Characterization of a novel pathway for xanthene degradation by the engineered strain Sphingobium yanoikuyae B1DR

: Polyaromatic hydrocarbons (PAHs) are a group of aromatic compounds that contain at least two rings. These compounds are found naturally in petroleum products and are considered the most prevalent pollutants in the environment. The lack of microorganism capable of degrading some PAHs led to their accumulation in the environment which usually causes major health problems as many of these compounds are known carcinogens. Xanthene is one of the small PAHs which has three rings. Many xanthene derivatives are useful dyes that are used for dyeing wood and cosmetic articles. However, several studies have illustrated that these compounds have toxic and carcinogenic effects. The first step of the bacterial degradation of xanthene is conducted by dioxygenase enzymes that introduces two oxygen atoms in the structure of the aromatic rings. In this study we focused on the bacterial bioremediation of xanthene via Sphingobium yanoikuyae B1DR, an engineered strain carrying the dioxin angular dioxygenase from Sphingomonas wittichii RW1. HPLC analysis of supernatant from resting cells of S. yanoikuyae B1DR grown on xanthene and succinate showed the ability of this strain to transform xanthene to 2-hydroxyphenylacetate that was not produced by the wild type of Sphingobium yanoikuyae B1. Production of 2-hydroxyphenylacetate was confirmed by GC-MS. Our results show the importance of this strain in reducing the toxic effects of xanthene in the environment and showed for the first time that ring-hydroxylation enzymes and hydrolases for biphenyl degradation in S. yanoikuyae B1 may function on metabolites generated from the degradation pathway of xanthene. By analyzing our results we were able to draw a novel pathway for xanthene degradation in S. yanoikuyae B1DR.

Polyaromatic hydrocarbons (PAHs) are a group of aromatic compounds that contain at least two rings. These compounds are found naturally in petroleum products and are considered the most prevalent pollutants in the environment. The lack of microorganism capable of degrading some PAHs led to their accumulation in the environment which usually causes major health problems as many of these compounds are known carcinogens. Xanthene is one of the small PAHs which has three rings. Many xanthene derivatives are useful dyes that are used for dyeing wood and cosmetic articles. However, several studies have illustrated that these compounds have toxic and carcinogenic effects. The first step of the bacterial degradation of xanthene is conducted by dioxygenase enzymes that introduces two oxygen atoms in the structure of the aromatic rings. In this study we focused on the bacterial bioremediation of xanthene via Sphingobium yanoikuyae B1DR, an engineered strain carrying the dioxin angular dioxygenase from Sphingomonas wittichii RW1. HPLC analysis of supernatant from resting cells of S. yanoikuyae B1DR grown on xanthene and succinate showed the ability of this strain to transform xanthene to 2hydroxyphenylacetate that was not produced by the wild type of Sphingobium yanoikuyae B1. Production of 2-hydroxyphenylacetate was confirmed by GC-MS. Our results show the importance of this strain in reducing the toxic effects of xanthene in the environment and showed for the first time that ringhydroxylation enzymes and hydrolases for biphenyl degradation in S. yanoikuyae B1 may function on metabolites generated from the degradation pathway of xanthene. By analyzing our results we were able to draw a novel pathway for xanthene degradation in S. yanoikuyae B1DR.

Introduction:
Polyaromatic hydrocarbons (PAHs) are a group of organic compounds that are biochemically stable and contain at least two aromatic rings. The major sources of PAHs are petroleum products and the incomplete combustion of fossil fuels 1 . Also, PAHs have five emission sources; agricultural, industrial, mobile, domestic, and natural. These compounds are considered as widespread pollutants in the environment and some of them are known carcinogens, where many studies on rabbits showed that exposure to PAH-containing compounds can cause tumors. However, PAHs are still used in the manufacture of plastic, pesticides, and dyes 2,3 . The accumulation of these compounds in the environment is due to the difficulty of microorganism to degrade them and to the fact that most PAHs are insoluble in water. Each compound of PAHs has a unique UV spectrum which can be used for their identification 4 . PAHs are classified based on their size into small simple compounds, which contain up to six rings, and complex large compounds, which contain more than six rings 5 . Xanthene is an example of small PAHs as it has three aromatic rings with the formula CH2[C6H4]2O. Many xanthene derivatives are useful dyes such as rhodamines, eosins, rose bengal and fluorescein. These dyes are used for dyeing wood, cosmetic articles, and fountain pen inks, therefore, are unintentionally excluded to the environment [6][7][8] . Several studies have tested the toxicity and the carcinogenicity of xanthene dyes, for instance: acridine red, rhodamine 6G, fluorescein sodium, and eosine yellowish. Multiple studies have illustrated that most of these dyes, especially those containing o-carboxy-phenyl group, caused sarcoma in rats 9 .
Several physical and chemical methods were used to reduce the hazardous effects of PAHs including xanthene dyes from the environment 10 . However, bioremediation, the treatment of pollutants by the use of microorganisms, has been found to be the most effective as it is less expensive and has low impacts on the environment 11,12 . The microorganisms that can degrade PAHs have special enzymes called dioxygenases which initiate the degradation process by introducing the oxygen atoms into the aromatic rings. The rate of the PAHs degradation depends on the molecular weight of these compounds and the type of the microorganisms that involved in the process. PAHs with low molecular weight are degraded faster and easier than the PAHs that have high molecular weight. The reason for that is the low molecular weight PAHs has high water solubility which increases the accessibility to the degrading microorganisms. Also, there are other factors that affect the PAHs bioremediation such as oxygen availability, moisture, soil type, temperature, pH, and PAH concentration 13,14 .
Although there are many microorganisms that were biochemically and genetically characterized for their ability to degraded PAHs such as sphingomonads and pseudomonads, yet to our knowledge no bacterium has been isolated for its ability to degrade xanthenes 15,16 . Through our search in literature we were only able to detect a unique white rot fungus (Coriolus versicolor) that has the ability to degrade xanthene and its derivatives 17 . Xanthone is another simple polyaromatic hydrocarbon that is similar to xanthene in structure. Our search found a single mutant bacteria (Arthrobacter GFB100) that was capable of degrading xanthone and use it as a sole source for carbon and energy 18 . We predicted that their steps for degradation would also be similar so that we can use the degradation pathway for xanthone to predict how xanthene is degraded. Xanthone degradation pathway by Arthrobacter GFB100 is similar to the degradation pathways of some polyaromatic heterocyclic compounds such as biphenyl, DBF, and DD [19][20][21][22] . The first step begins with the oxidation of xanthone to 3,4dihydroxyxanthone (DHX) via a dihydroxylating dioxygenase complex. The second step is catalyzed by (DHX) dioxygenase which stimulates an aromatic ring cleavage reaction and forms a yellow metabolite known as 4-hydroxy-3-(2'-oxo-3-transbutenoic acid)-coumarin (RFM). The third step is catalyzed by an NADPH dependent reductase which reduces the RFM to gentisic acid. The last step involves a dioxygenase that catalyzes the cleavage of gentisate which goes through sequential intermediates and ultimately forms fumarate and pyruvate that enters the TCA cycle to generate energy 18 .
Sphingobium yanoikuyae B1 was first isolated from polluted stream by Gibson in 1973 and was known as Beijerinckia species 19 . Sphingobium yanoikuyae is gram negative, obligate aerobes, nonmotile, nonsporulating, rod-shaped bacteria that produce deep-yellow colonies. The species that belong to sphingomonas has a unique outer membrane which contain glycosphingolipids instead of lipopolysaccharides 20 . The wild type S. yanoikuyae B1 is a biphenyl degrader that is incapable of degrading xanthene, DD, or DBF probably due to lack of an angular dioxygenase. However, previous studies showed that replacing its lateral dioxygenase with the angular dioxygenase of S. wittichii RW1 led to its growth on biphenyl and DD. Due to the fact that the angular dioxygenase of RW1 is capable of oxygenating xanthene at an angular position 23 . We predicted that this strain will be capable of metabolizing the further downstream intermediates formed. In this study, we have tested the ability of the engineered strain S. yanoikuyae B1DR to degraded xanthene, identified the intermediates generated using GC-MS, and were able to draw a proposed pathway for the degradation of xanthene in this strain.

Materials and methods: Bacterial strains and cultivation conditions
Sphingobium yanoikuyae B1DR is an engineered strain constructed from the substitution of the biphenyl lateral dioxygenase of S. yanoikuyae B1 with the dioxin angular dioxygenase from S. wittichii RW1. The wild type S. yanoikuyae B1 strain was kindly donated by Dr. Gerben J. Zylstra (Rutgers University, USA) and used as a control. S. yanoikuyae B1DR was grown on Luria Bertani (LB) media or Mineral Salts Basal (MSB) minimal media containing 50µg/ml kanamycin and an appropriate carbon source when needed and incubated at 30 o C for 24-48 hours. E. coli BL21/ pET_DBFDOS is an expression vector used as a positive control to produce THDM, this vector contains the dioxygenase system of S. wittichii RW1 and was grown on LB media containing 50µg/ml kanamycin at 37 o C for 24 hours 23 . Preparation of resting cells B1DR stain was cultured on LB plates containing 50 μg/ml kanamycin. The growing colonies were subcultured into 100 ml LB broth and incubated for 16 hours. Then, 2ml of this culture was used to inoculate 100 ml of LB broth which was incubated at 37°C with shaking (180 rpm). After reaching an OD600 of 1, the cells were pelleted and washed twice with phosphate buffer (50 mM sodium-potassium phosphate buffer) at pH 6.8. They were resuspended in phosphate buffer and 3mM of the xanthene plus 20mM succinate were added to the flasks and incubated at 30°C for 24 hours, the supernatant was then collected and analyzed by HPLC 24 . The supernatant from S. yanoikuyae B1DR strain grown on succinate alone and E. coli BL21/ pET_DBFDOS were used as controls were processed in a similar method. Supernatants were then filtered and analyzed with a Beckman (CA, USA) liquid chromatography system.

HPLC analysis
In order to examine the oxygenated intermediates, a reverse phase C18 column (4.6mm by 25mm) and a gradient of 0-50% methanol in water were used under acidic conditions (0.1% acetic acid) and a flow rate of 1ml/min. Peaks detection were monitored at both 254nm and 280nm wavelength 25 .

Analysis by GC-MS
The water-soluble products of the samples that showed positive results on the HPLC were extracted from the aqueous phase of the bacterial suspension by ethyl acetate as previously described 26 . Then, a mixture that contained 1ml of each extracted sample with 50μl of Sylon BFT reagent (contained trimethylsilyl (TMS) used for derivitization) was made followed by incubation for 1 hour at 80°C.
GC-MS analysis was done using a Varian 3400 GC machine with a capillary column (30 meter × 0.32 mm) Guardian-ZB-5MS (Phenomonex) (5% phenyl, polydimethylphenylsiloxane) with a 0.25μm film thickness. One microliter was the injection volume at 300°C. The temperature of the GC column was set to start at 50°C for 3min and then rised up to 320°C at a rate of 10°C /min with a 10 min hold at the upper limit. The transfer line temperature of the GC-MS was 320°C. EI mode (70 eV) scanning masses 35-750 m/z was used to operate the TSQ-7000 mass spectrometer once each second. In order to have data, peak list, and area integration values on spreadsheet, total ion current (TIC) chromatograms were integrated with the system software.

Results and Discussion: Testing growth on xanthene and resting cells experiments
S. yanoikuyae B1DR lacked the ability to grow on MSB plates supplemented with xanthene, however, the culture produced a brown intermediate when incubated for 48 hours on this substrate unlike the S. yanoikuyae B1 wild type. Therefore, we hypothesized that the presence of the dioxin dioxygenase of RW1 in this strain is being involved in metabolizing xanthene to a further extent. To determine the pathway that this organism is taking to degrade xanthene, two different supernatants from resting cells of S. yanoikuyae B1DR growing on succinate+xanthene and another growing on succinate alone (negative control), were collected and analyzed by Hplc and compared to a supernatant from E.coli pET_DBFDOS grown with xanthene (positive control). As illustrated in Fig. 1, our results detected a huge peak at the minute 33 from the supernatant of E.coli pET_DBFDOS grown with xanthene that represents the initial intermediate formed from the oxygenation of xanthene, 2,2'3-trihydroxydiphenylmethane (THDM). A previous study proved that this intermediate was 2,2'3trihydroxydiphenylmethane after GC-MS, 1H NMR, and gCOSY analysis 23 . Interestingly, the peak for THDM disappeared from the supernatant of S. yanoikuyae B1DR even though this organism still contains the same dioxygenase system found in E.coli pET_DBFDOS (originally the S. wittichii RW1 dioxygenase). In addition, S. yanoikuyae B1DR growing on succinate+xanthene contained extra peaks in the Hplc spectra that were not found neither in S. yanoikuyae B1DR growing on succinate (negative control) nor E.coli pET_DBFDOS grown with xanthene (positive control). This indicates the ability of S. yanoikuyae B1DR to further metabolize xanthene to simpler intermediates that follow THDM. Detection of degradation pathways depending on the structure of intermediates generated has been used to study the degradation of fluoranthene in Celeribacter baekdonensis B30 27 .
More precisely, S. yanoikuyae B1DR should contain a meta cleavage dioxygenase that is capable of metabolizing THDM. Genetic studies have diagnosed two important ring cleavage dioxygenases in S. yanoikuyae B1 designated BphC (2,3 dihydroxybiphenyl 1,2 dioxygenase) and xylE (catechol 2,3 dioxygenase) that are involved in biphenyl and xylene degradation, respectively 28 . Gene knockout experiments of bphC have showed that the second ring cleavage dioxygenase, xylE, can take place bphC in 2,3 dihydroxybiphenyl degradation thus complement growth on biphenyl 375 when bphC is missing. Even though the growth was slower, however xylE was successfully capable of complementing bphC mutants 29 . Therefore, we suggest that one of these enzymes, more likely BphC, is involved in THDM metabolism as this enzyme was found to be more specific towards 2,3 dihydroxybiphenyl which is somehow structurally similar to THDM unlike XylE which was more specific for single ring aromatics particularly catechol degradation.

GS-MS analysis
Ring cleavage products formed from the degradation pathways of aromatic hydrocarbons are known to have a yellow color 30 . Thus, if S. yanoikuyae B1DR contained an enzyme that cleaves one of the rings in THDM, a yellow intermediate should accumulate. However, this was not what we noticed with our accumulated metabolite from xanthene degradation. This led us to hypothesize that the ring cleavage product for xanthene can be further metabolized by S. yanoikuyae B1DR to a simpler compound. For this purpose, the supernatant from S. yanoikuyae B1DR growing on xanthene and succinate was extracted and analyzed by GC-MS to identify the molecular weight of the accumulated product. Results from GC-MS analysis detected a product that carried a molecular weight of 296 which is equivalent to the molecular weight of hydroxyphenyl acetate (152) with two TMS molecules attached (total of 144) as shown in (Fig.  2). phenyl)-6-oxo-2,4-octadieneoic acid) to 2hydroxyphenyl acetate and 2-hydroxypenta 2,4dienoate (Fig. 3). The latter was not seen in the extract of S. yanoikuyae B1DR growing on xanthene and succinate which is probably due to its conversion to simpler products. The best candidate we believe is involved in this hydrolysis step is the biphenyl hydrolase, BphD, as this enzyme has been proven to function on ring cleavage products formed from the biphenyl and the dibenzo-dioxin pathway, therefore we believe it may be the enzyme functioning on this step due to the similarities of the substrates from these pathways 23 . To our knowledge, we expect the degradation pathway for xanthene in S. yanoikuyae B1DR to follow the proposed pathway shown in Fig. 3.
Although 2-hydroxyphenylacetate has a lower level of toxicity than xanthene, yet it can cause eye and skin irritation. However, there have been evidence for the presence of many bacterial strains that are known for their ability to degrade 2hydroxyphenylacetate, the best example is Geobacillus thermodenitrificans NG80-2 31 . On the other hand, 2-hydroxyphenylacetate was found to be one of the precursors of benzylpenicillin production via filamentous fungi (e.g. Penicillium rubens and Aspergillus nidulans), thus these fungal species may help to remove this byproduct from the environment thus removing the toxicity of xanthenes in the environent 32 .
Organic synthetic dyes, including xanthenes, have been used extensively as colors in paper, plastic, cosmetics, and ink. During the synthesis and processing of these dyes, about 15% are lost in effluent. Some of these dyes are toxic compounds and can cause organic pollution as well as they are very recalcitrant against biodegradable processes 17,33 .

Conclusion:
In this study we have shown the ability of the engineered strain S. yanoikuyae B1DR to transform xanthene to the less toxic compounds, 2hydroxyphenylacetate and 2-hydroxypenta 2,4dienoate. These end products are easily degraded by other environmental organisms 31 . In addition, this study is successfully able to prove that S. yanoikuyae B1DR contains a ring cleavage enzyme and a hydrolase that metabolize THDM formed from the dioxygenation of xanthene. By analyzing our results, we are able to draw a proposed pathway for xanthene degradation in this strain. Our results identify for the first time a novel pathway in S. yanoikuyae B1DR that is used to degrade xanthene to fewer toxic products. This strain can be used as a bacterial candidate to eliminate xanthene from contaminated sites.