Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor.

BACKGROUND: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose. RESULTS: Pichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield. CONCLUSIONS: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

Bioaugmenting the poplar rhizosphere to enhance treatment of 1,4-dioxane.

1,4-Dioxane is a highly mobile and persistent groundwater pollutant that often forms large dilute plumes. Because of this, utilizing aggressive pump-and-treat and ex-situ technologies such as advanced oxidation can be prohibitively expensive. In this study, we bioaugmented the poplar rhizosphere with dioxane-degrading bacteria Mycobacterium dioxanotrophicus PH-06 or Pseudonocardia dioxanivorans CB1190 to enhance treatment of 1,4-dioxane in bench-scale experiments. All treatments tested removed 10 mg/L dioxane to near health advisory levels (<4 µg/L). However, PH-06-bioaugmented poplar significantly outperformed all other treatments, reaching <4 µg/L in only 13 days. Growth curve experiments confirmed that PH-06 could not utilize root extract as an auxiliary carbon source for growth. Despite this limitation, our findings suggest that PH-06 is a strong bioaugmentation candidate to enhance the treatment of dioxane by phytoremediation. In addition, we confirmed that CB1190 could utilize both 1,4-dioxane and root extract as substrates. Finally, we demonstrated the large-scale production of these two strains for use in the field. Overall, this study shows that combining phytoremediation and bioaugmentation is an attractive strategy to treat dioxane-contaminated groundwater to low risk-based concentrations (~1 µg/L).

Direct conversion of theophylline to 3-methylxanthine by metabolically engineered E. coli.

BACKGROUND: Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monomethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonas putida, ndmA, ndmB, and ndmD, provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3-methylxanthine by a metabolically engineered strain of E. coli. RESULTS: Here we report the construction of E. coli strains with ndmA and ndmD, capable of producing 3-methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3-methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3-methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-methylxanthine were also produced, attributed to slight activity of NdmA at the N 3 -position of theophylline. The 1- and 3-methylxanthine products were separated by preparative chromatography with less than 5% loss during purification and were identical to commercially available standards. Purity of the isolated 3-methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chromatography-mass spectrophotometry or nuclear magnetic resonance. CONCLUSIONS: We were able to biologically produce and separate 100 mg of highly pure 3-methylxanthine from theophylline (1,3-dimethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD. This microbial conversion represents a first step to develop a new biological platform for the production of methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.

Genetic characterization of caffeine degradation by bacteria and its potential applications.

The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.

Validation of caffeine dehydrogenase from Pseudomonas sp. strain CBB1 as a suitable enzyme for a rapid caffeine detection and potential diagnostic test.

Excess consumption of caffeine (>400 mg/day/adult) can lead to adverse health effects. Recent introduction of caffeinated products (gums, jelly beans, energy drinks) might lead to excessive consumption, especially among children and nursing mothers, hence attracting the Food and Drug Administration's attention and product withdrawals. An "in-home" test will aid vigilant consumers in detecting caffeine in beverages and milk easily and quickly, thereby restricting its consumption. Known diagnostic methods lack speed and sensitivity. We report a caffeine dehydrogenase (Cdh)-based test which is highly sensitive (1-5 ppm) and detects caffeine in beverages and mother's milk in 1 min. Other components in these complex test samples do not interfere with the detection. Caffeine-dependent reduction of the dye iodonitrotetrazolium chloride results in shades of pink proportional to the levels in test samples. This test also estimates caffeine levels in pharmaceuticals, comparable to high-performance liquid chromatography. The Cdh-based test is the first with the desired attributes of a rapid and robust caffeine diagnostic kit.

Two distinct pathways for metabolism of theophylline and caffeine are coexpressed in Pseudomonas putida CBB5.

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.

A novel caffeine dehydrogenase in Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid.

A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)(+) dependent; coenzyme Q(0) was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.

Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(II)/copper(I) redox cycling.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and procarcinogens that require activation by host metabolism. Metabolic activation of PAHs by aldo-keto reductases (AKRs) leads to formation of reactive and redox active o-quinones, which may cause oxidatively generated DNA damage. Spectrophotometric assays showed that NADPH caused PAH o-quinones to enter futile redox cycles, which result in the depletion of excess cofactor. Copper(II) amplified NADPH-dependent redox cycling of the o-quinones. Concurrent with NADPH oxidation, molecular oxygen was consumed, indicating the production of ROS. To determine whether PAH o-quinones can cause 8-oxo-dGuo formation in salmon testis DNA, three prerequisite experimental conditions were satisfied. Quantitative complete enzymatic hydrolysis of DNA was achieved, adventitious oxidation of dGuo was eliminated by the use of chelex and desferal, and basal levels of less than 2.0 8-oxo-dGuo/10(5) dGuo were obtained. The HPLC-ECD analytical method was validated by spiking the DNA with standard 8-oxo-dGuo and demonstrating quantitative recovery. HPLC-ECD analysis revealed that in the presence of NADPH and Cu(II), submicromolar concentrations of PAH o-quinones generated >60.0 8-oxo-dGuo adducts/10(5) dGuo. The rank order of 8-oxo-dGuo generated in isolated DNA was NP-1,2-dione > BA-3,4-dione > 7,12-DMBA-3,4-dione > BP-7,8-dione. The formation of 8-oxo-dGuo by PAH o-quinones was concentration-dependent. It was completely or partially inhibited when catalase, tiron, or a Cu(I) specific chelator, bathocuproine, was added, indicating the requirement for H(2)O(2), O(2)(-), and Cu(I), respectively. Methional, which is a copper-hydroperoxo complex [Cu(I)OOH] scavenger, also suppressed 8-oxo-dGuo formation. By contrast, mannitol, sodium benzoate, and sodium formate, which act as hydroxyl radical scavengers, did not block its formation. Sodium azide, which can act as both a hydroxyl radical and a (1)O(2) scavenger, abolished the formation of 8-oxo-dGuo. These data showed that the production of 8-oxo-dGuo was dependent on Cu(II)/Cu(I) catalyzed redox cycling of PAH o-quinones to produce ROS and that the immediate oxidant was not hydroxyl radical or Cu(I)OOH and that it is more likely (1)O(2), which can produce a 4,8-endoperoxide-dGuo intermediate.

Development of nonsteroidal anti-inflammatory drug analogs and steroid carboxylates selective for human aldo-keto reductase isoforms: potential antineoplastic agents that work independently of cyclooxygenase isozymes.

Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPARgamma). In human myeloid leukemia (HL-60) cells, ligand access to PPARgamma is regulated by AKR1C3, which diverts PGD(2) metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPARgamma-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC(50), K(I) values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC(50) values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH(2)(+) and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.

Human cytosolic 3alpha-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3beta-hydroxysteroid dehydrogenase activity: implications for steroid hormone metabolism and action.

The source of NADPH-dependent cytosolic 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5alpha-dihydrotestosterone (DHT) into inactive 3beta-androstanediol (3beta-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3alpha-/17beta-/20alpha-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3alpha- and 3beta-Diol (established by (1)H NMR spectroscopy). The rates of 3alpha- versus 3beta-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3alpha[17beta]-hydroxysteroid oxidase activity with 3alpha-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3beta-HSD/Delta(5-4) ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3alpha- and 3beta-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3alpha/3beta-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3alpha/3beta-HSD activities are achieved. The observation that AKR1Cs are a source of 3beta-tetrahydrosteroids is of physiological significance because: (i) the formation of 3beta-Diol (in contrast to 3alpha-Diol) is virtually irreversible, (ii) 3beta-Diol is a pro-apoptotic ligand for estrogen receptor beta, and (iii) 3beta-tetrahydrosteroids act as gamma-aminobutyric acid type A receptor antagonists.