Functional identification of fatty aldehyde dehydrogenase genes CtAld1 and CtAld2 from Candida tropicalis

Abstract:

[Objective] Candida tropicalis has become an important industrial strain to produce dicarboxylic acids due to its high ω-oxidation activity. Fatty aldehyde dehydrogenases (FALDHs) play important roles in the ω-oxidation pathway, converting fatty aldehydes to fatty acids. However, FALDHs characterization and their roles in the synthesis of dibasic acids have not been studied in depth. Therefore, we cloned two genes CtAld1 and CtAld2 encoding FALDHs and evaluated their function in cell phenotype, enzyme activity and dicarboxylic acids accumulation. [Methods] We screened two genes of CtAld1 and CtAld2 through genome mining and sequence alignment. Based on sequence analysis, we deleted CtAld1 and CtAld2 either separately or accumulatively by homologous recombination method, and generated various mutants. The effect of deletion of CtAld1 and CtAld2 on cell growth, FALDH activity and dodecanedioic acid (DCA12) production were evaluated and compared. [Results] XZX-1 (ΔCtAld1/ΔCtAld1), XZX-2 (ΔCtAld2/ΔCtAld2) and XZX-12 (ΔCtAld1/ΔCtAld1, ΔCtAld2/ΔCtAld2) were obtained. When using dodecane as sole carbon source, deletion of CtAld2 gene significantly inhibited cell growth, and the intracellular FALDH activity was only 30% of the parental strain. Deleting CtAld1 had a slight promotion of cell growth, however intracellular FALDH activity was decreased to some extent. Furthermore, simultaneous deletion of CtAld1 and CtAld2 significantly impaired the cell growth performance and decreased FALDH activity, thus causing a distinct lower DCA12 yield compared to the wild type strain. [Conclusion] The deletion of CtAld1 in C. tropicalis could reduce the yield of DCA12. And CtAld2 plays an important role in the growth on dodecane and production of DCA12. To our knowledge, they could be recruited as target genes for metabolic engineering of ω-oxidation pathway in C. tropicalis.