic resonance. Streptomyces tunisiensis DSM 42037 could have potential applications in the food, pharmaceutical and cosmetic industries thanks to its ability in biotransforming ferulic acid into 4-vinyl guaiacol and acetovanillone.

Non-alcoholic fatty liver disease (NAFLD), a growing health issue around the world, is defined as the presence of steatosis in the liver without any other detectable byproducts such as alcohol consumption, which includes a wide spectrum of pathologies, such as steatohepatitis, cirrhosis, and hepatocellular carcinoma. A growing body of evidence indicates that the reduction in the 5' adenosine monophosphate-activated protein kinase (AMPK) activity, which could be activated by the consumption of the drugs, hormones, cytokines, and dietary restriction, is related to some metabolic disorders such as obesity, diabetes, PCOS, and NAFLD. Vanillic acid (VA), as an anti-inflammatory, anti-oxidative, anti-angiogenic and anti-metastatic factor, has protective effects on the liver as in two animal models of liver damage, it reduces serum levels of transaminases, inflammatory cytokines, and the accumulation of collagen in the liver and also prevents liver fibrosis. Besides, it decreases body and adipose tissue weight in a mice model of obesity and, similar to the liver tissue, diminishes adipogenesis through the activation of AMPK. It has been reported that VA can target almost all of the metabolic abnormalities of NAFLD, such as hepatic steatosis, inflammation, and hepatic injury, at least partially through the activation of AMPK. Therefore, in this review, we will discuss the possible and hypothetical roles of VA in NAFLD, with a special focus on AMPK., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Genetic modifications of living organisms and proteins are made possible by a catalogue of molecular and synthetic biology tools, yet proper screening assays for genetic variants of interest continue to lag behind. Synthetic growth-coupling (GC) of enzyme activities offers a simple, inexpensive way to track such improvements. In this follow-up study we present the optimization of a recently established GC design for screening of heterologous methyltransferases (MTases) and related pathways in the yeast Saccharomyces cerevisiae . Specifically, upon testing different media compositions and genetic backgrounds, improved GC of different heterologous MTase activities is obtained. Furthermore, we demonstrate the strength of the system by screening a library of catechol O-MTase variants converting protocatechuic acid into vanillic acid. We demonstrated high correlation ( R 2 = 0.775) between vanillic acid and cell density as a proxy for MTase activity. We envision that the improved MTase GC can aid evolution-guided optimization of biobased production processes for methylated compounds with yeast in the future.

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA , ligM , and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX , located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes ( ferBA ), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA -SLG&#95;25010- desA gene cluster consists of the ferBA operon and the SLG&#95;25010- desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG&#95;25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX. IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA , encoding syringate O -demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB ), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6., (Copyright © 2020 American Society for Microbiology.)

This study aims to investigate the effects of fermentation on the phenolic components and their bioaccessibility in extruded brown rice (EBR). The saccharified solution of EBR (SS-EBR) depicted higher phenolics when fermented by single or co-culture of Lactobacillusplantarum, Lactobacillus fermentum and Saccharomyces cerevisiae for 24 h at 37 °C. The co-culture fermented SS-EBR more significantly enhanced free, conjugated and bound phenolics and flavonoids with total increment of 93.3% and 61.3%, respectively. Fermentation changed the contents and compositions of phenolics in each fraction with more than 10-fold increase in vanillic acid and quercetin contents. Ferulic, p-cumaric and chlorogenic acids were increased by 83.5%, 52.2% and 113.4%, respectively, while kaempferol and cinnamic acid were found only in fermented SS-EBR. Fermentation also improved the oxygen radical absorption capacity (ORAC) and the bioaccessible phenolics in SS-EBR. Hence, the co-culture fermented SS-EBR, can be utilized as a functional supplement to provide more bioaccessible antioxidants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

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., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli , the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter P vanCC was compared to the previously described rhamnose-inducible P rha , the anhydrotetracycline-inducible P L03 , and the Co 2+ -inducible P coaT . We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.

Aim: The present investigation was aimed at isolating and identifying bacterial strains from cured vanilla beans. Additionally, the study focused on evaluating bacterial processes pertaining to the aromatic compounds production (ACP)., Methods and Results: Three bacteria were isolated from Vanilla planifolia beans, previously subjected to the curing process. According to morphological, biochemical and 16S rRNA analysis, the strains were identified as Citrobacter sp., Enterobacter sp. and Pseudomonas sp. The polygalacturonase activity (PGA) was determined using the drop, cup-plate and DNS methods. Aromatic compounds production was analysed by cup-plate method using FA as substrate and quantified by high performance liquid chromatography (ppm), the functional groups of vanillic acid (VA) were identified by FT-IR and the aromatic compounds (AC) resistance was determined and reported as minimum inhibitory concentration. Citrobacter sp., Enterobacter sp. and Pseudomonas showed PGA (70·31 ± 364, 76·07 ± 12·47 and 51 ± 10·92 U ml -1 respectively), were producers of VA (3·23 ± 0·49, 324 ± 41 and 265·99 ± 11·61 ppm respectively) and were resistant to AC., Conclusions: The Gram-negative bacteria isolated from V. planifolia beans were responsible for ACP., Significance and Impact of the Study: This is the first evidence for the role of Gram-negative bacterial isolates from cured Mexican V. planifolia beans in the process related to ACP., (© 2019 The Society for Applied Microbiology.)

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. However, its recalcitrance and toxicity poses a challenge for ferulic acid-to-bioproducts bioconversion. Here, we develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-λ-Red system with pyrF as a selection marker, which maintains cell viability and genetic stability, increases mutation efficiency, and simplifies genetic manipulation. Via this method, four functional modules, comprised of nine genes involved in ferulic acid catabolism and polyhydroxyalkanoate biosynthesis, were integrated into the genome, generating the KTc9n20 strain. After metabolic engineering and optimization of C/N ratio, polyhydroxyalkanoate production was increased to ~270 mg/L, coupled with ~20 mM ferulic acid consumption. This study not only establishes a simple and efficient genome editing strategy, but also offers an encouraging example of how to apply this method to improve microbial aromatic compound bioconversion.

Employing isotope incubation studies, the biosynthetic pathway leading to a series of benzylic derivatives was elucidated in the fermentation broth of the edible mushroom Ischnoderma resinosum (P. Karst). Twenty-six hydroxy- and methoxy- benzylic derivatives were screened by gas chromatography-mass spectrometry (GC-MS) of which 13 were detected in the culture media. Results from the isotope incubation studies showed the transformation of both benzyl alcohol and benzoic acid into benzaldehyde. Benzaldehyde was then converted into 4-methoxybenzaldehyde via hydroxylation and subsequent methylation of the 4-C position. The resulting 4-methoxybenzaldehyde was then hydroxylated in the 3-C position followed by methylation into 3,4-dimethoxybenzaldehyde. Based on these findings, a novel metabolic scheme for the biosynthesis of benzylic derivatives in I. resinosum was proposed. The knowledge of the biosynthetic pathway was utilized to produce 4-hydroxy-3-methoxybenzaldehyde (vanillin) from 4-hydroxy-3-methoxybenzoic acid (vanillic acid). This is the first report to elucidate the biosynthetic pathway of benzyl derivatives and production of vanillin from I. resinosum .

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the van AB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95 ± 1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87 ± 0.38% molar yield. With fermentation time of less than 24 h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.

Vanillate and syringate are major intermediate metabolites generated during the microbial degradation of lignin. In Sphingobium sp. SYK-6, vanillate is O demethylated to protocatechuate by LigM; protocatechuate is then catabolized via the protocatechuate 4,5-cleavage pathway. Syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and then gallate is subjected to ring cleavage by DesB. Here, we investigated the transcriptional regulation of desA, ligM, and desB involved in vanillate and syringate catabolism. Quantitative reverse transcription-PCR analyses indicated that the transcription of these genes was induced 5.8-37-fold in the presence of vanillate and syringate. A MarR-type transcriptional regulator, SLG&#95;12870 (desR), was identified as the gene whose product bound to the desB promoter region. Analysis of a desR mutant indicated that the transcription of desB, ligM, and desR is negatively regulated by DesR. Purified DesR bound to the upstream regions of desB, ligM, and desR, and the inverted repeat sequences similar to each other in these regions were suggested to be essential for DNA binding of DesR. Vanillate and syringate inhibited DNA binding of DesR, indicating that these compounds are effector molecules of DesR. The transcription of desA was found to be regulated by an as-yet unidentified regulator.

Zhenjiang aromatic vinegar (ZAV) is one of the famous Chinese vinegars, which contains various physicochemical and bioactive compositions. In the present study, physicochemical properties and total antioxidant activity were detected in ZAV samples. The correlation between of organic acids, amino acids, phenolic compounds, and the antioxidant activity of ZAV were explored. The results showed that contents of total acids, soluble solids, reducing sugar and total antioxidant activity in ZAV were increased with aging time, and those in ZAV-5 were the highest. Organic acids and amino acids exhibited weak antioxidant activity, while phenolic compounds had higher antioxidant ability. In addition, amino acids had synergistic effect on the antioxidant activity of phenolic compounds, whereas organic acids inhibited the antioxidant activity of phenolic compounds. Moreover, it was found that phenolic compounds including catechin, vanillic acid and syringic acid showed higher contribution rates to antioxidant activities of mixed phenolic compounds. In conclusion, these findings would provide references to control the antioxidant characteristic of vinegar through regulating the main compositions, and further improve the quality of vinegar production.

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (T m ) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L -1 of vanillin. The space time yield of 0.029 g L -1  h -1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L -1 . In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rhodococcus opacus PD630 ( R. opacus ) is a nonmodel, Gram-positive bacterium that holds promise as a biological catalyst for the conversion of lignocellulosic biomass to value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. However, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus . Furthermore, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to the construction of AND and NAND genetic logic gates. Additionally, three isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoters were created by inserting LacI binding sites into newly characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Finally, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus , enabling complex and tunable gene regulation in this promising nonmodel host for bioproduction.

Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning process of vegetables and fruits. A series of veratric acid derivatives containing benzylidene-hydrazine moieties with different substitutions were synthesized and their inhibitory effect on mushroom tyrosinase and free radical scavenging activity were evaluated. The results indicated that N'-(4-chlorobenzylidene)-3,4-dimethoxybenzohydrazide (D5) and N'-(2,3-dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D12) showed the highest tyrosinase inhibitory activity with IC 50 values of 19.72 ± 1.84 and 20.63 ± 0.79 μM, respectively, that were comparable with the IC 50 value of kojic acid (19.08 ± 1.21 μM). D12 was also a potent radical scavenger with EC 50 value of 0.0097 ± 0.0011 mM. The free radical scavenging activity of D12 was comparable with the standard quercetin. The inhibition kinetic analyzed by Lineweaver-Burk plots revealed that compound D5 was a competitive tyrosinase inhibitor. Molecular docking study was carried out for the derivatives demonstrating tyrosinase inhibitory activity. D5 and D12 possessed the most negative estimated free energies of binding in mushroom tyrosinase active site. Therefore, D5 and D12 could be introduced as potent tyrosinase inhibitors that might be promising leads in medicine, cosmetics and food industry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In situ detoxification of lignocellulose-derived microbial inhibitory compounds is an economical strategy for the fermentation of lignocellulose-derived sugars to fuels and chemicals. In this study, we investigated homologous integration and constitutive expression of Cbei&#95;3974 and Cbei&#95;3904, which encode aldo-keto reductase and previously annotated short chain dehydrogenase/reductase, respectively, in Clostridium beijerinckii NCIMB 8052 (Cb), resulting in two strains: Cb&#95;3974 and Cb&#95;3904. Expression of Cbei&#95;3974 led to 2-fold increase in furfural detoxification relative to Cb&#95;3904 and Cb&#95;wild type. Correspondingly, butanol production was up to 1.2-fold greater in furfural-challenged cultures of Cb&#95;3974 relative to Cb&#95;3904 and Cb&#95;wild type. With 4-hydroxybezaldehyde and syringaldehyde supplementation, Cb&#95;3974 showed up to 2.4-fold increase in butanol concentration when compared to Cb&#95;3904 and Cb&#95;wild type. Syringic and vanillic acids were considerably less deleterious to all three strains of Cb tested. Overall, Cb&#95;3974 showed greater tolerance to furfural, 4-hydroxybezaldehyde, and syringaldehyde with improved capacity for butanol production. Hence, development of Cb&#95;3974 represents a significant progress towards engineering solventogenic Clostridium species that are tolerant to lignocellulosic biomass hydrolysates as substrates for ABE fermentation.

This work aimed to study the phenolic acid metabolism of sourdough lactic acid bacteria (LAB) in laboratory media, and in sourdough fermentation with single cultures and in co-fermentations. Lactobacilli were selected from isolates obtained from 35 sourdough samples. Isolates (114 strains) were screened for phenolic acid decarboxylase gene pdc and EPS production. Ferulic acid metabolism of the 18 pdc positive strains was evaluated in mMRS; all pcd positive strains converted ferulic acid by decarboxylation and/or reduction. Single whole wheat and rye malt dough fermentation fermented with lactobacilli or yeasts were characterized with respect to free, conjugated, or bound phenolic acids. Concentrations of free, conjugated, or bound phenolic acids were not altered substantially in chemically acidified sourdoughs, or in yeast fermented doughs. L. plantarum metabolized free ferulic acid in wheat and rye malt sourdoughs; L. hammesii DSM 16381 metabolized syringic and vanillic acids and reduced levels of bound ferulic acid in wheat sourdoughs. Co-fermentation of L. hammesii and L. plantarum achieved release of bound ferulic acid and conversion of the resultant free ferulic acid to dihydroferulic acid and volatile metabolites. Phenolic acid metabolism in sourdoughs was enhanced by co-fermentation with strains exhibiting complementary metabolic activities. Results may enable improvement of bread quality by targeted conversion of phenolic acids during sourdough fermentation., (Copyright © 2018. Published by Elsevier Ltd.)

The gut microbiome supplies essential metabolites such as short-chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota-derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN-M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose- and insulin-treated myotubes. One of the most effective was isovanillic acid 3 -O-sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3 -O-glucoside, a major compound in berry fruits. IVAS stimulated a dose-dependent increase in glucose transport through glucose transporter GLUT4- and PI3K-dependent mechanisms. IVAS also up-regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.-Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism.

Furanic and phenolic compounds are problematic compounds resulting from the pretreatment of lignocellulosic biomass for biofuel production. Microbial electrolysis cell (MEC) is a promising technology to convert furanic and phenolic compounds to renewable H 2 . The objective of the research presented here was to elucidate the processes and electron equivalents flow during the conversion of two furanic (furfural, FF; 5-hydroxymethyl furfural, HMF) and three phenolic (syringic acid, SA; vanillic acid, VA; 4-hydroxybenzoic acid, HBA) compounds in the MEC bioanode. Cyclic voltammograms of the bioanode demonstrated that purely electrochemical reactions in the biofilm attached to the electrode were negligible. Instead, microbial reactions related to the biotransformation of the five parent compounds (i.e., fermentation followed by exoelectrogenesis) were the primary processes resulting in the electron equivalents flow in the MEC bioanode. A mass-based framework of substrate utilization and electron flow was developed to quantify the distribution of the electron equivalents among the bioanode processes, including biomass growth for each of the five parent compounds. Using input parameters of anode efficiency and biomass observed yield coefficients, it was estimated that more than 50% of the SA, FF, and HMF electron equivalents were converted to current. In contrast, only 12 and 9% of VA and HBA electron equivalents, respectively, resulted in current production, while 76 and 79% remained as fermentation end products not further utilized in exoelectrogenesis. For all five compounds, it was estimated that 10% of the initially added electron equivalents were used for fermentative biomass synthesis, while 2 to 13% were used for exoelectrogenic biomass synthesis. The proposed mass-based framework provides a foundation for the simulation of bioanode processes to guide the optimization of MECs converting biomass-derived waste streams to renewable H 2 .

Background: Streptomyces sp. NL15-2K, previously isolated from the forest soil, features an extensive catabolic network for lignin-derived aromatic compounds, including pathways transforming ferulic acid to vanillin, vanillic acid, and protocatechuic acid. To successfully use Streptomyces sp. NL15-2K as a biocatalyst for vanillin production, it is necessary to characterize the vanillin dehydrogenase (VDH) that degrades the produced vanillin to vanillic acid, as well as the gene encoding this enzyme. Here, we cloned the VDH-encoding gene (vdh) from strain NL15-2K and comprehensively characterized its gene product., Results: The vdh open reading frame contains 1488 bp and encodes a 496-amino-acid protein with a calculated molecular mass of 51,705 Da. Whereas the apparent native molecular mass of recombinant VDH was estimated to be 214 kDa by gel filtration analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a subunit molecular mass of ca. 56 kDa, indicating that VDH is a homotetramer. The recombinant enzyme showed optimal activity at 45 °C and pH 9.5. The VDH substrate specificity followed this order: vanillin (100%) > protocatechualdehyde (91%) > benzaldehyde (79%) > p-hydroxybenzaldehyde (56%) > isovanillin (49%) ≈ salicylaldehyde (48%) > anisaldehyde (15%) ≈ veratraldehyde (12%). Although peptide mass fingerprinting and BLAST searches indicated that this enzyme is a salicylaldehyde dehydrogenase (SALDH), the determined kinetic parameters clearly demonstrated that the enzyme is a vanillin dehydrogenase. Lastly, phylogenetic analysis revealed that VDH from Streptomyces sp. NL15-2K forms an independent branch in the phylogenetic tree and, hence, is evolutionarily distinct from other VDHs and SALDHs whose activities have been confirmed experimentally., Conclusions: Our findings not only enhance the understanding of the enzymatic properties of VDH and the characteristics of its amino acid sequence, but also contribute to the development of Streptomyces sp. NL15-2K into a biocatalyst for the biotransformation of ferulic acid to vanillin.