White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

Biosensors based on allosteric transcription factors have been widely used in synthetic biology. In this study, we utilized the Acinetobacter ADP1 transcription factor PobR to develop a biosensor activating the PpobA promoter when bound to its natural ligand, 4-hydroxybenzoic acid (4HB). To screen for PobR mutants responsive to 4-hydroxyphenylpyruvate(HPP), we developed a dual selection system in E. coli. The positive selection of this system was used to enrich PobR mutants that identified the required ligands. The following negative selection eliminated or weakened PobR mutants that still responded to 4HB. Directed evolution of the PobR library resulted in a variant where PobRW177R was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobRWT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.

The composition of medium components is crucial for achieving the best performance of synthetic construction in genetically engineered cells. Which and how medium components determine the performance, e.g., productivity, remain poorly investigated. To address the questions, a comparative survey with two genetically engineered Escherichia coli strains was performed. As a case study, the strains carried the synthetic pathways for producing the aromatic compounds of 4-aminophenylalanine (4APhe) or tyrosine (Tyr), common in the upstream but differentiated in the downstream metabolism. Bacterial growth and compound production were examined in hundreds of medium combinations that comprised 48 pure chemicals. The resultant data sets linking the medium composition to bacterial growth and production were subjected to machine learning for improved production. Intriguingly, the primary medium components determining the production of 4PheA and Tyr were differentiated, which were the initial resource (glucose) of the synthetic pathway and the inducer (IPTG) of the synthetic construction, respectively. Fine-tuning of the primary component significantly increased the yields of 4APhe and Tyr, indicating that a single component could be crucial for the performance of synthetic construction. Transcriptome analysis observed the local and global changes in gene expression for improved production of 4APhe and Tyr, respectively, revealing divergent metabolic strategies for producing the foreign and native metabolites. The study demonstrated that ML-assisted medium optimization could provide a novel point of view on how to make the synthetic construction meet the designed working principle and achieve the expected biological function.

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

Extracellular polymeric substances (EPSs) constitute a largely global carbon pool that could participate in geochemical process of organic chemicals. Besides the chemical hydrolysis and redox of chemicals exerted by the EPS, weakly noncovalent interactions with dispersive EPS control the toxicity of numerous organic compounds. Nevertheless, there has been a lack of in-depth research on this issue. This work quantified a chain of links from bonding to detoxification using natural biofilms to explore the control behavior of fragile noncovalent bonding to the ecotoxicity of aromatic compounds. Such bonding decreases cell absorbability of m-phenylenediamine, 2-naphthol, and phenanthrene by 5.3-53.6%, resultantly increasing the indices of microbial diversity by 122.2-279.5%. Herein, the 60 kDa chaperonin in EPS acts as the most important contributor (16.4% of the top 20 proteins) to noncovalent interactions. Hydrophilic carboxyl groups in EPS bind with hydroxyl and amino groups of m-phenylenediamine and 2-naphthol via H-bonds, respectively. Methylene and carboxyl groups combine with hydrophobic phenanthrene via CH···π and H-bonding, respectively. A quantified chain was consequently established that weak interaction linearly controls ecotoxicity of aromatic compounds via the above suppressive cell absorbability of aromatic compounds (R2 =0.82). Considering ubiquitous EPS and prevailing aromatic compounds, our findings revealed a previously unnoticed phenomenon in which seemingly fragile noncovalent bonding profoundly alleviates the ecotoxicity of aromatic compounds in Earth\'s surface system.

This study aimed to evaluate the changes in aromatic components and other chemical properties of Tainong mango during fruit development, ripening, and storage. As the volatiles of Tainong mango and their related molecular mechanisms remain unclear, volatile profile, metabonomics, and transcriptome analyses were applied to investigate the molecular determinants of the synthesis of aroma components in mango during fruit development and storage. Total acids, total sugar, total carotenoids, enzyme activities of the mango pulp samples were also determined. Volatile components of the mango pulp samples were identified using a gas chromatography-mass spectrometric method. Ribonucleic acid (RNA) sequences of the samples were analyzed by real-time polymerase chain reaction. The results showed that 181 volatiles were isolated and identified in the fruit at seven stages. Compared to the other stages, mango collected on day 8 and day 12 had higher concentrations of 17 volatile components, especially (E,Z)-2,6-nonadienal, 53384 transcripts were also detected through RNA sequencing. The differentially expressed genes analyses included catalytic activity, transferase activity, adenosine diphosphate binding, transcription factor activity, and oxidoreductase activity. α-Pinene content and expression of the differentially expressed genes involved in terpenoid metabolism and enzyme activities in the terpenoid metabolic pathways gradually increased during the maturity of the fruit, and had maximum values at day 8 of storage. Moreover, the integrative analyses revealed potential molecular insights of mango development and aroma formation in the fruit.

Methane is an important energy resource internationally, and a large proportion of this methane is produced by microbial communities living in coal seams. Despite the value of this resource for human energy security, our understanding of the metabolic roles played by specific taxa during the biodegradation of coal to methane in situ is quite limited. In order to develop a greater understanding of microbial catabolism on coal, a community from a coal seam in the Surat Basin, Australia, was incubated on 10 different aromatic organic compounds: coronene, benzo[a]pyrene, pyrene, phenanthrene, naphthalene, ethylbenzene, phenol, benzoate, vanillate and syringate. Each of these aromatic compounds either occurs in coal or is a possible product of the coal biodegradation process. 16S rRNA sequencing revealed substantial changes to each community in response to each aromatic carbon substrate provided. Abundant taxa from these substrate-specific communities were identified and their probable catabolic roles proposed based on literature searches of related taxa. This study is the first to link specific coal seam taxa to aromatic substrates available in coal seam environments. Two conceptual models of the putative degradation pathways and key taxa responsible are proposed.

Volatile organic compounds (VOCs) are atmospheric pollutants that can affect human healthy and intensify some environmental problems. Among different techniques to degrade VOCs, heterogeneous photocatalysis has been highlighted. The aim of this research was to obtain high toluene degradation using heterogeneous photocatalysis in the ozone presence (TiO2/O3/UV) and analyze VOC degradation over the reactor length comparing with ozone concentration also over the reactor length. Ozone concentration has influence on toluene degradation; 75% of VOC degradation was reached with 69.0 mgL-1 of O3 meanwhile a degradation of 91% was obtained with 96.2 mgL-1 of O3. Toluene degradation reached a plateau over reactor length at flowrate of 565 mL min-1, which indicates the reactor was oversized in this case. However, it was not observed at 1425 mL min-1. In addition, it was evaluated that O3 concentration and toluene reaction rate decreased over the reactor length.

OBJECTIVE: With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter.
RESULTS: PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter.
CONCLUSIONS: Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.