New insights into the anti-inflammatory and anti-melanoma mechanisms of action of azelaic acid and other Fusarium solani metabolites via in vitro and in silico studies.

Metabolites exploration of the ethyl acetate extract of Fusarium solani culture broth that was isolated from Euphorbia tirucalli root afforded five compounds; 4-hydroxybenzaldehyde (1), 4-hydroxybenzoic acid (2), tyrosol (3), azelaic acid (4), malic acid (5), and fusaric acid (6). Fungal extract as well as its metabolites were evaluated for their anti-inflammatory and anti-hyperpigmentation potential via in vitro cyclooxygenases and tyrosinase inhibition assays, respectively. Azelaic acid (4) exhibited powerful and selective COX-2 inhibition followed by fusaric acid (6) with IC50 values (2.21 ± 0.06 and 4.81 ± 0.14 µM, respectively). As well, azelaic acid (4) had the most impressive tyrosinase inhibitory effect with IC50 value of 8.75 ± 0.18 µM compared to kojic acid (IC50 = 9.27 ± 0.19 µM). Exclusive computational studies of azelaic acid and fusaric acid with COX-2 were in good accord with the in vitro results. Interestingly, this is the first time to investigate and report the potential of compounds 3-6 to inhibit cyclooxygenase enzymes. One of the most invasive forms of skin cancer is melanoma, a molecular docking study using a set of enzymes related to melanoma suggested pirin to be therapeutic target for azelaic acid and fusaric acid as a plausible mechanism for their anti-melanoma activity.

Tetracosanoic acids produced by 3-ketoacyl-CoA synthase 17 are required for synthesizing seed coat suberin in Arabidopsis.

Very long-chain fatty acids (VLCFAs) are precursors for the synthesis of membrane lipids, cuticular waxes, suberins, and storage oils in plants. 3-Ketoacyl CoA synthase (KCS) catalyzes the condensation of C2 units from malonyl-CoA to acyl-CoA, the first rate-limiting step in VLCFA synthesis. In this study, we revealed that Arabidopsis KCS17 catalyzes the elongation of C22-C24 VLCFAs required for synthesizing seed coat suberin. Histochemical analysis of Arabidopsis plants expressing GUS (ß-glucuronidase) under the control of the KCS17 promoter revealed predominant GUS expression in seed coats, petals, stigma, and developing pollen. The expression of KCS17:eYFP (enhanced yellow fluorescent protein) driven by the KCS17 promoter was observed in the outer integument1 of Arabidopsis seed coats. The KCS17:eYFP signal was detected in the endoplasmic reticulum of tobacco epidermal cells. The levels of C22 VLCFAs and their derivatives, primary alcohols, α,ω-alkane diols, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids increased by ~2-fold, but those of C24 VLCFAs, ω-hydroxy fatty acids, and α,ω-dicarboxylic acids were reduced by half in kcs17-1 and kcs17-2 seed coats relative to the wild type (WT). The seed coat of kcs17 displayed decreased autofluorescence under UV and increased permeability to tetrazolium salt compared with the WT. Seed germination and seedling establishment of kcs17 were more delayed by salt and osmotic stress treatments than the WT. KCS17 formed homo- and hetero-interactions with KCR1, PAS2, and ECR, but not with PAS1. Therefore, KCS17-mediated VLCFA synthesis is required for suberin layer formation in Arabidopsis seed coats.

Glucose protects the cell membrane, Na+/K+-ATPase, nucleic acids, and proteins in Bacillus subtilis spores under high pressure thermal sterilization.

The extreme resistance of bacterial spores to sterilization makes them a major concern to the food industry and consumers. In this study, the effect of glucose on the inactivation of Bacillus subtilis spores by high pressure thermal sterilization (HPTS) was evaluated. The results showed that the protective effects of glucose increased with the increase in its concentration. Compared with the HPTS control (no addition of glucose), the activity of Na+/K+-ATPase was increased, the leakage of proteins and the release of 2,6-pyridine dicarboxylic acid (DPA) was decreased, and the vibrational strength of the functional group P = O was reduced by the addition of glucose. At the same time, glucose treatment increased the content of α-helix by 6%-22%, while decreased the random coil content by 5%-13% of the cellular protein. In conclusion, the addition of glucose protected the cell membrane, Na+/K+-ATPase, cellular nucleic acids and proteins of B. subtilis under HPTS treatment.

Organic acids and glucose prime late-stage fungal biotrophy in maize.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis. These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

The Emerging Plasticizer Alternative DINCH and Its Metabolite MINCH Induce Oxidative Stress and Enhance Inflammatory Responses in Human THP-1 Macrophages.

The use of the plasticizer bis(2-ethylhexyl)phthalate (DEHP) and other plasticizers in the manufacture of plastic products has been restricted due to adverse health outcomes such as obesity, metabolic syndrome, and asthma, for which inflammation has been described to be a driving factor. The emerging alternative plasticizer 1,2-cyclohexanedioic acid diisononyl ester (DINCH) still lacks information regarding its potential effects on the immune system. Here, we investigated the effects of DINCH and its naturally occurring metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to 10 nM-10 µM DINCH or MINCH for 4 h, 16 h, and 24 h. To decipher the underlying mechanism of action, we applied an untargeted proteomic approach that revealed xenobiotic-induced activation of immune-related pathways such as the nuclear factor κB (NF-κB) signaling pathway. Key drivers were associated with oxidative stress, mitochondrial dysfunction, DNA damage repair, apoptosis, and autophagy. We verified increased reactive oxygen species (ROS) leading to cellular damage, NF-κB activation, and subsequent TNF and IL-1ß release, even at low nM concentrations. Taken together, DINCH and MINCH induced cellular stress and pro-inflammatory effects in macrophages, which may lead to adverse health effects.

Structure-Activity Relationship Studies of Coumarin-like Diacid Derivatives as Human G Protein-Coupled Receptor-35 (hGPR35) Agonists and a Consequent New Design Principle.

A series of coumarin-like diacid derivatives were designed and synthesized as novel agonists of human G-protein-coupled receptor 35 (hGPR35). Active compounds were characterized to possess one acidic group on both sides of a fused tricyclic aromatic scaffold. Most of them functioned as full agonists selective to hGPR35 and exhibited excellent potency at low nanomolar concentrations. Substitution on the middle ring of the scaffold could effectively regulate compound potency. Structure-activity relationship studies and docking simulation indicated that compounds that carried two acidic groups with a proper special distance and attached to a rigid aromatic scaffold would most likely show a potent agonistic activity on hGPR35. Following this principle, we screened a list of known compounds and some were found to be potent GPR35 agonists, and compound 24 even had an EC50 of 8 nM. Particularly, a dietary supplement pyrroloquinoline quinone (PQQ) was identified as a potent agonist (EC50 = 71.4 nM). To some extent, this principle provides a general strategy to design and recognize GPR35 agonists.

Biomonitoring of emerging DINCH metabolites in pregnant women in charleston, SC: 2011-2014.

Due to the mounting evidence that phthalates, specifically di-2-ethylhexyl phthalate and dibutyl phthalate, produce adverse endocrine effects in humans and wildlife, the use of other chemicals as replacements has increased. One of the most commonly encountered phthalate replacements is di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH). Currently, little is known about the prevalence of human exposure, bioactivity, and endocrine disrupting potential of DINCH. We sampled urine from 100 pregnant women during the second trimester of pregnancy living in Charleston, SC between 2011 and 2014 and measured the following DINCH metabolites by LC-MS/MS: cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (OH-MINCH), cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (oxo-MINCH), and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (cx-MINCH). These metabolites were also tested on human estrogen receptor alpha and progesterone receptor beta transactivation assays in vitro. OH-MINCH was detected in 98% of urine samples. The specific gravity-adjusted median (interquartile range) OH-MINCH concentration was 0.20 (0.25) ng/mL, and concentrations were significantly higher in African American women compared to Caucasian women (p = 0.01). DINCH metabolite concentrations were consistent between years, and they did not exhibit estrogenic or progestogenic activity in vitro. Human exposure to these emerging compounds should continue to be monitored, especially in vulnerable populations, to ensure the replacement of phthalates by DINCH is not a case of regrettable substitution.

Engineering Stable Pseudomonas putida S12 by CRISPR for 2,5-Furandicarboxylic Acid (FDCA) Production.

FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). Pseudomonas putida S12 is promising for FDCA production, but generating stable P. putida S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable P. putida S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO2 and CaCO3 to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating HMFH and HMFT1 (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable P. putida S12 strains for FDCA production.

A Novel 2,5-Furandicarboxylic Acid Biosynthesis Route from Biomass-Derived 5-Hydroxymethylfurfural Based on the Consecutive Enzyme Reactions.

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block as a green alternative to petroleum-based terephthalate in polymer production. Most of FDCA is produced by the oxidation of 5-hydroxymethylfurfural (HMF), which is derived from hexose. Although the chemical conversion is widely applied, the biocatalytic conversion is expected due to the relatively mild condition and fewer toxic chemicals consumption. However, it's difficult to catalyze the conversion of HMF to FDCA by a single enzyme. Here, a newly enzymatic cascade reaction process was introduced with a yield of 94.0% by the combination of 5-hydroxymethylfurfural oxidase (HMFO) and lipase. Briefly, a flavine adenosine dinucleotide independent (FAD-independent) HMFO of Methylovorus sp. MP688 was used to convert HMF to 2,5-diformylfuran (DFF) and 5-formylfuroic acid (FFA), which consecutively transformed to FDCA by a lipase Novozym 435. To facilitate the purification, a coupled alkali precipitation was developed to recover FDCA from organic solvent with an improved purity from 84.4 to 99.0% and recovery of 78.1%. This work will help to construct the green biorefinery route for the bulk FDCA from biomass by enzymes.

Non-invasive urinary metabolomics reveals metabolic profiling of polycystic ovary syndrome and its subtypes.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder, which affects 4-10 % women of reproductive age. Though accumulating scientific evidence, its pathogenesis remains unclear. In the current study, metabolic profiling as well as diagnostic biomarkers for different phenotypes of PCOS was investigated using non-invasive urinary GCMS based metabolomics. A total of 371 subjects were recruited for the study. They constituted the following groups: healthy women, those with hyperandrogenism (HA), women with insulin-resistance (IR) in PCOS. Two cross-comparisons with PCOS were performed to characterize metabolic disturbances. A total of 23 differential metabolites were found. The altered metabolic pathways included glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions, and citrate cycle and butanoate metabolism. For differential diagnosis, a panel consisting of 9 biomarkers was found from the comparison of PCOS from healthy subjects. The area under the receiver operating characteristic (ROC) curve (AUC) was 0.8461 in the discovery phase. Predictive value of 89.17 % was found in the validation set. Besides, a panel of 8 biomarkers was discovered from PCOS with HA vs IR. The AUC for 8-biomarker panel was 0.8363, and a panel of clinical markers (homeostasis model assessment-insulin resistance and free androgen index) had 0.8327 in AUC. While these metabolites combined with clinical markers reached 0.9065 in AUC from the discovery phase, and 93.18 % in predictive value from the validation set. The result showed that differences of small-molecule metabolites in urine may reflect underlying pathogenesis of PCOS and serve as biomarkers for complementary diagnosis of the different phenotypes of PCOS.

Effect of organic acid on cyanidin-3-O-glucoside oxidation mediated by iron in model Chinese bayberry wine.

Cyanidin-3-O-glucoside (C3G) is principal anthocyanin in Chinese bayberry wine and its degradation is main problem with respect to wine color. Effect of five organic acids existing in bayberry wine on C3G oxidation mediated by iron was investigated in model wine. Fe(II) oxidation was found to follow a decreasing order in oxalate > citrate > tartrate > malate model wine whereas it hardly occurred in succinate model wine. The C3G oxidation mediated by iron followed an increasing order in citrate > oxalate > succinate > malate > tartrate model wine. More degradation products were observed in succinate, malate and tartrate model wine than in citrate and oxalate model wine. C3G degradation mediated by Fe(III) was faster than that mediated by Fe(II) in oxalate, succinate, malate, and tartrate model wine, but not in citrate model wine. C3G oxidation mediated by iron is probably not main mechanism of anthocyanin degradation in bayberry wine.

Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae.

Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion-sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.

Benzenedicarboxylic acid upregulates O48814 and Q9FJQ8 for improved nutritional contents of tomato and low risk of fungal attack.

BACKGROUND: Tomato is an important food item and a cocktail of phytonutrients. In the current study, metabolites from a non-pathogenic fungal species Penicillium oxalicum have been exploited to obtain nutritionally augmented tomato fruits from the plants to better withstand against Alternaria alternata infection. RESULTS: Initially, bioactivity-guided assay and chromatographic analyses identified the bioactive metabolites of P. oxalicum [benzenedicarboxylic acid (BDA) and benzimidazole]. Then, ≥3 times elevated quantities of vitamins and other nutritional elements (protein, fat, fibers, and carbohydrates) were achieved by the foliar application of BDA. The maximum increase (625.81%) was recorded in riboflavin contents; however, thiamine showed the second highest enhancement (542.86%). Plant metabolites analysis revealed that jasmonic acid contents were boosted 121.53% to significantly enhance guaiacyl lignin defenses along with the reduction in coumarin contents. The protein profile analysis explored three most actively responding protein species toward BDA applications, (i) palmitoyltransferase protein Q9FLM3; (ii) serine/threonine-protein kinase O48814; and (iii) E3 ubiquitin-protein ligase Q9FJQ8. The O48814 improved plant defenses; whereas, Q9FJQ8 protein was negatively regulating cysteine-type endopeptidase activity and assisted plant to resist schedule alterations. Tomato cultivar with more active innate metabolism was found to be more responsive toward BDA. Furthermore, the bioactive compounds were enriched by using the two-step extraction method of ethyl acetate and chloroform, respectively. CONCLUSION: Penicillium oxalicum a non-pathogenic fungal species, produced BDA, induced nutritional contents in tomato and protected it against Alternaria alternata. The current study is the first report on the bioactivity of BDA and benzimidazole concerning the nutritional enhancement and plant defense improvement. © 2019 Society of Chemical Industry.

Biotransformation of dicarboxylic acids from vegetable oil-derived sources: current methods and suggestions for improvement.

Sustainable manufacture of dicarboxylic acids (DCAs), which are used as raw materials for multiple commercial products, has been an area of considerable research interest in recent years. Traditional chemical-based manufacture of DCAs suffers from limitations such as harsh operational conditions and generation of hazardous by-products. Microbiological methods involving DCA production depend on the capability of alkane-assimilating microorganisms, particularly α, ω-oxidation, to metabolize alkanes. Alkanes are still used as the most common substrates for this method, but the use of renewable resources, such as vegetable oil-derived fatty acid methyl esters (FAMEs), offers multiple advantages for the sustainable production of DCA. However, DCA production using FAME, unlike that using alkanes, still has low productivity and process stability, and we have attempted to identify several limiting factors that weaken the competitiveness. This review discusses the current status and suggests solutions to various obstacles to improve the biotransformation process of FAMEs.

The spent culture supernatant of Pseudomonas syringae contains azelaic acid.

BACKGROUND: Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. RESULTS: A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 µg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule. CONCLUSIONS: This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling.

Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates.

The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides, and cofactors across the mitochondrial membrane. Here, two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified, and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteine sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate, and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. AtUCP1 and AtUCP2 catalyzed a fast counterexchange transport as well as a low uniport of substrates, with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts shows changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartateout/glutamatein exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.

Investigation of the Antifatigue Effects of Korean Ginseng on Professional Athletes by Gas Chromatography-Time-of-Flight-Mass Spectrometry-Based Metabolomics.

Ginseng is usually used for alleviating fatigue. The purpose of this paper was to evaluate the regulatory effect of Korean ginseng on the metabolic pattern in professional athletes, and, further, to explore the underlying mechanism of the antifatigue effect of Korean ginseng. GC-time-of-flight-MS was used to profile serum samples from professional athletes before training and after 15 and 30 day training, and professional athletes administered with Korean ginseng in the meanwhile. Biochemical parameters of all athletes were also analyzed. For the athlete control group, strength-endurance training resulted in an elevation of creatine kinase (CK) and blood urea nitrogen (BUN), and a reduction in blood hemoglobin, and a dynamic trajectory of the metabolomic profile which were related to fatigue. Korean ginseng treatment not only lead to a marked reduction in CK and blood urea nitrogen (BUN) in serum, but also showed regulatory effects on the serum metabolic profile and restored scores plots close to normal, suggesting that the change in metabolic profiling could reflect the antifatigue effect of Korean ginseng. Furthermore, perturbed levels of 11 endogenous metabolites were regulated by Korean ginseng significantly, which might be primarily involved in lipid metabolism, energy balance, and chemical signaling. These findings suggest that metabolomics is a potential tool for the evaluation of the antifatigue effect of Korean ginseng and for the elucidation of its pharmacological mechanism.

Biotechnological production of bio-based long-chain dicarboxylic acids with oleogenious yeasts.

Long-chain α,ω-dicarboxylic acids (DCAs) are versatile chemical intermediates of industrial importance used as building blocks for the production of polymers, lubricants, or adhesives. The majority of industrial long-chain DCAs is produced from petro-chemical resources. An alternative is their biotechnological production from renewable materials like plant oil fatty acids by microbial fermentation using oleogenious yeasts. Oleogenious yeasts are natural long-chain DCA producers, which have to be genetically engineered for high-yield DCA production. Although, some commercialized fermentation processes using engineered yeasts are reported, bio-based long-chain DCAs are still far from being a mass product. Further progress in bioprocess engineering and rational strain design is necessary to advance their further commercialization. The present article reviews the basic strategies, as well as novel approaches in the strain design of oleogenious yeasts, such as the combination of traditional metabolic engineering with system biology strategies for high-yield long-chain DCA production. Therefore a detailed overview of the involved metabolic processes for the biochemical long-chain DCA synthesis is given.

Production of dodecanedioic acid via biotransformation of low cost plant-oil derivatives using Candida tropicalis.

Dodecanedioic acid (DDA) is highly useful to the chemical industry as a versatile precursor for producing the polyamide nylon-6,12, which is used for many technical applications, such as heat and chemical-resistant sheaths. However, DDA synthesis has several drawbacks, such as high energy input and cost-intensive removal of by-products. Therefore, alternative bio-based production routes are required due to increasing industrial demand for green chemicals and renewable products. Candida tropicalis converts petrochemical-based n-dodecanes to the corresponding dicarboxylic acids by targeted functionalization. To increase sustainability of the DDA production process, we tested dodecanoic acid methyl ester, which can be easily obtained from transesterification of coconut oil, in whole-cell biotransformation by C. tropicalis. By modifying selected process parameters, a final DDA concentration of 66 g/L was achieved using a highly reliable, small-scale bioreactor system. Crucial process development included a gradual pH shift, an optimized substrate feeding strategy, and monitoring the transcriptional profile.

The malate sensing two-component system MaeKR is a non-canonical class of sensory complex for C4-dicarboxylates.

Microbial colonization of different environments is enabled to a great extent by the plasticity of their sensory mechanisms, among them, the two-component signal transduction systems (TCS). Here, an example of TCS plasticity is presented: the regulation of L-malate catabolism via malic enzyme by MaeRK in Lactobacillales. MaeKR belongs to the citrate family of TCS as the Escherichia coli DcuSR system. We show that the Lactobacillus casei histidine-kinase MaeK is defective in autophosphorylation activity as it lacks a functional catalytic and ATP binding domain. The cognate response regulator MaeR was poorly phosphorylated at its phosphoacceptor Asp in vitro. This phosphorylation, however, enhanced MaeR binding in vitro to its target sites and it was required for induction of regulated genes in vivo. Elucidation of the MaeR structure revealed that response regulator dimerization is accomplished by the swapping of α4-ß5-α5 elements between two monomers, generating a phosphoacceptor competent conformation. Sequence and phylogenetic analyses showed that the MaeKR peculiarities are not exclusive to L. casei as they are shared by the rest of orthologous systems of Lactobacillales. Our results reveal MaeKR as a non-canonical TCS displaying distinctive features: a swapped response regulator and a sensor histidine kinase lacking ATP-dependent kinase activity.