Functional Characterization Of The Fatty Aldehyde Dehydrogenase Genes CtALD1 And CtALD2 In Candida Tropicalis

C.tropicalis is a valuable strain in industry.It has been applied in the production of long chain dicarboxylic acids owing to its efficient ?-oxidation pathway.The oxidation of fatty aldehydes is essential in the ?-oxidation pathway,however,the fatty aldehyde dehydrogenase genes have not been characterized and identified.It is scientific important for understanding mechanism of ?-oxidation pathway and metabolic engineering of C.tropicalis to characterize fatty aldehyde dehydrogenase genes.In this thesis,two allelic putative fatty aldehyde dehydrogenase genes of C.tropicalis were cloned,heterologous expression and in vivo functional confirmation based on the whole genome DNA sequencing of the indicated strain.The critical results were showed as follows:(1)Four putative fatty aldehyde dehydrogenase genes(FALDH)were obtained from the genome of C.tropicalis and designated as CtALD1 a,CtALD1b,CtALD2 a,and CtALD2 b.Among them,the identity of amino acid sequences of CtALD1 a and CtALD1 b,CtALD2a and CtALD2 b were 95.95% and 97.06%,respectively.Sequence aligment and phylogenetic analysis showed that CtALD1 a and CtALD1 b,CtALD2a and CtALD2 b belong to two pairs of allelic genes.CtALD1 a and CtALD2 a were cloned and heterologously expressed in E.coli BL21(DE3),the emerging activity of fatty aldehyde dehydrogenase detected in the strains expressing CtALD1 a and CtALD2 a revealed that these genes are indeed FALDH.(2)C.tropicalis XZX was cultivated using glucose or methyl laurate as carbon source,respectively,and the transcription levels of the two pairs of genes were detected by qPCR.The qPCR results showed that the transcription of CtALD1 was induced when using methyl laurate as a sole carbon source,which was 70 times higher than that when using glucose as a sole carbon source.However,qPCR results showed the similar expression level for CtALD2 gene using glucose or methyl laurate as a sole carbon source.(3)CtALD1 and CtALD2 were knocked out separately or cumulatively in C.tropicalis XZX to construct XZX-1(?CtALD1),XZX-2(?CtALD2)and XZX-12(?CtALD1?CtALD2)strains,and the physiological function of mutants was evaluated.When glucose or methyl laurate was used as the sole carbon source,the growth rates of XZX-1,XZX-2,and XZX-12 were similar to those of the original strain XZX,and there was no significant difference in the final biomass.When dodecane was used as the sole carbon source,XZX-1 strain grew well,the growth rate of XZX-2 and XZX-12 cells was relatively slow,and the final biomass was about 30% and 20% of XZX,respectively.At the same time,the intracellular fatty aldehyde dehydrogenase activity of XZX-2 decreased by 71.6% compared with the original strain,and the intracellular enzyme activity of XZX-12 decreased 85.9% of the original strain.(4)At last,the effect of CtALD1 and CtALD2 disruption and overexpression on the DCA production was evaluated.The yield of dibasic acid was decreased when CtALD1 or CtALD2 gene was knocked out,and the ability to produce dicarboxylic acid was further impaired after cumulative knockout of CtALD1 and CtALD2 in 03 strain(?CAT).Whereas overexpression of CtALD1 a and CtALD2 a did not promote the synthesis of diacids.