Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl‐CoA intermediate during anaerobic phthalate degradation

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen‐independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from ‘Aromatoleum aromaticum’: a highly specific succinyl‐CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl‐CoA formed by SPT accumulated only to sub‐micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half‐life of around 7 min. Upon addition of excess phthaloyl‐CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl‐CoA formation. In conclusion, a massive overproduction of PCD in phthalate‐grown cells to concentrations >140 μM was observed that allowed for efficient phthaloyl‐CoA conversion at concentrations 250‐fold below the apparent K_m‐value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.     

A patchwork pathway for oxygenase‐independent degradation of side chain containing steroids

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen‐independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo‐, phyto‐ and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin‐containing steroid dehydrogenases are involved in C25‐hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26‐oic acids. Side chain degradation to androsta‐1,4‐diene‐3,17‐dione (ADD) via aldolytic C–C bond cleavages involves acyl‐CoA synthetases/dehydrogenases specific for the respective 26‐, 24‐ and 22‐oic acids/‐oyl‐CoAs and promiscuous MaoC‐like enoyl‐CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA‐ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase‐dependent reactions.     

A newly discovered xenobiotic metabolic pathway: ethyl ester formation.

Formation of etretinate, ethyl ester of acitretin, can be confirmed in vitro and in vivo using acitretin as the substrate. Etretinate was identified by LC/MS. The in vitro incubation was performed using rat and human liver 12,000 g supernatant, and the in vivo experiment was conducted in rats after oral dosing of acitretin. The ethyl ester formation was greatly enhanced by addition of or dosing with ethanol.     

Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.     

Evolution of novel metabolic pathways for the degradation of chloroaromatic compounds

Chlorobenzenes are substrates not easily metabolized by existing bacteria in the environment. Specific strains, however, have been isolated from polluted environments or in laboratory selection procedures that use chlorobenzenes as their sole carbon and energy source. Genetic analysis indicated that these bacteria have acquired a novel combination of previously existing genes. One of these gene clusters contains the genes for an aromatic ring dioxy-genase and a dihydrodiol dehydrogenase. The other contains the genes for a chlorocatechol oxidative pathway. Comparison of such gene clusters with those from other aromatics degrading bacteria reveals that this process of recombining or assembly of existing genetic material must have occurred in many of them. Similarities of gene functions between pathways suggest that incorporation of existing genetic material has been the most important mechanism of expanding a metabolic pathway. Only in a few cases a horizontal expansion, that is acqui sition of gene functions to accomodate a wider range of substrates which are then all transformed in one central pathway, is observed on the genetic level. Evidence is presented indicating that the assembly process may trigger a faster divergence of nearby gene sequences. Further fine-tuning, for example by developing a proper regulation, is then the next step in the adaptation.     

Organization of metabolic pathways and molecular-genetic mechanisms of xenobiotic degradation in microorganisms: A review

Contemporary data on the mechanism of biodegradation of aromatic hydrocarbons and biodegradation genes (genomic organization and pathways of evolution) in diverse groups of microorganisms have been reviewed. Studies of this problem are topical, in view of the need in identification and construction of new strains degrading xenobiotics, particularly those halogenated. For this reason, emphasis is placed on specific features of explored metabolic pathways that can be used for constructing new enzymatic systems not present in nature. Sections on the mechanisms of genomic rearrangements involving biodegradation determinants are presented from the same standpoint. Part of the review is devoted to analyzing methods used for studying the population dynamics of bacterial communities involved in xenobiotic degradation in natural biotopes or industrial waste disposal plants. Particular attention is given to methods of gene systematics.     

Measurement of the degradation capacity of activated sludge for a xenobiotic organic

Studies were conducted to evaluate the capacities of an activated sludge in degradation process for a xenobiotic. The results showed that during its acclimation to 2,4-D, the sludge sharply accumulated degradation capacities before degradation could proceed at a noticeable rate; moreover, during de-acclimation the sludge gradually lost some capacities in a manner resembling an exponential decay.     

Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli

The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.     

NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis

Fish embryo toxicity tests for chemical risk assessment have traditionally been based upon non-specific endpoints including morphological abnormalities, hatching success, and mortality. Here we extend the application of ¹H NMR-based metabolomics in environmental toxicology by adding a suite of metabolic endpoints to the Japanese medaka (Oryzias latipes) embryo assay, with the goal to provide more sensitive, specific and unbiased biomarkers of toxicity. Medaka were exposed throughout embryogenesis to five concentrations of trichloroethylene (TCE; 0, 8.76, 21.9, 43.8, 87.6, 175 mg/L) and the relative sensitivities of the traditional and metabolomic endpoints compared. While the no-observable-adverse-effect-level for hatching success, the most sensitive traditional indicator, was 164 mg/L TCE, metabolic perturbations were detected at all exposure concentrations. Principal components analysis (PCA) highlighted a dose-response relationship between the NMR spectra of medaka extracts. In addition, 12 metabolites that exhibited highly significant dose-response relationships were identified, which indicated an energetic cost to TCE exposure. Next, embryos were exposed to 0, 0.88, 8.76 mg/L TCE and sampled on each of the 8 days of development. Projections of 66 two-dimensional J-resolved NMR spectra were obtained, and PCA revealed developmental metabolic trajectories that characterized the basal and TCE-perturbed changes in the entire NMR-visible metabolome throughout embryogenesis. Although no significant increases in mortality, gross deformity or developmental retardation were observed relative to the control group, TCE-induced metabolic perturbations were observed on day 8. In conclusion, these results support the continued development of NMR-based metabolomics as a rapid and reproducible tool for biomarker discovery and environmental risk assessment.     

An interdependent metabolic patchwork in the nested symbiosis of mealybugs

Highly reduced genomes of 144-416 kilobases have been described from nutrient-provisioning bacterial symbionts of several insect lineages [1-5]. Some host insects have formed stable associations with pairs of bacterial symbionts that live in specialized cells and provide them with essential nutrients; genomic data from these systems have revealed remarkable levels of metabolic complementarity between the symbiont pairs [3, 4, 6, 7]. The mealybug Planococcus citri (Hemiptera: Pseudococcidae) contains dual bacterial symbionts existing with an unprecedented organization: an unnamed gammaproteobacteria, for which we propose the name Candidatus Moranella endobia, lives inside the betaproteobacteria Candidatus Tremblaya princeps [8]. Here we describe the complete genomes and metabolic contributions of these unusual nested symbionts. We show that whereas there is little overlap in retained genes involved in nutrient production between symbionts, several essential amino acid pathways in the mealybug assemblage require a patchwork of interspersed gene products from Tremblaya, Moranella, and possibly P.?citri. Furthermore, although Tremblaya has the smallest cellular genome yet described, it contains a genomic inversion present in both orientations in individual insects, starkly contrasting with the extreme structural stability typical of highly reduced bacterial genomes [4, 9, 10].     

A novel metabolic pathway for creatinine degradation in Pseudomonas putida 77

Abstract A simple and rapid method is described to determine the plasmid content of cyanobacteria. This procedure is a modification of the Eckhardt in-well lysis and agarose gel electrophoresis technique and can be used for both unicellular and filamentous cyanobacteria.     

Extracellular metabolomics: a metabolic footprinting approach to assess fiber degradation in complex media

This work reports the implementation and optimization of a method for high-throughput analysis of metabolites produced by the breakdown of natural polysaccharides by microorganisms. Our simple protocol enables simultaneous separation and quantification of more than 40 different sugars and sugar derivatives, in addition to several organic acids in complex media, using 50-mul samples and a standard gas chromatography-mass spectrometry platform that was fully optimized for this purpose. As an implementation proof-of-concept, we assayed extracellular metabolite levels of three bacterial strains cultivated on complex medium rich in polysaccharides and under identical growth conditions. We demonstrate that the metabolic footprinting profile data distinguish among sample types such as typical metabolomics data. Moreover, we demonstrate that the differential metabolite-level data provide insight on specific fibrolytic activity of the different microbial strains and lay the groundwork for integrated proteome-metabolome studies of fiber-degrading microorganisms.     

Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae

Dynamic regulation has been widely applied to optimize metabolic flux distribution. However, compared with prokaryotes, quorum sensing-mediated pathway control is still very limited in Saccharomyces cerevisiae. In this study, we designed quorum sensing-regulated protein degradation circuits for dynamic metabolic pathway control in S. cerevisiae. The synthetic quorum sensing circuits were developed by integration of a plant hormone cytokinin system with the endogenous yeast Ypd1-Skn7 signal transduction pathway and the positive feedback circuits were optimized by promoter engineering. We then constructed an auxin-inducible protein degradation system and used quorum sensing circuits to regulate auxin synthesis to achieve dynamic control of protein degradation. As a demonstration, the circuits were applied to control Erg9 degradation to produce α-farnesene and the titer of α-farnesene increased by 80%. The population-regulated protein degradation system developed here extends dynamic regulation to the protein level in S. cerevisiae and is a promising approach for metabolic pathway control.     

Meta-All: a system for managing metabolic pathway information

Background  

Many attempts are being made to understand biological subjects at a systems level. A major resource for these approaches are biological databases, storing manifold information about DNA, RNA and protein sequences including their functional and structural motifs, molecular markers, mRNA expression levels, metabolite concentrations, protein-protein interactions, phenotypic traits or taxonomic relationships. The use of these databases is often hampered by the fact that they are designed for special application areas and thus lack universality. Databases on metabolic pathways, which provide an increasingly important foundation for many analyses of biochemical processes at a systems level, are no exception from the rule. Data stored in central databases such as KEGG, BRENDA or SABIO-RK is often limited to read-only access. If experimentalists want to store their own data, possibly still under investigation, there are two possibilities. They can either develop their own information system for managing that own data, which is very time-consuming and costly, or they can try to store their data in existing systems, which is often restricted. Hence, an out-of-the-box information system for managing metabolic pathway data is needed.     

Evolution of a secondary metabolic pathway from primary metabolism: shikimate and quinate biosynthesis in plants

The shikimate pathway synthesizes aromatic amino acids essential for protein biosynthesis. Shikimate dehydrogenase (SDH) is a central enzyme of this primary metabolic pathway, producing shikimate. The structurally similar quinate is a secondary metabolite synthesized by quinate dehydrogenase (QDH). SDH and QDH belong to the same gene family, which diverged into two phylogenetic clades after a defining gene duplication just prior to the angiosperm/gymnosperm split. Non‐seed plants that diverged before this duplication harbour only a single gene of this family. Extant representatives from the chlorophytes (Chlamydomonas reinhardtii), bryophytes (Physcomitrella patens) and lycophytes (Selaginella moellendorfii) encoded almost exclusively SDH activity in vitro. A reconstructed ancestral sequence representing the node just prior to the gene duplication also encoded SDH activity. Quinate dehydrogenase activity was gained only in seed plants following gene duplication. Quinate dehydrogenases of gymnosperms, represented here by Pinus taeda, may be reminiscent of an evolutionary intermediate since they encode equal SDH and QDH activities. The second copy in P. taeda maintained specificity for shikimate similar to the activity found in the angiosperm SDH sister clade. The codon for a tyrosine residue within the active site displayed a signature of positive selection at the node defining the QDH clade, where it changed to a glycine. Replacing the tyrosine with a glycine in a highly shikimate‐specific angiosperm SDH was sufficient to gain some QDH function. Thus, very few mutations were necessary to facilitate the evolution of QDH genes.     

Hyaluronan catabolism: a new metabolic pathway

A new pathway of intermediary metabolism is described involving the catabolism of hyaluronan. The cell surface hyaluronan receptor, CD44, two hyaluronidases, Hyal-1 and Hyal-2, and two lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase, are involved. This metabolic cascade begins in lipid raft invaginations at the cell membrane surface. Degradation of the high-molecular-weight extracellular hyaluronan occurs in a series of discreet steps generating hyaluronan chains of decreasing sizes. The biological functions of the oligomers at each quantum step differ widely, from the space-filling, hydrating, anti-angiogenic, immunosuppressive 10(4)-kDa extracellular polymer, to 20-kDa intermediate polymers that are highly angiogenic, immuno-stimulatory, and inflammatory. This is followed by degradation to small oligomers that can induce heat shock proteins and that are anti-apoptotic. The single sugar products, glucuronic acid and a glucosamine derivative are released from lysosomes to the cytoplasm, where they become available for other metabolic cycles. There are 15 g of hyaluronan in the 70-kg individual, of which 5 g are cycled daily through this pathway. Some of the steps in this catabolic cascade can be commandeered by cancer cells in the process of growth, invasion, and metastatic spread.     

Community analysis and metabolic pathway of halophilic bacteria for phenol degradation in saline environment

A moderately halophilic bacterial enrichment was able to degrade 120 mg/L of phenol in the presence of 1–2 M of NaCl within 3 d or 2.5–3 M of NaCl within 6 d. The optimal degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. PCR-DGGE profile of the enrichment showed that the Acidobacterium sp. and Chloroflexus sp. dominated the community. The phenol-biodegradation pathways consisted of an initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. Nuclear magnetic resonance (NMR) spectroscopy profiles showed that ectoine and hydroxyectoine were the main compatible solutes to adjust the bacterial osmotic pressure. This study provides further information on the understanding of phenol-degradation over a wide range of salinity and remediation of phenol as a pollutant in the environment.     

Loss of degradation capacity of activated sludge for a xenobiotic after a period without its influent

Xenobiotic shock experiments were conducted on lab-scale continuous-flow activated sludge systems to examine activated sludge treatment performance and to determine the xenobiotic degrader loss after periods of xenobiotic absence. The systems were operated with normal influent of a xenobiotic and a biogenic substrate until steady state, and were then artificially disturbed by removing and re-adding the xenobiotic in the influent. Substantial xenobiotic leaks were found when xenobiotic absent time was approximately one mean cell residence time (theta(c)), and the system failed when xenobiotic absent time was longer than a theta(c). Amount of degrader at the time of dual substrate steady state was estimated to be approximately 6% of the total sludge. As the xenobiotic absence time was lengthened, degrader amount in the system was reduced exponentially at a half life of approximately three days. The loss rate could be attributed mainly to the rate of displacement by theta(c) operation, followed by endogenous decay and de-acclimation loss.