Molecular Mechanism Of Carotenoid Synthesis And Oxidative Stress Analysis In The Degradation Of Heterocycles By A Sphingobium Yanoikuyae

Microbial oxidative degradation is a potential way of removingpollutants such as heterocycles from the environment. During this process,reactive oxygen species （ROS） or other oxidants are inevitably produced, andmay cause damages to DNA, proteins, and membranes, thereby decreasingthe degradation rate. Hence, reacting quickly enough to ward off ROS attackis important for the survival of bacteria. Thus, the toxicity in bacteria due toexcessive ROS might contribute to the recalcitrance and persistence ofcontaminants in the environment. Carotenoids can serve asmembrane-integrated antioxidants, protecting cells from oxidative stress.Based on the genome sequence and annotation of Sphingobium yanoikuyaeXLDN2-5, several genes, designated as crtE, crtB, crtI, crtY, crtZ and crtW,showed sequence homologies to geranylgeranyl pyrophosphate （GGPP）synthase, phytoene synthase, phytoene dehydrogenase, lycopene cyclase,β-carotene hydroxylase, and β-carotene ketolase respectively. Most of thecarotenoid synthetic genes in strain XLDN2-5locate separately from eachother, except for crtY, crtI, and crtB. This might be attributed to a distincttime course for genomic integration （as a result of horizontal gene transfer） as compared to the tightly linked crtY and crtI genes.The above-mentioned genes were amplified using PCR, and were clonedinto the corresponding sites of a pUC19vector to construct plasmids. Thepigments accumulated in E. coli transformants carrying two distinct plasmidswere separately extracted and analyzed by HPLC and HPLC-MS. Theseresults indicate that the hypothetical gene crtI encodes the phytoenedesaturase, which catalyzes lycopene synthesis from phytoene. Thehypothetical gene crtY encodes lycopene cyclase, which catalyzes bothcyclization steps required for the production of β-carotene from lycopene.The hypothetical gene crtZ encodes β-carotene hydroxylase that catalyzes thesynthesis of zeaxanthin from β-carotene through β-cryptoxanthin. However,the predicted phytoene were not detected when the hypothetical genes crtEand crtB were co-expressed. And the plasmid carrying crtW gene could notcatalyze zeaxanthin into astaxanthin.Using HPLC coupled with an atmospheric pressure chemical ionizationmass spectrometer （HPLC-MS）, we analyzed the pigments extracted fromSphingobium yanoikuyae XLDN2-5and concluded that strain XLDN2-5accumulates zeaxanthin, which is synthesized from β-carotene throughβ-cryptoxanthin. To characterize carotenoid biosynthesis in strain XLDN2-5,the species and amount of carotenoids produced were analysis by HPLC andHPLC-MS. During the biodegradation of heterocycles, strain XLDN2-5justaccumulated zeaxanthin as the main carotenoid, while the amount ofzeaxanthin in strain XLDN2-5was significantly enhanced （CA <CA+BT <CA+DBT）.Furthermore, the levels H₂O₂in living bacterial cells with or without heterocycles （CA, BT, and DBT） was measured by using the oxidativestress-sensitive probe2,7-dichlorofluorescein-diacetate （DCFH-DA）.During the degradation of heterocycles, excessive H₂O₂are generated inbacterial cells, and that bacterial cells were exposed to much more oxidativestress than the control cells were （strain XLDN2-5grown with glucose）.During the process of long-term degradation of heterocycles, the amounts ofbacteria in per unit decreased as the increasement of the oxidative stress.After several additions of hetercycles, the degradative capabilities has beenremarkably weakened. Therefore, the ROS formation mechanism in thebacterium is likely associated with the degradation. It is reasonable to suggestthat the ROS-generated toxicity during the degradation may contribute to therecalcitrance and persistence of the heterocycles in the environment.As a possible survival adaptation mechanism, strain XLDN2-5accumulated massive amounts of zeaxanthin; in addition, the transcriptionallevel of crtI, the gene encodes the phytoene desaturase, one of the keyenzymes for carotenoid biosynthesis, showed a pronounced rise. On the otherhand, the transcriptional level of crtZ decreased relative to the controlexperiment, and no obvious regular change was observed for the crtZtranscripts with the degradation of the heterocycles. The resultant higherenzyme levels of CrtI and lower enzyme levels of CrtZ may ensure the rapidaccumulation of β-carotene, the precursor of zeaxanthin, which ultimatelyleads to the increased production of zeaxanthin.In summary, the higher production levels of zeaxanthin and H₂O₂accumulated in Sphingobium yanoikuyae XLDN2-5were consistent with thetranscriptional increase of the gene encoding phytoene desaturase （CA < CA+BT <CA+DBT）. The accumulations of carotenoids would increase theamount of strains. Our results suggest that carotenoids might play a positiverole in the degradation of heterocycles. When bacterial cells are exposed tothe oxidative stress as a result of cellular metabolism, zeaxanthinaccumulated in the bacterial cell membrane might quench the ROS, reducethe space into which heterocycles can insert and decrease the extent ofinteraction. It might also decrease the membrane fluidity during thebiodegradation; enhance the diffusion barrier of the membrane to balance thegeneration and quenching of ROS; thus ultimately protect this bacteriumfrom oxidative damage, resulting in an improved strain XLDN2-5survivalrate and more efficient biodegradation.Additionally, the genomes of two efficient carbazole-degradingpseudomonad strains, Pseudomonas luteola XLDN4-9and Pseudomonasstutzeri XLDN-R were sequenced using the Illumina HiSeq2000system.These whole-genome shotgun projects have been deposited atDDBJ/EMBL/GenBank under the accession numbers ALAT00000000andAKYE00000000for strains XLDN4-9and XLDN-R. The reads wereassembled into232and167contigs, respectively, using VELVET1.2.03. Thedraft genome sequence of strain XLDN4-9consists of4,627,073bases with aG+C content of54.2%, while the draft genome sequence of strain XLDN-Rcontains4,695,416bases with a G+C content of63.9%, which is inaccordance with other P. stutzeri. As expected, the genomes of strainsXLDN4-9and XLDN-R encode a diverse array of related proteins withpredicted roles in the metabolism of aromatic compounds, such as biphenyl,benzoate and chloroaromatic compounds. With respect to carbazoledegradation, the car genes in strains XLDN4-9and XLDN-R were found to be clustered in an arrangement similar to that of the car cluster inPseudomonas sp. strain CA10, which is different from that of Sphingobiumyanoikuyae XLDN2-5. However, the carotenoid biosynthetic pathway geneshave not been found in these two strains.Based on comparative genomics of five heterocycles-degraingbacterium and proteomic analysis of strain XLDN2-5, we found that therewere several enzymes or proteins involved in stress response, especially tooxidative stress, such as alkyl hydroperoxide reductase, catalase, glutathione/glutaredoxin, and thioredoxin. The antioxidant intracellular systerms is hencevital for an improved strain XLDN2-5survival rate under environmentalstress. They will play an important role in the long-term degradation ofheterocycles in the environment. This study implies a new research topicwhich would shed light on the advance of bioremediation or biocatalysis,especially on the bioremediation of the recalcitrance and persistence ofcontaminants in the environment.