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Molecular genetic investigation of the abietane diterpenoid degradation pathway of pseudomonas abietaniphila BKME-9

University of British Columbia

Pseudomonas abietaniphila BKME-9 is able to degrade abietane diterpenoids, via a dioxygenolytic pathway. Tn5 transposon mutagenesis and inverse PCR were used to clone and sequence the dit gene cluster which encodes enzymes and a transcription regulator of the catabolic pathway for abietane diterpenoid degradation in P. abietaniphila BKME-9. The gene cluster is located on a 16.7 kb EcoRI-EcoRI DNA fragment containing 13 complete and 1 partial open reading frames (ORFs). The genes ditAl ditA2 and ditA3 encode the a and p subunits and the ferredoxin of a new class of ring-hydroxylating dioxygenases. Sequence analysis of the ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin, and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in E. coli of ditAl A2 and dit A3, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxodehydroabietic acid but not dehydroabietic acid (DhA), at C-l 1 and C- 12, producing 7-oxo-ll,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 {ditAl::ln5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxoDhA in cell suspensions assays, demonstrating that the dioxygenase is essential and central to the abietane catabolic pathway. Using xylE reporter gene transcriptional fusions, it was shown that abietic, dehydroabietic, 7-oxodehydroabietic, isopimaric and 12,14-dichlorodehydroabietic acids induce the expression of ditAl and ditA3. In addition to the aromatic ring-hydroxylating dioxygenase genes, the dit cluster encodes the extradiol ring cleavage dioxygenase ditC and the IclR-type transcriptional regulator ditR. The ditC gene is required for the growth of P. abietaniphila on abietanes. Cell suspensions of the ditC mutant strain produced a yellow colored supernatant and accumulated several DhA metabolites. Although ditR is not required for the growth of strain BKME-9 on abietanes, catechol-2,3-dioxygenase activity of xylE reporter strains with a dilR.Km* mutation demonstrated that it encodes a transcriptional activator of ditA3 and possibly a repressor of ditAl. A second Tn5 transposon mutant with an insertion in a gene with similarity to reductases of cytochrome P-450 and alkane hydroxylases was identified. However, further experiments aimed at confirming the presence of a cytochrome P-450 in P. abietaniphila provided inconclusive results.

Thesis/Dissertation

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2009-07-02

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http://hdl.handle.net/2429/10019

Microbiology and Immunology

University of British Columbia

UBCV

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