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theses

Sphingomonas wittichii RW1 is one of three strains known to degrade dibenzo-pdioxin (DXN) and dibenzofuran (DBF). Due to the toxic, carcinogenic, and endocrine disruption characteristics of these compounds molecular and biochemical studies of the
enzymes involved in the DXN and DBF degradative pathways has been of great interest. Dibenzofuran 4,4a-dioxygenase (DBFDO) is the first enzyme involved in the DXN and DBF degradation pathways. This enzyme is a heterodimer of two polypeptides DxnA1 (45KDa) and DxnA2 (23KDa) which functions to add two atoms of molecular oxygen at two adjacent carbon atoms where one of the carbons is a bridge atom between the two benzene rings. This enzyme is thus often called an angular dioxygenase. Based on studies with the purified enzyme DBFDO is known to be capable of hydroxylating other aromatic compounds, however, the exact product made and the location where the dioxygenation occurs on these aromatics are unknown. For that purpose, we cloned the four genes necessary for DBFDO into E. coli in the pET30a expression vector. This included the genes for the two oxygenase subunits dxnA1 and dxnA2, the reductase redA2, and the ferredoxin fdx3. Results based on the HPLC, GC-MS, and NMR analysis showed that E. coli BL21 strains harboring this clone when induced had the ability to perform three types of oxygenations: angular dioxygenation towards DBF, DXN, 2-hydroxyDBF, xanthene, and xanthone; cis-dihydroxylation towards biphenyl, phenanthrene, anthracene, and xanthone; and monooxygenation to the benzylic methylenic group in fluorene as well as monooxygenation and dioxygenation to the sulfur heteroatom in dibenzothiophene. On the other hand, no oxygenation was seen for diphenylmethane, diphenyl ether, carbazole, chrysene, naphthalene, pyrene, anthrone, salicylate, and toluene.
Our next goal was to engineer a DXN and DBF degrading strain which was achieved through cloning the S. wittichii RW1 dxnA1-dxnA2-redA2-fdx3 genes into the biphenyl degrading organism S. yanoikuyae B1 in place of the bphA1f-bphA2f genes thus placing the RW1 oxygenase under control of the B1 biphenyl pathway promoter. This allowed us to examine if the enzymatic activity of DBFDO towards biphenyl is sufficient to allow growth on biphenyl as the sole carbon source. More importantly, the engineered S. yanoikuyae B1 (B1DR, DR for dioxygenase replacement) will now have the ability to perform angular dioxygenation towards DXN and DBF which will allow us to identify if S. yanoikuyae B1 contains genes that metabolize these aromatics. Our results showed that B1DR had the ability to grow on biphenyl and DXN but not DBF indicating the presence of downstream DXN degrading enzymes in this organism and showing that the enzymes do not function on DBF. Gene knockout and gene insertion experiments showed that the biphenyl extradiol dioxygenase, BphC, and the HOPDA hydrolase, BphD, from S. yanoikuyae B1 are the enzymes that showed activity on DXN metabolites, as deleting bphC abolished the ability of B1DR to metabolize DXN but not biphenyl. However, deletion of bphD abolished growth of B1DR on both DXN and biphenyl. On the other hand complementing B1DRΔbphC with the specific DXN extradiol dioxygenase, SWIT3046, from RW1 retained growth on DXN, while complementing B1DRΔbphD with RW1 hydrolase, dxnB, retained growth on both DXN and biphenyl to similar growth rates as of B1DR. These results show that bphC and bphD from S. yanoikuyae B1 carry similar activity to specific DXN metabolizing genes from S. wittichii RW1 and shows that more than one extradiol dioxygenase is involved in biphenyl degradation in S. yanoikuyae B1 but only BphC functions on DXN metabolites. This work also shows that S. yanoikuyae B1 uses a single hydrolase, BphD, for biphenyl degradation which also has activity towards DXN. DBF was metabolized by B1DR only after inserting both an extradiol dioxygenase (dbfB or SWIT3046) and a hydrolase (dxnB) from S. wittichii RW1, indicating the lack of DBF degrading genes in S. yanoikuyae B1.
Even though S. wittichii RW1 is unable to use biphenyl as a sole carbon source, the present work showed that it contains an angular dioxygenase that can attack biphenyl at a lateral position producing cis-2,3-dihydro-2,3-dihydroxybiphenyl. Also, a previous study showed that its 2,2’,3-trihydroxybiphenyl dioxygenase involved in the DBF pathway, dbfB, showed activity towards 2,3-dihydroxybiphenyl. Finally, its hydrolase, dxnB, is able to hydrolyze HOPDA generated from biphenyl degradation. All these facts collectively led us to hypothesize that the only missing gene for S. witichii RW1 to grow on biphenyl would be the cis-dihydrodiol dehydrogenase. To prove our hypothesis, the gene for cis-dihydrodiol dehydrogenase, bphB, from the biphenyl degrader Sphingobium yanaikuyae B1 was placed downstream of the fdx3 gene under the control of the constitutive promoter of the dxn locus in S. wittichii RW1. Interestingly, this engineered strain grew on biphenyl revealing a hidden pathway for biphenyl degradation in RW1. Using a series of knockout mutant strains we showed the involvement of two different ring-cleavage dioxygenases and two different hydrolases in the biphenyl degradation pathway. This work demonstrates that the enzymes in the upper pathway for DBF and DXN degradation have a wide substrate range with activity towards other aromatic hydrocarbons.

ETD_10118

Ph.D.

Includes bibliographical references

doi:10.7282/t3-cct8-q279

ETD doctoral

The author owns the copyright to this work.