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Microbial metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9 and Burkholderia xenovorans LB400 Smith, Daryl James

Abstract

This dissertation has elucidated initial steps in the degradation of abietane diterpenoids by Pseudomanas abietaniphila BKME-9 and Burkholderia xenovorans LB400. A 10.4-kbp extension of the dit cluster in BKME-9 containing genes involved in abietane diterpenoid degradation has been sequenced. The ditQ gene was found to encode a cytochrome P450 monooxygenase. Knocking out ditQ had little effect on growth of BKME-9 on abietic acid (AbA) but impaired growth on dehydroabietic acid (DM) and palustric acid (PaA). A xylE transcriptional fusion showed that a range of diterpenoids induced transcription of ditQ. Substrate binding assays of DitQ revealed that DhA binds to the enzyme (Kd = 0.43 ± 0.03 μM). These results indicate that DitQ is involved in the metabolism of DhA and PaA and are consistent with its putative role in converting DhA to 7-hydroxy-DhA. The genome of LB400 was found to contain a large cluster of genes with high similarity to the BKME-9-dit cluster. Microarray transcriptional analysis revealed that of the 72 genes encoded by an 80.5-kb cluster, 43 are up-regulated at least 2-fold in expression during growth on DhA versus on succinate. This cluster has been named the LB400 dit cluster. Through 2D gel proteomic analysis, we have determined that a key difference in the catabolism of the abietane diterpenoids, DhA and AbA lies in the differential expression of a cytochrome P450, DitU (CYP226A2) encoded by the dit cluster. DitQ was expressed during growth on both DhA and AbA, whereas DitU expression was only detectable during growth on AbA. Phenotypic studies of knockout mutants in LB400 containing insertion mutations of ditQ or ditU showed that ditQ was required for growth on DhA, whereas ditU was required for growth on AbA. In cell suspension assays, patterns of metabolite accumulation confirmed the role of DitU in AbA transformation and DitQ in DhA transformation. Substrate binding assays revealed that DitQ binds both DhA (Kd = 0.98 ± 0.01 μM) and PaA (Kd = 1.6 ± 0.1 μM). An in vitro P450 assay confirmed that DitQ transforms DhA to 7-hydroxy-DhA. These results demonstrate distinct roles of DitQ and DitU in the transformation of DhA and AbA to the central intermediate, 7-oxo-DhA, in a convergent degradation pathway.

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