Design and development of novel self-assembled catechol-modified bile acid conjugates as pH-responsive apical sodium-dependent bile acid transporter targeting nanoparticles.

Catechol-based biomaterials demonstrate biocompatibility, making them suitable for a wide range of therapeutic applications when integrated into various molecular frameworks. However, the development of orally available catechol-based biomaterials has been hindered by significant pH variations and complex interactions in the gastrointestinal (GI) tract. In this study, we introduce a novel catechol-modified bile acid (CMBA), which is synthesized by anchoring the FDA-approved drug, ursodeoxycholic acid to the neurotransmitter dopamine. This modification could form a new apical sodium-dependent bile acid transporter (ASBT) inhibitor (ASBTi) due to the bile acid moiety. The computational analysis using the TRAnsient Pockets in Proteins (TRAPP) module, coupled with MD simulations, revealed that CMBA exhibits a strong binding affinity at residues 51-55 of ASBT with a low inhibitory constant (Ki) value. Notably, in slightly alkaline biological conditions, CMBA molecules self-assemble into carrier-free nanoparticles with an average size of 240.2 ± 44.2 nm, while maintaining their ability to bind with ASBT. When administered orally, CMBA accumulates in the ileum and liver over 24 h, exhibiting significant therapeutic effects on bile acid (BA) metabolism in a high-fat diet (HFD)-fed mouse model. This study underscores the therapeutic potential of the newly developed catechol-based, pH-responsive ASBT-inhibiting nanoparticles presenting a promising avenue for advancing therapy.

A genome assembly of ginger (Zingiber officinale Roscoe) provides insights into genome evolution and 6-gingerol biosynthesis.

Ginger is cultivated in tropical and subtropical regions and is one of the most crucial spices worldwide owing to its special taste and scent. Here, we present a high-quality genome assembly for 'Small Laiwu Ginger', a famous cultivated ginger in northern China. The ginger genome was phased into two haplotypes, haplotype A (1.55Gb), and haplotype B (1.44Gb). Analysis of Ty1/Copia and Ty3/Gypsy LTR retrotransposon families revealed that both have undergone multiple retrotransposon bursts about 0-1 million years ago. In addition to a recent whole-genome duplication event, there has been a lineage-specific expansion of genes involved in stilbenoid, diarylheptanoid, and gingerol biosynthesis, thereby enhancing 6-gingerol biosynthesis. Furthermore, we focused on the biosynthesis of 6-gingerol, the most important gingerol, and screened key transcription factors ZoMYB106 and ZobHLH148 that regulate 6-gingerol synthesis by transcriptomic and metabolomic analysis in the ginger rhizome at four growth stages. The results of yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter gene assays showed that both ZoMYB106 and ZobHLH148 bind to the promoters of the key rate-limiting enzyme genes ZoCCOMT1 and ZoCCOMT2 in the 6-gingerol synthesis pathway and promote their transcriptional activities. The reference genome, transcriptome, and metabolome data pave the way for further research on the molecular mechanism underlying the biosynthesis of 6-gingerol. Furthermore, it provides precious new resources for the study on the biology and molecular breeding of ginger.

Oxidation of dopamine and related catechols in dopaminergic brain regions in Parkinson's disease and during ageing in non-Parkinsonian subjects.

The present study was performed to examine if catechol oxidation is higher in brains from patients with Parkinson's disease compared to age-matched controls, and if catechol oxidation increases with age. Brain tissue from Parkinson patients and age-matched controls was examined for oxidation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylalanine (DOPA) to corresponding quinones, by measurement of 5-S-cysteinyl-dopamine, 5-S-cysteinyl-DOPAC and 5-S-cysteinyl-DOPA. The cysteinyl catechols are assumed to be biomarkers for DA, DOPAC and DOPA autoxidation and part of the biosynthetic pathway of neuromelanin. The concentrations of the 5-S-cysteinyl catechols were lower, whereas the 5-S-cysteinyl-DA/DA and 5-S-cysteinyl-DOPAC/DOPAC ratios tended to be higher in the Parkinson group compared to controls, which was interpreted as a higher degree of oxidation. High 5-S-cysteinyl-DA/DA ratios were found in the substantia nigra of a sub-population of the Parkinson group. Based on 5-S-cysteinyl-DA/DA ratios, dopamine oxidation was found to increase statistically significantly with age in the caudate nucleus, and non-significantly in the substantia nigra. In conclusion, the occurrence of 5-S-cysteinyl-DA, 5-S-cysteinyl-DOPAC and 5-S-cysteinyl-DOPA was demonstrated in dopaminergic brain areas of humans, a tendency for higher oxidation of DA in the Parkinson group compared to controls was observed as well as a statistically significant increase in DA oxidation with age. Possibly, autoxidation of DA and other catechols are involved in both normal and pathological ageing of the brain. This study confirms one earlier but small study, as well as complements one study on non-PD cases and one study on both PD cases and controls on NM bound or integrated markers or catechols.

Biodegradation of chloroethene compounds under microoxic conditions.

The biodegradation of chloroethene compounds under oxic and anoxic conditions is well established. However, the biological reactions that take place under microoxic conditions are unknown. Here, we report the biostimulated (BIOST: addition of lactate) and natural attenuated (NAT) degradation of chloroethene compounds under microoxic conditions by bacterial communities from chloroethene compounds-contaminated groundwater. The degradation of tetrachloroethene was significantly higher in NAT (15.14% on average) than in BIOST (10.13% on average) conditions at the end of the experiment (90 days). Sporomusa, Paracoccus, Sedimentibacter, Pseudomonas, and Desulfosporosinus were overrepresented in NAT and BIOST compared to the source groundwater. The NAT metagenome contains phenol hydrolase P1 oxygenase (dmpL), catechol-1,2-dioxygenase (catA), catechol-2,3-dioxygenases (dmpB, todE, and xylE) genes, which could be involved in the cometabolic degradation of chloroethene compounds; and chlorate reductase (clrA), that could be associated with partial reductive dechlorination of chloroethene compounds. Our data provide a better understanding of the bacterial communities, genes, and pathways potentially implicated in the reductive and cometabolic degradation of chloroethene compounds under microoxic conditions.

Characterization of a novel aromatic substrate-processing microcompartment in Actinobacteria.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

Conjunctive Analyses of BSA-Seq and BSR-Seq to Identify Candidate Genes Controlling the Black Lemma and Pericarp Trait in Barley.

Black barley seeds are a health-beneficial diet resource because of their special chemical composition and antioxidant properties. The black lemma and pericarp (BLP) locus was mapped in a genetic interval of 0.807 Mb on chromosome 1H, but its genetic basis remains unknown. In this study, targeted metabolomics and conjunctive analyses of BSA-seq and BSR-seq were used to identify candidate genes of BLP and the precursors of black pigments. The results revealed that five candidate genes, purple acid phosphatase, 3-ketoacyl-CoA synthase 11, coiled-coil domain-containing protein 167, subtilisin-like protease, and caffeic acid-O-methyltransferase, of the BLP locus were identified in the 10.12 Mb location region on the 1H chromosome after differential expression analysis, and 17 differential metabolites, including the precursor and repeating unit of allomelanin, were accumulated in the late mike stage of black barley. Phenol nitrogen-free precursors such as catechol (protocatechuic aldehyde) or catecholic acids (caffeic, protocatechuic, and gallic acids) may promote black pigmentation. BLP can manipulate the accumulation of benzoic acid derivatives (salicylic acid, 2,4-dihydroxybenzoic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid, protocatechuic aldehyde, and syringaldehyde) through the shikimate/chorismite pathway other than the phenylalanine pathway and alter the metabolism of the phenylpropanoid-monolignol branch. Collectively, it is reasonable to infer that black pigmentation in barley is due to allomelanin biosynthesis in the lemma and pericarp, and BLP regulates melanogenesis by manipulating the biosynthesis of its precursors.

Bioremediation of Catechol and Concurrent Accumulation of Biocompounds by the Microalga Crypthecodinium cohnii.

Burgeoning commercial applications of catechol have led to its excessive accumulation in the environment, thereby posing a severe ecological threat. Bioremediation has emerged as a promising solution. The potential of the microalga Crypthecodinium cohnii to degrade catechol and use the byproduct as a carbon source was investigated in this study. Catechol significantly increased C. cohnii growth and was rapidly catabolized within 60 h of cultivation. Transcriptomic analysis highlighted the key genes involved in catechol degradation. Real-time polymerase chain reaction (RT-PCR) analysis showed that transcription of key genes CatA, CatB, and SaID involved in the ortho-cleavage pathway was remarkably increased by 2.9-, 4.2-, and 2.4- fold, respectively. Key primary metabolite content was also markedly altered, with a specific increment in polyunsaturated fatty acids. Electron microscopy and antioxidant analysis showed that C. cohnii could tolerate catechol treatment without morphological aberrations or oxidative stress. The findings provide a strategy for C. cohnii in the bioremediation of catechol and concurrent polyunsaturated fatty acids (PUFA) accumulation.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae.

BACKGROUND: Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology. RESULTS: With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days. CONCLUSIONS: Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.

Does the presence of heavy metal and catechol contaminants in organic waste challenge the physiological performance of the bioconverter Hermetia illucens?

The increased human activities and the worldwide population growth are constantly increasing the production of solid wastes. Over the years, waste management has thus become a prominent issue for several companies and municipalities, and several engineering techniques have been developed over the years in order to convert wastes into other solid materials or fuels. Yet, several techniques are important contributors to environmental pollution, and biological-based solutions have thus become progressively very popular. In particular, insect-based conversion of organic wastes represent eco-friendly tools, and the growth and development of insect species such as the black soldier fly have been tested and improved for a large diversity of organic wastes. However, organic wastes, including food wastes, may contain several pollutants such as heavy metals and catechol which could affect the bioconversion efficiency by incurring physiological costs that would be undetectable at the organismal level, i.e. have null to little effects on the life cycle of Hermetia illucens. In this context, assessments of antioxidant capacities can provide a rapid and low-cost evaluation of the capability of insects to handle exposure to heavy metals and catechol. Here, we aimed at measuring the physiological responses of the black soldier fly H. illucens grown on food wastes (kitchen, fruit or vegetable wastes) contaminated by cadmium, iron, lead or catechol. Biomarkers of oxidative stress (concentrations of hydrogen peroxide and protein carbonyls), non-enzymatic total antioxidant capacity (ascorbic acid amounts) and activity of enzymatic antioxidants (activities of superoxide dismutase and polyphenoloxidase) were measured from the gut of the larvae. We found no evidence of deleterious impacts of food waste contamination by catechol or heavy metals on H. illucens. In most experimental treatments, the array of physiological endpoints we measured for evaluating the degree of oxidative stress experienced by the larvae remained similar to controls. Possible physiological effects were reported for cadmium and catechol only, which tended to increase the oxidation of proteins and hydrogen peroxide in the larvae. Finally, our results suggested that the nature of the food waste could equally affect the physiological responses of the insect.

Unraveling the skatole biodegradation process in an enrichment consortium using integrated omics and culture-dependent strategies.

3-Methylindole (skatole) is regarded as one of the most offensive compounds in odor emission. Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic. In this study, an efficient aerobic skatole-degrading consortium was obtained. Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses. Bioinformatic analyses indicated that the skatole downstream degradation was mainly via the catechol pathway, and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase. Genome binning and gene analyses indicated that Pseudomonas, Pseudoclavibacter, and Raineyella should cooperate with Rhodococcus for the skatole degradation process. Moreover, a pure strain Rhodococcus sp. DMU1 was successfully obtained which could utilize skatole as the sole carbon source. Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium. Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway. Collectively, our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader, and the degradation mainly proceeded via the catechol pathway.

Systems metabolic engineering upgrades Corynebacterium glutamicum to high-efficiency cis, cis-muconic acid production from lignin-based aromatics.

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis-muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p- hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated.

Transcriptomic profiling reveals the molecular responses of Rhodococcus aetherivorans DMU1 to skatole stress.

Skatole is a typical malodor compound in animal wastes. Several skatole-degrading bacterial strains have been obtained, whereas the molecular response of strains to skatole stress has not been well elucidated. Herein, the skatole degradation by a Gram-positive strain Rhodococcus aetherivorans DMU1 was investigated. Strain DMU1 showed high efficiency in skatole degradation under the conditions of 25-40 °C and pH 7.0-10.0. It could utilize various aromatics, including cresols, phenol, and methylindoles, as the sole carbon source for growth, implying its potential in the bioremediation application of animal wastes. Transcriptomic sequencing revealed that 328 genes were up-regulated and 640 genes were down-regulated in strain DMU1 when grown in the skatole-containing medium. Skatole increased the gene expression levels of antioxidant defense systems and heat shock proteins. The expression of ribosome-related genes was significantly inhibited which implied the growth inhibition of skatole. A rich set of oxidoreductases were changed, and a novel gene cluster containing the flavoprotein monooxygenase and ring-hydroxylating oxygenase genes was highly up-regulated, which was probably involved in skatole upstream degradation. The upregulation pattern of this gene cluster was further verified by qRT-PCR assay. Furthermore, skatole should be mainly degraded via the catechol ortho-cleavage pathway with cat25170 as the functional gene. The gene cat25170 was cloned and expressed in E. coli BL21(DE3). Pure enzyme assays showed that Cat25170 could catalyze catechol with Km 9.96 µmol/L and kcat 12.36 s-1.

Catechol-functionalized sulfobetaine polymer for uniform zwitterionization via pH transition approach.

The study aims to develop a modification strategy to facilitate uniform catechol-assisted zwitterionization on nitinol alloy for bio-compatibility and fouling resistance. Catechol-functionalized polysulfobetaine methacrylate (pSBMA/DA) is synthesized via dopamine-initiated photo-polymerization. Under UV irradiation, semiquinone radicals from dopamine (DA) can be generated, and prevented loss of one electron to intramolecular cyclization and intermolecular dimerization in a solution at pH 2. Pseudo-first-order polymerization kinetics, and relations of apparent rate constant and number average molecular weight with the molar ratio of DA in photopolymerization for pSBMA/DA are unveiled. In a solution at pH 3, PSBMA/DA begins aggregation, kept catechol moieties from premature oxidization, and enabled even deposition on the nitinol substrate. After pH regulation to 8.5, pSBMA/DA extends, and concurrently catechol moieties are activated to interact with the nitinol surface via the formation of bidentate binding. X-ray photoelectron spectroscopy (XPS) analysis revealed that a shorter pSBMA/DA chain with higher catechol content provides more anchoring sites to enhance zwitterionic moieties coverage on substrates. Interestingly, atomic force microscopy (AFM) images revealed a smooth and uniform deposition of pSBMA/DA using the pH-transition method. Strong ionic hydration of pSBMA/DA coating on nitinol surface repels non-specific adsorption of bio-foulants, permitting excellent antifouling properties. Zwitterion-modified nitinol achieved a reduction rate of 99.9% against Escherichia coli and Staphylococcus aureus attachment. In addition, pSBMA/DA exhibits a robust antifouling performance to NIH 3T3 mouse fibroblasts in culture media after incubation for 24 h. Overall, the pSBMA/DA coating via pH transition approach opens up a promising strategy to facilitate uniform surface functionalization for antifouling and coating technology.

Comparative Metabolomics Reveals a Bifunctional Antibacterial Conjugate from Combined-Culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596.

To investigate the potential for secondary metabolite biosynthesis by Streptomyces species, we employed a coculture method to discover natural bioactive products and identified specific antibacterial activity from a combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. Molecular networking using ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) data revealed a specific clade of metabolites in this combined-culture that were not detected in both monocultures. Using the chemical profiles, a previously unidentified conjugate between FabF inhibitor and catechol-type siderophore was successfully identified and named harundomycin A. Harundomycin A was a conjugate between the 2,4-dihydroxy-3-aminobenzoate moiety of platensimycin and N,N'-bis(2,3-dihydroxybenzoyl)-O-seryl-cysteine (bisDHBA-Ser-Cys) with a thioester linkage. Along with the production of harundomycin A, platensimycin, its thiocarboxylic acid form thioplatensimycin, enterobactin, and its degradation product N,N'-bis(2,3-dihydroxybenzoyl)-O-l-seryl-dehydroalanine (bisDHBA-Ser-Dha) were also induced in the combined-culture. Genomic data of S. hygroscopicus HOK021 and T. pulmonis TP-B0596 indicated that strain HOK021 possessed biosynthetic gene clusters for both platensimycin and enterobactin, and thereby revealed that T. pulmonis stimulates HOK021 and acts as an inducer of both of these metabolites. Although the harundomycin A was modified by bulky bisDHBA-Ser-Cys, responsible for the binding to the target molecule FabF, it showed a similar antibacterial spectrum to platensimycin, including against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, suggesting that the pharmacophore is platensimycin. Additionally, Chrome Azurol S assay showed that harundomycin A possesses ferric iron-chelating activity comparable to that of enterobactin. Our study demonstrated the transformation of existing natural products to bifunctional molecules driven by bacterial interaction.

[Colonization performance and pyrene degradation characteristics of Stenotrophomonas maltophilia PX1]. / å—œéº¦èŠ½å¯¡å »å•èƒžèŒPX1的降解芘特性及定殖效能.

We screened and identified an endophytic bacterium that could efficiently degrade PAHs, which would expand the library of polycyclic aromatic hydrocarbons (PAHs) degrading microorganisms and reduce the pollution risk of crops. Its degradation mechanism and colonization performance were preliminarily examined. The results showed that strain PX1 belonged to Stenotrophomonas maltophilia. The strain had broad spectrum ability to remove PAHs. In PAH mineral salt (MS) media, almost 100% naphthalene was degraded by strain PX1 after 7-d incubation. In a cultivation system solely containing phenanthrene of 50.0 mg·L-1, pyrene of 20.0 mg·L-1, fluoranthene of 20.0 mg·L-1 or benzo[a]pyrene of 10.0 mg·L-1, the degradation efficiency of phenanthrene, pyrene, fluoranthene and benzo[a]pyrene by strain PX1 reached 72.6%, 50.7%, 31.9%, and 12.9%, respectively. Pyrene was selected as PAHs model to study the degradation characteristics of strain PX1. Enzyme activity tests showed that the activities of phthalate dioxygenase, catechol-1,2-dioxygenase, and catechol-2,3-dioxygenase in strain PX1 were induced by pyrene. Some metabolic intermediates such as 4,5-epoxypyrene, 4,5-dihydroxypyrene, gentilic acid/protocatechuic acid, salicylic acid, cis-hexadienedioic acid/2-hydroxymyxofuroic acid semialdehyde, cis-2'-carboxyphenylpyruvic acid, 1-hydroxy-2-naphthoic acid, and salicylaldehyde were detected during the degradation of pyrene by strain PX1. Results of the seed soaking experiment showed that strain PX1 could efficiently colonize in Ipomoea aquatic and Triticum aestivum. After inoculated with strain PX1, the growth of I. aquatic and T. aestivum was significantly increased, and the pyrene concentration in I. aquatic, T. aestivum and MS media was reduced by 29.8%-50.7%, 52.4%-67.1% and 8.0%-15.3%, respectively. Our results suggested that strain PX1 degraded pyrene mainly through 'salicylate pathway' and 'phthalate pathway', and could be colonized into plants and promote plant growth.

In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii.

Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central l-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of l-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of l-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of l-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of l-Ser, DHB, and l-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (l-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

Biodegradation kinetics and metabolism of Benzo(a)fluorene by Pseudomonas strains isolated from refinery effluent.

The final sinkers of polyaromatic hydrocarbons are water sources, where they undergo bioaccumulation and biomagnification, leading to adverse mutagenic, carcinogenic, and teratogenic effects on exposure in flora, fauna, and humans. Two indigenous strains, Pseudomonas sp. WDE11 and Pseudomonas sp. WD23, isolated from refinery effluent, degraded over 97.5% of benzo(a)fluorene (10 mg/L) in 7 days. On growth at concentration dependent amounts (50 mg/L and 100 mg/L), the degradation reduced to approximately 90% and 80% respectively in 56 days. Degradation kinetics was concentration dependent, as degradation followed first-order and second-order kinetics for 50 mg/L and 100 mg/L respectively. The half-life for degradation of benzo(a)fluorene ranged between 11.64 - 12.26 days and 13.11-14.5 days for strains WDE11 and WD23 respectively. The values of Andrew-Haldane kinetic parameters i.e. µmax, Ks, and Ki were 0.306 day-1, 11.11 mg/L, and 120.41 mg/L for strain WDE11 respectively, while for strain WD23, the respective values were 0.312 day-1, 9.97 mg/L, and 152 mg/L. Degradation metabolites were identified by their MS patterns as 3,4-dihydroxy fluorene, 2-(1-oxo-2,3-dihydro-1H-inden-2-yl) acetic acid, 3,4-dihydrocoumarin, salicylic acid, catechol, and oxalic acid. Metabolic pathway of degradation constructed, revealed that benzo(a)fluorene was metabolized via the formation of fluorene, further metabolized by salicylate pathway forming catechol. The catechol formed was degraded into simpler metabolites by meta-cleavage pathway, which was validated by catechol 2,3 dioxygenase enzyme activity.

Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects.

The shikimate pathway is a necessary pathway for the synthesis of aromatic compounds. The intermediate products of the shikimate pathway and its branching pathway have promising properties in many fields, especially in the pharmaceutical industry. Many important compounds, such as shikimic acid, quinic acid, chlorogenic acid, gallic acid, pyrogallol, catechol and so on, can be synthesized by the shikimate pathway. Among them, shikimic acid is the key raw material for the synthesis of GS4104 (Tamiflu®), an inhibitor of neuraminidase against avian influenza virus. Quininic acid is an important intermediate for synthesis of a variety of raw chemical materials and drugs. Gallic acid and catechol receive widespread attention as pharmaceutical intermediates. It is one of the hotspots to accumulate many kinds of target products by rationally modifying the shikimate pathway and its branches in recombinant strains by means of metabolic engineering. This review considers the effects of classical metabolic engineering methods, such as central carbon metabolism (CCM) pathway modification, key enzyme gene modification, blocking the downstream pathway on the shikimate pathway, as well as several expansion pathways and metabolic engineering strategies of the shikimate pathway, and expounds the synthetic biology in recent years in the application of the shikimate pathway and the future development direction.

Polycatechols with Robust Efficiency in Cytosolic Peptide Delivery via Catechol-Boronate Chemistry.

Cytosolic delivery of peptides remains a challenging task because of the limited binding sites on peptides and the existence of multiple intracellular barriers. Here, we proposed the use of polycatechols with a high cell permeability to deliver peptides of different physicochemical properties using the catechol-boronate chemistry. Peptides were decorated with boronate moieties via three strategies, and the introduced boronate groups greatly increased the binding affinity of cargo peptides with polycatechols. The loading peptides could be released under the endolysosomal acidity. When the cargo peptide was modified with boronate moiety via a p-hydroxybenzylcarbamate self-immolative spacer, it could be loaded by polycatechols and released in a traceless manner triggered by reactive oxygen species. The proposed strategies greatly promote the cytosolic delivery efficiency of different peptides into various cell lines and restored their biofunctions after intracellular delivery and release. This study provides a general and robust platform for the intracellular delivery of membrane-impermeable peptides.

Comparing in planta accumulation with microbial routes to set targets for a cost-competitive bioeconomy.

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.