代谢工程改造热带假丝酵母高效合成中链ω-羟基脂肪酸

Efficient synthesis of medium-chainω-hydroxy fatty acids by metabolic engineering in Candida tropicalis

【目的】ω-羟基脂肪酸(ω-hydroxyfatty acid,ω-HFAs)是一种绿色安全无毒,具有良好生物相容性的理想生物降解材料,广泛应用于化工、食品、药学等方面,微生物发酵法生产ω-羟基脂肪酸具有重要的研究意义和应用前景。【方法】为了得到高产ω-羟基脂肪酸的代谢工程菌株,通过同源重组技术,连续敲除二倍体热带假丝酵母β-氧化途径中酰基辅酶A氧化酶基因(ACO)的同工酶POX基因,考察基因缺失对细胞生长以及合成ω-羟基脂肪酸能力的影响。【结果】基于脂肪醇氧化酶(FAO)基因缺陷菌株,成功构建了重组菌株F4PT(ΔPOX4,ΔPOX5),在碳源为葡萄糖的培养基中,重组菌株出现生长减缓,但在碳源为脂肪酸甲酯(C12–C14)的培养基中生长明显减缓,说明删除POX4、POX5基因使脂肪酸甲酯的代谢能力降低,其次通过摇瓶发酵实验,12-羟基十二酸和14-羟基十四酸的产量分别为3.01 g/L和8.69 g/L,转化率分别为11.44%和33.14%,达到积累ω-羟基脂肪酸的目的,并且代谢十四酸甲酯的能力是明显强于代谢十二酸甲酯。【结论】FAO、POX基因双缺失的热带假丝酵母可以明显积累ω-羟基脂肪酸,不同碳链的积累效率有差异,其中14-羟基十四酸的产量及转化率更高,重组菌株有定向转化十四酸甲酯为14-羟基十四酸的潜能。

[Objective]ω-hydroxy fatty acid(ω-HFAs)is a green,safe,non-toxic,ideal biodegradable material with good biocompatibility.It is widely used in chemical industry,food,pharmacy.Microbial fermentation,the method for producingω-HFAs has important research significance and application prospects.[Methods]In order to obtain high-yield metabolically engineered strains ofω-hydroxy fatty acids,the POX gene in theβ-oxidation of Candida tropicalis was continuously knocked out through homologous recombination technology.The effect of gene deletion on cell growth and the ability to synthesizeω-hydroxy fatty acids was investigated.[Results]The recombinant strain F4PT(ΔPOX4,ΔPOX5)was successfully constructed.Growth in the medium with fatty acid methyl esters(C12~C14)as the carbon source was significantly slower than that in glucose,indicating that the deletion of the POX4 and POX5 genes reduced the metabolic ability of fatty acid methyl esters.Secondly,through the fermentation experiment,the concentration of 12-hydroxydodecanoic acid and 14-hydroxytetradecanoic acid were 3.01 g/L and 8.69 g/L,the conversion rates were 11.44%and 33.14%,respectively.The ability to metabolize fourteen-carbon fatty acids is significantly stronger than that of twelve-carbon fatty acids.[Conclusion]Candida tropicalis with double deletion of FAO and POX genes can obviously accumulateω-hydroxy fatty acids,but the accumulation efficiency of different carbon chains is different.The production and conversion rate of 14-hydroxytetradecanoic acid are higher,and the recombinant strain has the potential to convert methyl tetradecanoate to 14-hydroxytetradecanoic acid.