热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2的功能评价

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

【目的】热带假丝酵母是发酵法生产二元酸的重要工业菌株,具有较高的ω-氧化活性。脂肪醛脱氢酶在ω-氧化途径中起重要作用,催化脂肪醛生成脂肪酸,但其具体催化功能及对细胞生理影响还未被系统研究。本文通过删除脂肪醛脱氢酶基因CtAld1和CtAld2鉴定了其在ω-氧化途径中的功能。【方法】通过基因组信息挖掘获得热带假丝酵母脂肪醛脱氢酶基因CtAld1和CtAld2序列,在此基础上,通过同源重组敲除CtAld1和CtAld2基因。考察突变株的生长和胞内脂肪醛脱氢酶活性变化,并评价CtAld1和CtAld2基因敲除对细胞二元酸合成能力的影响。【结果】分别获得了热带假丝酵母突变株XZX-1(ΔCtAld1/ΔCtAld1)、XZX-2(ΔCtAld2/ΔCtAld2)和XZX-12(ΔCtAld1/ΔCtAld1,ΔCtAld2/ΔCtAld2)。在以十二烷为唯一碳源的培养基中,敲除CtAld2基因显著抑制细胞的生长,胞内脂肪醛脱氢酶活性降低为出发菌株的30%;敲除CtAld1基因尽管会使细胞损失一部分醛脱氢酶活性,但能够一定程度地提升细胞在十二烷中的生长性能。敲除CtAld1或CtAld2会降低菌株二元酸产量,组合敲除CtAld1和CtAld2严重削弱菌株十二碳二元酸的合成能力。【结论】CtAld2对热带假丝酵母细胞的生长和十二碳二元酸的合成具有重要作用,缺失CtAld1或CtAld2基因降低细胞的二元酸合成能力。CtAld1和CtAld2可作为热带假丝酵母ω-氧化途径代谢工程改造的潜在靶点。

[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.