Biotransformation Processes Research Articles

Biotransformation of Low-Quality Wastewater into the Production of Water of Economic Value

In recent years, many countries, especially after the increase in population and agricultural land, suffered from a freshwater shortage. This research obtained the positive role of using biotransformation processes to treat contaminated water and reuse it in agriculture. The results indicated that bioremediation of wastewater by using "bacterial consortium” has a positive effect on reducing some biological and non-biological pollutants. There was excellent potential to decrease pathogenic microbes. In addition, the release of some important compounds during the growth of these bacteria in the developing environments, such as some enzymes, amino acids (1.74-fold), organic acids (20.16-fold), gibbrillic acid (2.5-fold), indole acetic acid (1.8-fold), total carbohydrates (10.8-fold), total lipids (18.3-fold) and total phenols (1.68-fold) compared with control.

Effects of terminal electron acceptors on the biodegradation of waste motor oil using Chlorella vulgaris-Rhodococcus erythropolis consortia: Kinetic and thermodynamic windows of opportunity analysis

The Chlorella vulgaris-Rhodococcus erythropolis consortia was constructed for the biodegradation of waste motor oil (WMO), combined with thermodynamic calculations and stoichiometric analyses. The microalgae-bacteria consortium was constructed as C. vulgaris: R. erythropolis = 1:1 (biomass, cell/mL), pH = 7, 3 g/L WMO. Under the same condition, the terminal electron acceptors (TEAs) play a crucial role in the WMO biodegradation, which follows Fe3+ >SO42- > none. The biodegradation of WMO fitted well with the first-order kinetic model under experimental temperatures with different TEAs (R2 >0.98). The WMO biodegradation efficiency reached 99.2 % and 97.1 % with Fe3+ and SO42−as TEAs at 37 °C, respectively. Thermodynamic methanogenesis opportunity windows with Fe3+ as TEA are 2.72 times fold as large as those with SO42-. Microorganism metabolism equations demonstrated the viability of anabolism and catabolism on WMO. This work lays the groundwork for the implementation of WMO wastewater bioremediation and supports research into the biochemical process of WMO biotransformation.

Influence of deoxynivalenol and zearalenone on the immunohistochemical expression of oestrogen receptors and liver enzyme genes in vivo in prepubertal gilts

Deoxynivalenol (DON) and zearalenone (ZEN) are often detected in plant materials used to produce feed for pre-pubertal gilts. Daily exposure to small amounts of these mycotoxins causes subclinical conditions in pigs and affects various biological processes (e.g. mycotoxin biotransformation). The aim of this preclinical study was to evaluate the effect of low monotonic doses of DON and ZEN (12 µg/kg body weight—BW—and 40 µg/kg BW, respectively), administered alone or in combination to 36 prepubertal gilts for 42 days, on the degree of immunohistochemical expression of oestrogen receptors (ERs) in the liver and the mRNA expression of genes encoding selected liver enzymes during biotransformation processes. The level of expression of the analysed genes proves that the tested mycotoxins exhibit variable biological activity at different stages of biotransformation. The biological activity of low doses of mycotoxins determines their metabolic activity. Therefore, taking into account the impact of low doses of mycotoxins on energy-intensive processes and their endogenous metabolism, it seems that the observed situation may lead to the activation of adaptation mechanisms.

Variation in photochemical properties of dissolved black carbon during bio-transformation and iron mineral fractionation process

The excellent photochemical properties of dissolved black carbon (DBC) have been proven to be a significant contributor to the removal of organic pollutants in environment. However, the photochemical properties of DBC will inevitably be changed during biotic and abiotic processes. Herein, the structures and compositions of DBC during bio-transformation and goethite adsorption processes were comprehensively studied, and their corresponding photochemical properties were also evaluated. Bio-transformed DBC (B-DBC) contained more aromatic, high molecular weight, and phenolic substances compared with pristine DBC (P-DBC). The photodegradation of 17α-ethynylestradiol (EE2) was significantly promoted by B-DBC because of its superior capacity for producing 3DBC*. Moreover, the subsequent goethite fractionation selectively reduced the parts of components with high aromaticity and carboxylic functional groups in B-DBC. The interaction between B-DBC and goethite resulted in the release of Fe2+ into goethite-fractionated DBC (G-DBC), which induced the photodegradation mechanism of EE2 shifting from a single-electron transfer driven by 3DBC⁎ to the oxidation of •OH. This study provides valuable insights into the changes in photochemical behavior of DBC resulting from biotic or abiotic processes, and enhances our understanding of the role of DBC in the fate of organic pollutants.

The influence of the configurations of multiple-impeller on canrenone bioconversion using resting cells of Aspergillus ochraceus

Abstract 11 α-Hydroxycanrenone is a key intermediate in the synthesis of eplernone which is a drug that protects the cardiovascular system. It can be obtained by microbial transformation of canrenone using Aspergillus ochraceus. The impeller configuration has a great impact on the microbial transformation efficiency. In this study, three kinds of multiple-impeller including six-blade Rushton turbine (lower) and six-blade Rushton turbine (upper) (RT + RT), six-blade Rushton turbine (lower) and six-arrow blade turbine (upper) (RT + ABT), six-blade Rushton turbine impeller (lower) and six-blade Chemineer CD6 impeller (upper) (RT + CD6) were employed to carry out the microbial conversion process, which was investigated by experiments and computational fluid dynamic (CFD) simulations. The CFD simulation was performed only for the hydrodynamic part of the bioreactor in this article. The results showed that RT + CD6 gave better conversion ratio compared to the other two multiple impellers. It had higher axial flow and better air volume fraction distribution which was benefit for the biotransformation process. A certain amount of cell content should be guaranteed in order to obtain a good substrate conversion (45 % approximately). The final conversion ratio of canrenone was proportional to the content of mycelium at the late stage of conversion, while the content of mycelium at the early stage had a subtle effect. Besides, A. ochraceus resting cells could tolerate the maximum and average shear strain rate in the order of 2598 s−1 and 52 s−1, respectively. The research results provided a guide for the selection of impeller for the biotransformation of canrenone in biopharmaceutical industry.

Biotechnologies in Perfume Manufacturing: Metabolic Engineering of Terpenoid Biosynthesis.

The fragrance industry is increasingly turning to biotechnology to produce sustainable and high-quality fragrance ingredients. Microbial-based approaches have been found to be particularly promising, as they offer a more practical, economical and sustainable alternative to plant-based biotechnological methods for producing terpene derivatives of perfumery interest. Among the evaluated works, the heterologous expression of both terpene synthase and mevalonate pathway into Escherichia coli has shown the highest yields. Biotechnology solutions have the potential to help address the growing demand for sustainable and high-quality fragrance ingredients in an economically viable and responsible manner. These approaches can help compensate for supply issues of rare or impermanent raw materials, while also meeting the increasing demand for sustainable ingredients and processes. Although scaling up biotransformation processes can present challenges, they also offer advantages in terms of safety and energy savings. Exploring microbial cell factories for the production of natural fragrance compounds is a promising solution to both supply difficulties and the demand for sustainable ingredients and processes in the fragrance industry.

Diastereomer-specific transformation of hexabromocyclododecane by soil bacterial communities

For decades, the brominated fire retardant hexabromocyclododecane (HBCD) has been extensively used to reduce fire risk. Due to the inherent properties associated with environmental and biological persistence of HBCD, the legacy environmental problems caused by HBCD require investigation. In particular, the stereoisomer-specific biochemical processes that occur during microbial transformation of HBCD remain unclear. In this study, biotransformation of HBCD isomers by soil bacterial communities was systematically investigated. After 30 days, the removal rate of technical HBCD was 57.0%, and the composition of diastereomers changed due to the preference for β-HBCD transformation. The removal rates of each isomer were in the order β-HBCD > γ-HBCD > α-HBCD. High-throughput Illumina sequencing of 16S rRNA genes revealed that soil bacterial communities responded to the stress of α-HBCD and β-HBCD by increasing Proteobacteria dominance, while they responded to the stress of γ-HBCD by enhancing species diversity. Moreover, Acinetobacter hemolyticum strain HW-2 was isolated and demonstrated the same isomer specificity, attributed to the differential expression of the four key genes (had1, had1, had1, dehH). The strain successfully colonized the soil bacterial communities during bioaugmentation and enhanced the HBCD removal rates. These findings reveal that the diastereomer-specific biotransformation process is expedited by bioaugmentation with strain HW-2, minimizing ecotoxicities caused by HBCD. These results improve our understanding of the HBCD biotransformation process and provide a theoretical basis for the control of HBCD in ecosystems.

Brain CoA and acetyl CoA system in mechanisms of neurodegeneration

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, the key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, the main substrate of acetylation processes, including the formation of N-acetylaspartate and acetylcholine, post-translational modification of histones, determines the protection of neurons from degenerative signals and cholinergic neurotransmission. The biochemical mechanisms of neurodegenerative syndromes in PANK and CoASY defects and the possibility of correcting the development of CoA biosynthesis in knockout models for these enzymes are described. The data of a post-mortem study of the brain of patients with Huntington’s and Alzheimer’s diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in pathognomonic neurostructures. In the frontal cortex of patients with Parkinson’s disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. The redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro on synaptosomal membranes and mitochondria in the modeling of aluminum neurotoxicosis, accompanied by a decrease in the level of CoA in the CNS. The ability of CoA biosynthesis precursors to stabilize the glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism when exposed to neurotoxins, the development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA - redox-dependent processes of CoAlation of proteins, the possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology, the study of the bioavailability and biotransformation of Pan derivatives, in particular, D-panthenol, 4′-phospho-pantetheine, its acylated derivatives and compositions with redox pharmacological compounds are promising as potential etiopathogenetic agents.

Cometabolic biotransformation mediates the detoxification of oxytetracycline in Flavobacterium-enriched microbiome

Oxytetracycline (OTC), a widely used antibiotic that is readily detectable in the environment, potentially has a number of negative ecological consequences. The present study revealed the key role of the cometabolic breakdown of OTC in activated sludge microbiomes, which led to a high removal efficiency and detoxification of both OTC and its derivatives. An AS bioreactor was established to treat environmentally relevant levels of OTC. The bioreactor achieved an overall OTC removal efficiency of 94–97% at the steady state, with cometabolic biotransformation as the primary contributor to this ahead of other removal routes such as direct metabolic biodegradation, biosorption, and phosphorolysis. A novel biotransformation pathway for OTC proposed in this study involved dehydration, hydroxylation, ring cleavage, and alcohol dehydrogenation, distinguishing it from previously reported pathways. Although transformation products with potentially higher toxicity than OTC formed during the biotransformation process, the AS microbiome eliminated the overall toxicity of both OTC and its derivatives, as illustrated by both molecular structure-based toxicity simulations and antimicrobial susceptibility testing. Analysis of microbiome sequence data revealed the bacterial populations potentially facilitating cometabolic (e.g., Tolumonas) and direct metabolic (e.g., Flavobacterium) biotransformation. Overall, our study provides new insights into the removal routes and kinetics for OTC in AS microbiomes and the key species that potentially promote OTC transformation and detoxification.

Characterization of modified rice straw biochar in immobilizing Bacillus subtilis 168 and evaluation on its role as a novel agent for zearalenone-removal delivery

Microbial remediation of environmental pollutants can be advanced by carrier based cells immobilization. Whereas the effects of microorganisms immobilized on biochar for removal of zearalenone (ZEN) still remain unknown. Herein, this work presented the characterization of rice straw biochar (RSB) around modification in immobilizing Bacillus subtilis 168 and the role in fighting ZEN in vitro. Specifically, 10% of RSB with pH 5 condition were optimal for bearing cells, where majority of cells loaded inside the pore and minority on surface with agglomeration or scattering status. Octadecyl trimethyl ammonium chloride-inclusion RSB showed better performances including over 93% of ZEN detoxification rate (32.48% in free cells), cells preservation, and stability of detoxification in simulated gastrointestinal environment. RSB treated with sulphuric acid made nutrients adsorption generally less than 6.5%. No residues of α-ZEL and α-ZAL were found in ZEN biotransformation process whether by free cells or composites. Mechanism discussion implied that predominant monolayer chemisorption by RSB and subsequent biodegradation by extracellular enzymes from microorganism involved in ZEN-removal process. Collectively, these findings contribute to provide an applying strategy for coordination of biochar and microorganisms as potentially mycotoxin detoxifying agent in agricultural feed bioremediation and environmental decontamination processes.

Brain CoA and Acetyl CoA Metabolism in Mechanisms of Neurodegeneration

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, which is the main substrate of acetylation processes including formation of N-acetyl aspartate and acetylcholine, post-translational modification of histones, predetermines protection of the neurons against degenerative signals and cholinergic neurotransmission. Biochemical mechanisms of neurodegenerative syndromes in the cases of PANK and CoASY defects, and the possibility of correcting of CoA biosynthesis in the models with knockouts of these enzymes have been described. The data of a post-mortem study of the brains from the patients with Huntington's and Alzheimer's diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in the pathognomonic neurostructures. In the frontal cortex of the patients with Parkinson's disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. Redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro with synaptosomal membranes and mitochondria during modeling of aluminum neurotoxicity accompanied by the decrease in the level of CoA in CNS. The ability of CoA biosynthesis precursors to stabilize glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism during exposure to neurotoxins, development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA (redox-dependent processes of CoAlation of proteins, possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology), it seems promising to study bioavailability and biotransformation of Pan derivatives, in particular of D-panthenol, 4'-phospho-pantetheine, its acylated derivatives, and compositions with redox pharmacological compounds, are promising for their potential use as etiopathogenetic agents.

Enantioselectivity and origin of enhanced efficiency in polyethylene terephthalate hydrolases catalyzed depolymerization

Biotechnology is one of the most promising strategies to resolve the global crisis of plastic pollution. A clear understanding of the core enzyme mechanisms in the biotransformation process is critical for rational enzyme engineering and for practical, industrial-scale applications. Herein, we systematically examined and evidenced a largely unexplored piece in the depolymerization mechanism catalyzed by polyethylene terephthalate (PET) hydrolases: their enantioselectivity. We found that all the short-lived tetrahedron intermediates (IM3 and IM8) possess S-type chirality in six representative PET hydrolases. For instance, the binding percentage ratio of pro-S:pro-R is 57:21 in FAST-PETase, while pro-S binding leads to a much lower average energy barrier (5.2 kcal/mol) than pro-R binding (33.1 kcal/mol). Key structural features (e.g. the angle for Ser@H1-His@N1-PET@O2 and distance for His@N1-PET@O2) that significantly modulate the enantioselectivity were identified. The origin of the energy landscape variation between wild-type IsPETase and mutant FAST-PETase was also unveiled via analysis of key features, the distortion/interaction energy, and non-covalent bond interactions. This study supplies the missing piece in the mechanism for depolymerization catalyzed by PET hydrolases, and will aid in the rational design of enzymes for industrial recycling of PET plastic waste.

Growth kinetics of Lactobacillus acidophilus During the Anaerobic Biotransformation Process of Agro-Sugarcane Waste

Growth kinetics of Lactobacillus acidophilus During the Anaerobic Biotransformation Process of Agro-Sugarcane Waste

Fermentation of Robinia pseudoacacia flower for improving the antioxidation: optimized conditions, active composition, mechanism, and biotransformation process

Robinia pseudoacacia flower is a natural product with many biological activities, including antioxidation. To further develop its antioxidation, the extract was fermented by Aspergillus niger FFCC 3112 in the medium with carbon to nitrogen ratio of 1.4:1 and initial pH of 4.2 for 3.5 days to form the best antioxidant activity of the fermentation product by strain screening, single factor optimization, and response surface methodology. Further analysis, isolation and activity determination showed that a main chemical component, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, in the extract was completely hydrolyzed to kaempferol-7-O-α-L-rhamnopyranoside and kaempferol with better antioxidant activity through biotransformation, which was the basis for improving the antioxidant activity of fermentation products. Moreover, the mechanism of antioxidant and the contribution of phenolic hydroxyl groups were investigated by density functional theory. The result indicated that the antioxidant capacity of kaempferol-7-O-α-L-rhamnopyranoside and kaempferol increased with the increase of solvent polarity. In high-polarity solvents, they mainly scavenge free radicals through single electron transfer followed by proton transfer.

Influence of anaerobic biotransformation process of agro-industrial waste with Lactobacillus acidophilus on the rheological parameters: case of study of pig manure

This study evaluated the rheological behavior of the pig waste biotransformation process to produce lactic acid (LA) and biomass with Lactobacillus acidophilus in a stirred reactor. In addition, cell growth, carbohydrate consumption, and LA production were measured at three different agitation speeds, 100, 150, and 200rpm at 37°C, with a reaction time of 52h. During the development of the process, the kinetic and rheological parameters were obtained using the logistic, Gompertz, generalized Gompertz, Ostwald de Waele, and Herschel-Bulkley mathematical models, respectively. The substrate used was pig manure, to which molasses was added at 12% v/v to increase the concentration of carbohydrates. The results suggest that mass exchange is favorable at low agitation speeds. Nevertheless, the presence of molasses rich in carbohydrates as a carbon source modifies the characteristics of the fluid, dilatant (n > 1) at the beginning of the process to end up as pseudoplastic (n < 1) due to the addition of exopolysaccharides and the modification of the physical structure of the substrate. This effect was confirmed by the Herschel-Bulkley model, which presented a better fit to the data obtained, in addition to finding a direct relationship between viscosity and pH that can be used as variables for the control of bioconversion processes of pig manure into biomass rich in Lactobacillus acidophilus.

High-rate and -yield continuous fluidized-bed bioconversion of glucose-to-gluconic acid for enhanced metal leaching

Continuous low-cost bulk biolixiviant production remains as one of the main challenges of heterotrophic bioleaching towards large scale application. This study aimed at developing non-aseptic Gluconobacter oxydans-amended fluidized-bed reactor (FBR) process for continuous production of gluconic acid for efficient leaching of rare earth elements (REEs) and base metals from spent nickel-metal-hydride (NiMH) batteries. In preliminary experiments, the FBR became contaminated and massively overgrown by air-borne fungus, Leptobacillium leptobactrum. In a series of batch bioassays, operational conditions were investigated to discourage the fungal activity i.e., an ecologically engineered niche for gluconic acid production. High gluconate concentration (≥100 g/l) and/or low pH (≤2.5) gave a selective preference for G. oxydans growth over L. leptobactrum and controlled the activity of possible contaminants during FBR continuous operation. The highest gluconic acid production rate of 390 g/l∙d with corresponding glucose-to-gluconic acid conversion yield of 94% was obtained at hydraulic retention time (HRT) of 6.3 h and 380 g/l∙d glucose loading rate. Using the FBR effluents as leaching agents, respectively, total base metals and REEs leaching yields of up to 82% and 55% were achieved within 7 days at 1% (w/v) spent battery pulp density. The obtained glucose-to-gluconic acid conversion rates and yields were one of the highest reported for any glucose biotransformation process. The REE leaching yields were higher than those reported for similar high metal-grade REE secondary sources. The high-rate glucose-to-gluconic acid bioconversion in the non-aseptic system utilizing microbial ecology based FBR operation strategy rather than aseptic chemostats indicates industrial feasibility of gluconic acid production and thus, the applicability of heterotrophic bioleaching.

Biotransformation of gluten-free composite flour mediated by probiotics via solid-state fermentation process conducted under different moisture contents.

Staple foods produced from composite flour are considered feasible to alleviate protein-energy malnutrition (PEM). However, one of the major limitations of composite flour is poor protein digestibility. The biotransformation process mediated by probiotics via solid-state fermentation (SSF) holds a promising potential to address the poor protein digestibility in composite flour. Yet, there is no report established in this regard to the best of our knowledge. Therefore, 4 strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus UP2 isolated from Malaysian foods that were previously reported to produce versatile extracellular hydrolytic enzymes were employed to biotransform gluten-free composite flour derived from rice, sorghum, and soybean. The SSF process was performed under 30-60% (v/w) moisture content for 7 days, where samples were withdrawn at 24 h intervals for various analyses such as pH, total titratable acidity (TTA), extracellular protease activity, soluble protein concentration, crude protein content, and in vitro protein digestibility. The pH of the biotransformed composite flour showed a significant reduction from the initial range of pH 5.98-6.67 to the final pH of 4.36-3.65, corresponding to the increase in the percentage of TTA in the range of 0.28-0.47% to 1.07-1.65% from days 0 to 4 and remained stable till day 7 of the SSF process. The probiotics strains exhibited high extracellular proteolytic activity (0.63-1.35 U/mg to 4.21-5.13 U/mg) from days 0 to 7. In addition, the treated composite flour soluble protein increased significantly (p ≤ 0.05) (0.58-0.60 mg/mL to 0.72-0.79 mg/mL) from days 0 to 7, crude protein content (12.00-12.18% to 13.04-14.39%) and protein digestibility (70.05-70.72% to 78.46-79.95%) from days 0 to 4 of SSF. The results of biotransformation of 50% (v/w) moisture content were mostly comparable to 60% (v/w) moisture content, implying 50% (v/w) moisture content was the most suitable moisture content for the effective biotransformation of gluten-free composite flour mediated by probiotics via SSF since flour quality is better at lower moisture content. As for the overall performance, L. plantarum RS5 was ranked the best strain, attributed to the general improvement in the physicochemical properties of composite flour.

Characterizations of food-derived ellagic acid-Undaria pinnatifida polysaccharides solid dispersion and its benefits on solubility, dispersity and biotransformation of ellagic acid

The current study was aimed to enhance the solubility, dispersibility and biotransformation efficacy of ellagic acid (EA) by preparing food-derived ellagic acid-Undaria pinnatifida polysaccharides solid dispersion (EA/UPP SD). The results demonstrated that the solubility of EA/UPP SD was improved from 0.014 mg/mL to 0.383 mg/mL, and the enhancement was related to converting to a more amorphous state and restraining its self-aggregation during the mechanochemical process. The structure of EA/UPP SDs was mostly maintained by hydrogen bonds and hydrophobic interactions between EA and UPP. Moreover, the result of in vitro anaerobic incubations showed the biotransformation process was improved with EA/UPP SD addition to substrate due to the advance of microbial accessibility in EA dispersion. Altogether, these results indicated that the EA/UPP SDs expanded the application of EA by increasing the solubility and dispersity, and provided a theoretical basis for bioconversion efficiency enhancement.

Improved terephthalic acid production from p-xylene using metabolically engineered Pseudomonas putida

Terephthalic acid (TPA) is an important commodity chemical used as a monomer of polyethylene terephthalate (PET). Since a large quantity of PET is routinely manufactured and consumed worldwide, the development of sustainable biomanufacturing processes for its monomers (i.e. TPA and ethylene glycol) has recently gained much attention. In a previous study, we reported the development of a metabolically engineered Escherichia coli strain producing 6.7 g/L of TPA from p-xylene (pX) with a productivity and molar conversion yield of 0.278 g/L/h and 96.7 mol%, respectively. Here, we report metabolic engineering of Pseudomonas putida KT2440, a microbial chassis particularly suitable for the synthesis of aromatic compounds, for improved biocatalytic conversion of pX to TPA. To develop a plasmid-free, antibiotic-free, and inducer-free biocatalytic process for cost-competitive TPA production, all heterologous genes required for the synthetic pX-to-TPA bioconversion pathway were integrated into the chromosome of P. putida KT2440 by RecET-based markerless recombineering and overexpressed under the control of constitutive promoters. Next, TPA production was enhanced by integrating multiple copies of the heterologous genes to the ribosomal RNA genes through iteration of recombineering-based random integration and subsequent screening of high-performance strains. Finally, fed-batch fermentation process was optimized to further improve the performance of the engineered P. putida strain. As a result, 38.25 ± 0.11 g/L of TPA was produced from pX with a molar conversion yield of 99.6 ± 0.6%, which is equivalent to conversion of 99.3 ± 0.8 g pX to 154.6 ± 0.5 g TPA. This superior pX-to-TPA biotransformation process based on the engineered P. putida strain will pave the way to the commercial biomanufacturing of TPA in an industrial scale.

Congener-Specific Uptake and Metabolism of Bisphenols in Carrot Cells: Dissipation Kinetics, Biotransformation, and Enzyme Responses.

Food consumption has been considered a key pathway of bisphenol compound (BP) exposure for humans. However, there is a lack of evidence concerning their congener-specific behavior and metabolism in plants. Herein, we examined the uptake and metabolism of five BPs in plants using carrot cells. Bisphenol S (BPS) and bisphenol AF (BPAF) exhibited substantially lower dissipation rates in the cells than the other BPs, indicating a strong selectivity in the uptake and metabolism among bisphenol congeners. For a total of 23 metabolites of BPs, the predominant biotransformation pathways were found to be glycosylation, methoxylation, and conjugation, while hydroxylation, methylation, and glutathionylation were only observed for some BPs. The changes in the mRNA expression of cytochrome P450 (P450) and the activities of glycosyltransferase and glutathione S-transferase were remarkably higher in cells exposed to bisphenol F, bisphenol A, and bisphenol B than in cells exposed to BPS and BPAF, indicating congener specificity in their effects on enzymes and the associated biotransformation processes. Consequently, the potential congener-specific differences in plant uptake, metabolism, and accumulation must be considered when assessing the environmental risks posed by these commonly used plasticizers.