Hydroxylation and Further Oxidation of Δ9-Tetrahydrocannabinol by Alkane-Degrading Bacteria

The microbial biotransformation of Δ⁹-tetrahydrocannabinol was investigated using a collection of 206 alkane-degrading strains. Fifteen percent of these strains, mainly gram-positive strains from the genera Rhodococcus, Mycobacterium, Gordonia, and Dietzia, yielded more-polar derivatives. Eight derivatives were produced on a mg scale, isolated, and purified, and their chemical structures were elucidated with the use of liquid chromatography-mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and two-dimensional NMR (¹H-¹H correlation spectroscopy and heteronuclear multiple bond coherence). All eight biotransformation products possessed modified alkyl chains, with hydroxy, carboxy, and ester functionalities. In a number of strains, β-oxidation of the initially formed C₅ carboxylic acid led to the formation of a carboxylic acid lacking two methylene groups.Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the decarboxylated product of the corresponding Δ⁹-THC acid, the major cannabinoid present in the cannabis plant (Cannabis sativa L., Cannabaceae). This compound is officially registered as a drug for the stimulation of appetite and antiemesis in patients under chemotherapy and human immunodeficiency virus therapy regimens. Other biological activities ascribed to this compound include lowering intraocular pressure in glaucoma, acting as an analgesic for muscle relaxation, immunosuppression, sedation, bronchodilation, and neuroprotection (11).Δ⁹-THC and many of its derivatives are highly lipophilic and poorly water soluble. Calculations of the n-octanol/water partition coefficient (K_o/w) of Δ⁹-THC at neutral pH vary between 6,000, using the shake flask method (15), and 9.44 × 10⁶, by reverse-phase high-performance liquid chromatography estimation (19). The poor water solubility and high lipophilicity of cannabinoids cause their absorption across the lipid bilayer membranes and fast elimination from blood circulation. In terms of the “Lipinsky rule of 5” (14), the high lipophilicity of cannabinoids hinders the further development of these compounds into large-scale pharmaceutical products.To generate more water-soluble analogues, one can either apply de novo chemical synthesis (as, e.g., in reference 16) or modify naturally occurring cannabinoids, e.g., by introducing hydroxy, carbonyl, or carboxy groups. Chemical hydroxylation of compounds such as cannabinoids is difficult (Δ⁹-THC is easily converted into Δ⁸-THC under mild conditions), and therefore microbial biotransformation of cannabinoids is potentially a more fruitful option to achieve this goal.So far, studies on biotransformation of Δ⁹-THC were mainly focused on fungi, which led to the formation of a number of mono- and dihydroxylated derivatives. Previous reports on the biotransformation of cannabinoids by various microorganisms are summarized in Table ​Table1.1. The aim of the present study was to test whether bacterial strains are capable of transforming Δ⁹-THC into new products (with potentially better pharmaceutical characteristics) at a higher yield and specificity than previously found for fungal strains. For this purpose, we have chosen to use a collection of alkane-degrading strains, since it was shown in previous studies (8, 18, 20) that alkane oxygenases often display a broad substrate range. Production of novel cannabinoid derivatives that might have interesting pharmacological activities was another objective of this project.

TABLE 1.

Previous biotransformation experiments conducted using various microorganisms to transform cannabinoids

Elimination of d-Lactate Synthesis Increases Poly(3-Hydroxybutyrate) and Ethanol Synthesis from Glycerol and Affects Cofactor Distribution in Recombinant Escherichia coli

The effect of eliminating d-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP⁺ ratio was higher than the NADH/NAD⁺ ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP⁺. In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.Poly(3-hydroxybutyrate) (PHB) is the best-known and most common polyhydroxyalkanoate (PHA). PHAs are polymers with thermoplastic properties that are totally biodegradable by microorganisms present in most environments and that can be produced from different renewable carbon sources (38). Accumulated as intracellular granules by many bacteria under unfavorable conditions (1, 21), PHAs are carbon and energy reserves and also act as electron sinks, enhancing the fitness and stress resistance of bacteria and contributing to redox balance (12, 30). Escherichia coli offers a well-defined physiological environment for the construction and manipulation of various metabolic pathways to produce different bioproducts, such as PHB, from cost-effective carbon sources.In recent years, a significant increase in the production of biodiesel has caused a sharp fall in the cost of glycerol, the main by-product of biodiesel synthesis. As a result, glycerol has become a very attractive substrate for bacterial fermentations (10), specially for reduced products, such as PHB (36). The E. coli strain used in this work, K24K, carries phaBAC, the structural genes responsible for PHB synthesis, from Azotobacter sp. strain FA8 (23) (Table ​(Table1).1). The pha genes in K24K are expressed from a chimeric promoter and consequently are not subject to the genetic regulatory systems present in natural PHA producers. Because of this, it can be assumed that regulation of PHA synthesis in the recombinants is restricted by enzyme activity levels, modulated principally by substrate availability. In most natural producers, and also in PHB-producing E. coli recombinants, PHB is synthesized through the condensation of two molecules of acetyl-coenzyme A (acetyl-CoA), catalyzed by an acetoacetyl-CoA transferase or 3-ketothiolase, resulting in acetoacetyl-CoA. This compound is subsequently reduced by an NAD(P)H-dependent acetoacetyl-CoA reductase to R-(−)-3-hydroxybutyryl-CoA, which is then polymerized by a specific PHA synthase (34).

TABLE 1.

E. coli strains, plasmids, and oligonucleotides used in this study

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.Succinic acid is valued as one of the key basic chemicals used in the preparation of biodegradable polymers or as raw material for chemicals of the C₄ family (8, 19). The fermentative production of succinic acid from renewable resources is environmentally acceptable and sustainable (3). A breakthrough in genetically engineering Escherichia coli (6, 7, 11, 18) for succinate production was the isolation of strain AFP111 (1, 4), a mutant of NZN111 with a spontaneous ptsG mutation (pflAB ldhA double mutant). The process involves a two-stage fermentation, with aerobic cell growth followed by anaerobic conditions for succinate production (16, 21, 22). The aerobically induced enzymes can maintain their activity during the anaerobic phase and significantly affect succinate fermentation (22, 23). Using the best transition time based on the activities of the key enzymes and other physiological states, a two-stage fermentation using the recombinant AFP111 strain harboring pTrc99A-pyc achieved a final succinic acid concentration and productivity of 99.2 g·liter⁻¹ and 1.3 g·liter⁻¹·h⁻¹, respectively (21).Aerobic cell growth is essential for the subsequent anaerobic fermentation. However, few studies have focused on the regulation of aerobic cell growth. As a regulation method, gluconeogenic carbon sources were used instead of glucose for the aerobic growth of Escherichia coli NZN111 and the activities of enzymes that are favorable for the anaerobic synthesis of succinate were enhanced (23, 24). Unfortunately, a gluconeogenic carbon source (e.g., sodium acetate) might increase the osmotic pressure of culture media, which would be detrimental to succinate production (23). As another regulation method, a glucose feeding strategy controlling the glucose concentration at about 0.5 g·liter⁻¹ up to 1 g·liter⁻¹ was reported to prevent excessive formation of acetic acid (16).In this study, we investigated different glucose feeding strategies for the aerobic growth phase of the two-phase process for succinate production by E. coli AFP111. Specifically, we compared several growth rates by using glucose limitation in addition to maximum growth under conditions of excess glucose.E. coli AFP111 [F⁺ λ⁻ rpoS396(Am) rph-1 ΔpflAB::Cam ldhA::Kan ptsG] (4, 16), which was a kind gift from D. P. Clark (Southern Illinois University), was the only strain used in this study. Luria-Bertani (LB) medium (60 ml) was used for inoculum culture in 1,000-ml flasks, and 3 liters of chemically defined medium (13, 14) was used for two-stage culture in a 7-liter fermentor. Two-stage fermentations were divided into three types, based on the glucose feeding strategy used during the aerobic stage. For type I culture, the glucose concentration was maintained at about 20 g·liter⁻¹ during aerobic cell growth. Type II and III cultures comprised a batch process and subsequent glucose-limited fed-batch process (Fig. ​(Fig.1).1). The batch process initially contained 13 g/liter of glucose. The fed-batch process began when the dry cell weight (DCW) reached about 6 g/liter, with type II and type III cultures using a 600 g/liter glucose feed to achieve cell growth rates of 0.15 h⁻¹ and 0.07 h⁻¹, respectively (10). When the DCW reached 12 g·liter⁻¹, the aerobically grown cells were directly transferred to anaerobic conditions (Fig. ​(Fig.1).1). For the anaerobic process, oxygen-free CO₂ was sparged at 0.5 liter·min⁻¹, the pH was controlled between 6.4 and 6.8 with intermittent supplementation of solid magnesium carbonate hydroxide, and the glucose concentration was maintained at about 20 g·liter⁻¹ by supplying glucose in an 800-g·liter⁻¹ solution.Open in a separate windowFIG. 1.Concentrations of glucose (circles), DCW (triangles), and succinic acid (squares) in the three types of two-stage fermentation by AFP111. μ, growth rate.The optical density at 600 nm was used to monitor cell growth, and this value was correlated to DCW. The concentration of glucose was assayed with an enzyme electrode analyzer, and organic acids were quantified by high-performance liquid chromatography (HPLC). The intracellular concentrations of NADH and NAD⁺ were assayed with a cycling method (12). The activities of isocitrate lyase (ICL) (20), pyruvate kinase (PYK) (17), phosphoenolpyruvate (PEP) carboxykinase (PCK) (20, 23), PEP carboxylase (PPC) (23), and malate dehydrogenase (MDH) (23) were measured spectrophotometrically at the end of the aerobic phase and 12 h after the onset of the anaerobic phase.All three types of fermentations were terminated when the succinate concentration increased less than 1 g·liter⁻¹ in 5 h. Type III fermentation was terminated at a final succinic acid concentration of 101.2 g·liter⁻¹ and an anaerobic-phase productivity of 1.89 g·liter⁻¹·h⁻¹ (Fig. ​(Fig.1).1). Trace amounts of by-products (such as acetate, ethanol, and pyruvate) accumulated and did not follow any trend in the anaerobic phase (data not shown).At the end of the aerobic culture phase, the specific enzyme activities of PCK, PYK, and ICL in type III culture were 2.9, 2.5, and 11.4 times higher, respectively, than the activities in type I culture (Table ​(Table1)1) . This phenomenon is consistent with published reports that suggest that the expression of enzymes involved in anaplerotic metabolism and the glyoxylate shunt (5, 15) is elevated in E. coli grown under glucose-limited conditions. These enzymes maintained their activities in the subsequent anaerobic phase (Table ​(Table1)1) and would be central to succinate production (22, 23). The elevated levels of PCK and PPC would provide the reductive branch of the tricarboxylic acid (TCA) cycle with oxaloacetate (OAA) at a higher rate (9), thereby supplying both malate and citrate (Table ​(Table11).

TABLE 1.

Activities of enzymes at the end of the aerobic culture phase and 12 h after the onset of the anaerobic phase

Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.The aim of microbial source tracking (MST) methods is to identify, and in some cases quantify, the dominant sources of fecal contamination in surface waters and groundwater (2, 16). One of the most promising library- and cultivation-independent approaches utilizes fecal Bacteroidales bacteria and quantitative PCR (qPCR) assays to measure gene copies of host-specific genetic markers for 16S rRNA (4, 5, 10, 14). Currently, molecular assays do not directly discriminate between viable and nonviable cells since DNA of both live and dead cells and extracellular DNA can be amplified. Consequently, source tracking data based on detection of genetic markers by PCR cannot distinguish between recent and past contamination events since DNA of selected pathogens can persist after cell death for more than 3 weeks (6). Hence, it would be preferable to detect host-specific markers in viable cells of Bacteroidales bacteria, which are strictly anaerobic microorganisms and unlikely to survive in water.Previous studies have suggested the use of intercalating DNA-binding chemicals combined with PCR to inhibit PCR amplification of DNA derived from dead cells (8, 9, 11, 15). For example, ethidium monoazide (EMA) has been investigated as a means of reducing the PCR signal from DNA originating from dead bacterial cells (7, 15, 19). However, the use of EMA prior to DNA extraction has been found to result in a significant loss of the genomic DNA of viable cells in the case of Escherichia coli 0157:H7, Campylobacter jejuni, and Listeria monocytogenes (3, 7). Recently propidium monoazide (PMA) has been proposed as a more selective agent, penetrating only dead bacterial cells but not viable cells with intact membranes (8). EMA/PMA in combination with PCR or qPCR has been applied to identify viable food-borne pathogens in a simple matrix (3, 7, 8, 11), and possible restrictions in the use of PMA in environmental samples were reported (9, 19). Yet the feasibility of applying PMA in environmental samples or MST studies using fecal Bacteroidales bacteria has not been systematically studied. Any meaningful application of EMA or PMA in stool or natural water samples must consider potential interferences due to particulate matter present in the environmental matrix. Similarly, procedures for the concentration of large volumes of water samples to simultaneously monitor pathogens and MST identifiers can lower the limit of detection (4, 12), but they concentrate solids or other inhibitors of quantitative PCR (qPCR) as well, which might interfere in the covalent binding of PMA to DNA.The objectives of this study were, therefore, the following: (i) to evaluate the applicability of PMA-qPCR methods to detect culturable Bacteroides fragilis, (ii) to determine the feasibility of PMA-qPCR analysis for environmental samples containing different concentrations of solids, and (iii) to validate the utility of the PMA-qPCR method for the detection of fecal Bacteroidales bacteria in defined live and heat-treated mixtures of human feces and in wastewater treatment plant influent and effluent.Pure cultures of Bacteroides fragilis (ATCC 25285) were grown in thioglycolate broth (Anaerobe System, Morgan Hill, CA) under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD). The solids were obtained by hollow-fiber ultrafiltration as described previously (12, 13). Ultrasonification and heat sterilization in an autoclave were used for removing attached bacteria or DNA from solids and inactivating residual DNA. Finally, the solids were resuspended with 1× phosphate-buffered saline (PBS) solution to 100 mg liter⁻¹ or 1,000 mg liter⁻¹ of suspended solids. The concentration of total suspended solids (TSS) was measured using method 2450 C (1). Next, 1 ml of broth medium containing 2 × 10⁹ viable or 2 × 10⁸ heat-treated B. fragilis cells, which had been exposed at 80°C for 20 min, was spiked into 1× PBS buffer solutions containing 0 mg liter⁻¹, 100 mg liter⁻¹, or 1,000 mg liter⁻¹ of TSS. Before the cells were spiked, 1 ml of Bacteroides fragilis cell suspension was enumerated with the Live/Dead BacLight bacterial viability kit (Molecular Probes Inc., Eugene, OR) using a hemacytometer and an Axioskop 2 Plus epifluorescence microscope (Zeiss, Thornwood, NY) equipped with two filter sets (fluorescein isothiocyanate and Texas Red). The inoculated samples were incubated under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD) for 4 h at 20°C to allow sufficient time for the cells to sorb to solids.A fresh human fecal specimen was obtained from a healthy adult. Two grams of feces was suspended in 25 ml 1× PBS. The fecal suspension was diluted 1:10 and 1:100 in a 1× PBS solution, and aliquots were subjected to heat treatment at 80°C for 20 min. The heat-treated fecal portions were mixed with fresh diluted samples (1:10 and 1:100 dilutions) in defined ratios, with fresh feces representing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% of the total, respectively. Effluent and influent water samples were collected in sterile 2-liter bottles from the University of California, Davis, wastewater treatment plant. The effluent samples were concentrated to approximately 200 ml by hollow-fiber ultrafiltration (12).PMA (Biotium Inc., Hayward, CA) was prepared, stored, and used as described in previous studies (8, 9), but PMA concentrations and light exposure time were varied to determine the optimal condition of PMA-qPCR; the PMA concentrations were 2 μM, 6 μM, 20 μM, and 100 μM. Light exposure times were 1 min, 5 min, 10 min, and 20 min. Genomic DNA was extracted using the FastDNA spin kit for soil (Biomedicals, Solon, OH). Cell lysis was achieved by bead beating using a bead mill Minibread beater (Biospec Products Inc., Bartlesville, OK) at 2,400 rpm for 20 s. Otherwise, DNA extraction was performed according to the manufacturer''s instructions. TaqMan probe and primer assays targeting the rRNA genes of all fecal Bacteroidales bacteria (BacUni-UCD) and mixed human-specific Bacteroidales bacteria (BacHum-UCD), developed by Kildare et al. (4), were used to detect and quantify fecal Bacteroidales bacteria present in fecal and (waste)water samples.We explored the ability of PMA-qPCR to discriminate between viable and heat-killed cells at different solids concentrations using Bacteroides fragilis cultures (Fig. ​(Fig.1).1). PMA did not influence the PCR amplification of DNA derived from viable cells when no solids were present (TSS = 0 mg liter⁻¹) (Fig. ​(Fig.1A).1A). The level of PMA concentration slightly affected the mean cycle threshold differences (ΔC_T) of viable cells at higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹) (Fig. 1C and E). The signal reductions in the amplification of heat-killed cells were a function of both the PMA concentration and exposure time (Fig. 1B, D, and F). Lower solids concentrations did not inhibit the efficacy of discrimination from heat-killed cells. However, solids at 1,000 mg liter⁻¹ affected the amplification of DNA derived from heat-killed cells. Higher solids concentrations affected the suppression of PCR amplification from heat-treated cells by interfering with the cross-linking of PMA. In agreement with previous reports, the number of viable Bacteroides fragilis cells was underestimated in our study when EMA-treated and untreated samples containing only viable cells were compared because mean ΔC_T values were as high as 10 (data not shown). In contrast to EMA, PMA seems to not penetrate live cells, since higher selectivity of PMA is most probably associated with the higher charge of the molecule (8).Open in a separate windowFIG. 1.Effect of PMA on amplification of BacUni-UCD universal marker in viable and dead Bacteroides fragilis cells with different concentrations of solids. The contour lines represented ΔC_T values and were generated by the Origin Pro 8 software program. The mean cycle threshold differences (ΔC_T) were calculated by subtracting C_T values obtained without PMA treatment from C_T values obtained with PMA treatment. (A and B) ΔC_T for viable cells (A) or dead cells (B) in the absence of added solids. (C and D) ΔC_T for viable cells (C) or dead cells (D) at a solids concentration of 100 mg liter⁻¹. (E and F) ΔC_T for viable cells (E) or dead cells (F) at a solids concentration of 1,000 mg liter⁻¹.A factorial three-way analysis of variance including the PMA concentration, exposure time, and TSS concentration was performed to determine the interferences of solids and the optimal PMA-qPCR condition in the differentiation of viable cells from dead cells (Table ​(Table1).1). The mean ΔC_T of viable cells in the PMA experiments was slightly influenced by the PMA concentration (P = 0.05) in the absence of solids (TSS = 0 mg liter⁻¹), but the effect was biologically insignificant (mean ΔC_T = 0.004). The PMA concentration had a significant effect on ΔC_T values for both viable and dead cells in the presence of higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹), as shown in Table ​Table1.1. However, the effect of exposure time in PMA treatment was insignificant at a TSS concentration of 1,000 mg liter⁻¹ (P > 0.4). The solids concentration caused significantly different ΔC_T values for viable and dead cells in the PMA treatments (P < 0.001) as determined by factorial three-way analysis. The greatest differences in the mean ΔC_T values between viable and dead cells were seen at 100 μM of PMA and with a 10-min exposure time, as determined by Tukey''s comparison test, for TSS concentrations of 100 mg liter⁻¹ and 1,000 mg liter⁻¹. Ideally, shorter light exposure and a lower concentration of dye can minimize the penetration of live cells. However, these conditions were not compatible with sufficient inhibition of amplification of DNA from dead cells for PMA treatment.

TABLE 1.

Statistical analysis for differences (ΔC_T) between nontreatment and PMA treatment for experiments where Bacteroides fragilis was spiked^a