Kinetics and key enzyme activities of phenanthrene degradation by Pseudomonas mendocina

Phenanthrene degradation by Pseudomonas mendocina CGMCC 1.766, a new phenanthrene-degrading strain, was investigated in this work. When cells were grown on 20, 50, 100 and 200 mg l⁻¹ of phenanthrene, the doubling time was 18.3, 19.8, 21.0 and 20.3 h and the growth yield during exponential phase was 242, 271, 221 and 206 mg protein (g phenanthrene)⁻¹, respectively. High level accumulation of the intermediate metabolite 1-hydroxy-2-naphthoic acid (1H2N) up to ≈94% of its theoretical yield was observed. Dynamic profiles of the activities of two key enzymes, i.e. polycyclic aromatic hydrocarbon (PAH) dioxygenase (PDO) and catechol-2,3-oxygenase (C23O), during the biodegradation were revealed and the results suggest a delicate mechanism in the regulation of these phenanthrene-degrading enzymes in this strain.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.     

5-Chloropicolinic acid is produced by specific degradation of 4-chlorobenzoic acid by Sphingomonas paucimobilis BPSI-3

We have previously shown that the bacterium Sphingomonas paucimobilis BPSI-3, isolated from PCB-contaminated soil, can degrade halogenated biphenyls, naphthalenes, catechols and benzoic acids. However, before such an organism can be used in bioremediation, it is important to characterise the degradation products and determine the degradation pathways to ensure that compounds more toxic or mobile than the original contaminants are not produced. In the degradation of 4-chlorobiphenyl, S. paucimobilis BPSI-3 produces a novel chlorinated picolinic acid. In this paper, we show that 4-chlorobenzoate is an intermediate in this degradation and, through ¹⁵N-labelling, that 5-chloropicolinate is the only nitrogenous metabolite isolated under the extraction conditions used. The position of the chlorine indicates that degradation of 4-chlorocatechol occurs exclusively via a 2,3-extradiol cleavage. These data allow us to postulate a more definitive catabolic pathway for the biodegradation of 4-chlorobiphenyl to 5-chloro-2-hydroxymuconic acid semialdehyde via 4-chlorobenzoate in S. paucimobilis BPSI-3. Received 19 April 1999/ Accepted in revised form 23 July 1999     

Choice of microbial host for the naphthalene dioxygenase bioconversion

The use of whole cell biotransformations for single and multistep enzyme conversions is gaining widespread application. In this study the naphthalene dioxygenasenah A gene was transferred intoPseudomonas aeruginosa PAC 1R,Escherichia coli JM107 andPseudomonas putida PpG 277. The effect of ethanol on these genetically engineered Gram-negative bacteria was studied by measurement of enzyme activity, stability and cell integrity. Ethanol has been used in biotransformations as a co-substrate carbon source for co-factor recycling and as a co-solvent increasing dissolved substrate and product levels. Ethanol increased the dissolved substrate (naphthalene) concentration slightly and dissolved product ((+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene) by approximately 30% at 4% (w/v) ethanol. BothP. aeruginosa PAC 1R andP. putida PpG 277 showed decreased activity with increasing ethanol concentration whilstE. coli enzyme activity increased with increasing ethanol concentration being comparable to that when glucose was used as a carbon source. This project highlighted the many factors involved in the selection of microbial hosts for whole cell biotransformation processes.     

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

Differences in effects of norharman with various classes of chemical mutagens and amounts of S-9.

The mutagenicities of aniline, $\text{o}$-toluidine and yellow OB were demonstrated only in the presence of the β-carboline compound, norharman. The effect of norharman increased linearly with increase in the amount of S-9. The mutagenicity of 4-dimethylaminoazobenzene was greatly enhanced by the presence of norharman, and again dose-dependency on the amount of S-9 was observed. In the presence of a large amount of S-9, norharman caused several fold enhancement of the mutagenicities of $\text{N}$-2-fluorenylacetamide, benzo(a)-pyrene, and 1,4-dimethyl-3-amino-5$\text{H}$-pyrido(4,3$\text{b}$) indole, isolated from a tryptophan pyrolysate. However, norharman suppressed the mutagenicities of these compounds in the presence of a small amount of S-9. The mutagenicity of kaempferol, a flavonoid, was inhibited by norharman with either a large or small amount of S-9.     

Degradation of biphenyl by methanogenic microbial consortium

Biphenyl was readily degraded and mineralized to CO₂ and CH₄ by a PCB-dechlorinating anaerobic microbial consortium. Degradation occurred when biphenyl was supplied as a sole source of carbon or as a co-metabolic substrate together with glucose and methanol. p-Cresol was detected and confirmed by mass spectroscopy as a transient intermediate. Production of ¹⁴ C-CO₂ and ¹⁴C-CH₄ from ¹⁴C-biphenyl was observed in the approximate ratio of 1:2. The results indicated the existence of novel pathways for biphenyl degradation in a natural anaerobic microbial community.     

Ethylene biosynthesis: The role of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, and its possible evolutionary origin

Recent developments in our knowledge of the biochemistry of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, the enzyme responsible for the final stage in the biosynthesis of ethylene, are reviewed. Particular reference is made to the role of carbon dioxide as an essential cofactor, the activity of ACC oxidase in the plant cell, the enzyme catalytic centre, and the role of ACC oxidase in the evolutionary development of ethylene biosynthesis in plants. Evidence is marshalled to support a proposal that the membrane requirement for ACC oxidase that is observed in vivo is attributable to a need for a charged plasma membrane to maintain ascorbate in the reduced state for an ACC oxidase located in the apoplast. It is argued on biochemical grounds that the acquisition of the ACC oxidase was the crucial evolutionary step in the development by seed plants of an ethylene biosynthesis pathway that could easily be regulated, and that signalled the plant's response to stress and pathogen attack.     

Cytochrome P450–redox partner fusion enzymes

The cytochromes P450 (P450s) are a broad class of heme b-containing mono-oxygenase enzymes. The vast majority of P450s catalyse reductive scission of molecular oxygen using electrons usually derived from coenzymes (NADH and NADPH) and delivered from redox partner proteins. Evolutionary advantages may be gained by fusion of one or more redox partners to the P450 enzyme in terms of e.g. catalytic efficiency. This route was taken by the well characterized flavocytochrome P450_BM3 system (CYP102A1) from Bacillus megaterium, in which soluble P450 and cytochrome P450 reductase enzymes are covalently linked to produce a highly efficient electron transport system for oxygenation of fatty acids and related molecules. However, genome analysis and ongoing enzyme characterization has revealed that there are a number of other novel classes of P450–redox partner fusion enzymes distributed widely in prokaryotes and eukaryotes. This review examines our current state of knowledge of the diversity of these fusion proteins and explores their structural composition and evolutionary origins.     

Purification and properties of ferredoxinBPH, a component of biphenyl 2,3-dioxygenase of Pseudomonas sp strain LB400

The ferredoxin component (ferredoxin_BPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography. The protein was a monomer with a molecular weight of 15000 and contained 2 gram-atoms each of iron and acid-labile sulfur. Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm. The spectrum was partially bleached in the visible region upon reduction by reductase_BPH with NADPH as the source of electrons. Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein. Upon reduction with sodium dithionite, signals with g_x = 1.82, g_y = 1.92 and g_z = 2.02 were detected. These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center. Ferredoxin_BPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductase_BPH to the terminal oxygenase during catalysis. Received 01 November 1996/ Accepted in revised form 27 May 1997