过表达基因elo1和ole1增强光滑球拟酵母△med15B菌株的低pH耐受能力

Enhancing low pH tolerance of Candida glabrata △med15B by overexpressing genes elo1 and ole1

【目的】研究中介体亚基Med15B(ORF CAGL0H06215g)介导的脂肪酸代谢影响光滑球拟酵母(Candida glabrata)耐受低pH胁迫的生理机制。【方法】在菌株Δmed15B中过量表达脂肪酸延伸酶基因elo1(ORF CAGL0L08184g)和Δ9去饱和酶基因ole1(ORF CAGL0I00418g),构建过表达菌株Δmed15B/elo1-ole1,然后与菌株Δmed15B和亲本菌株ATCC55对比分析基因elo1和ole1的表达水平、脂肪酸组分比例、细胞膜完整性和耐受能力在pH 2.0和pH 6.0条件下的差异。【结果】发现,在pH 2.0条件下过表达菌株Δmed15B/elo1-ole1:(1)基因elo1和ole1表达水平比菌株Δmed15B分别上调了4.1倍和3.3倍,与亲本菌株ATCC55相比分别上调了2.5倍和2.2倍;(2)脂肪酸平均链长延长至17.4,高于菌株Δmed15B的16.8和亲本菌株ATCC55的17.1;不饱和脂肪酸与饱和脂肪酸的比值比菌株Δmed15B和亲本菌株ATCC55分别提高了53.1%和41.5%;(3)碘化丙啶(PI)染色细胞数占总检测细胞数的比例,与菌株Δmed15B和亲本菌株ATCC55比较分别下降了60.8%和37.7%;(4)细胞半抑制pH(胁迫)(IC_(50))值达到pH 2.3,比菌株Δmed15B的pH 3.7和亲本菌株ATCC55的pH 3.1具有更强的耐受能力。【结论】在菌株Δmed15B中过表达基因elo1和ole1能通过提高长链脂肪酸和不饱和脂肪酸含量,增强细胞对低pH胁迫的耐受能力。

[Objective]To investigate the effect of mediator subunit Med15B mediated fatty acid metabolism on Candida glabrata low pH tolerance.[Methods]Overexpression of genes elo1 and ole1,encoding fatty acid elongase andΔ9 desaturase,in strainΔmed15B we obtained an overexpressed strainΔmed15B/elo1-ole1.The expression level of genes elo1 and ole1,the membrane fatty acid composition,the membrane integrity and stress tolerance of parent strain ATCC55,strainΔmed15B and overexpression strainΔmed15B/elo1-ole1 were determined and compared at pH 2.0 and pH 6.0.[Results]At pH 2.0,the expression level of genes elo1 and ole1 in overexpression strainΔmed15B/elo1-ole1 was increased by 4.1-fold and 3.3-fold compared to those of strainΔmed15B,and increased by 2.5-fold and 2.2-fold compared to those of parent strain ATCC55,respectively.The average acyl-chain length of overexpression strainΔmed15B/elo1-ole1 reached 17.4 longer than that of the deletion strain(16.8)and parent strain(17.1),and the ratio of unsaturated fatty acid to saturated fatty acid(UFA/SFA ratio)increased by 53.1%and 41.5%,respectively.Furthermore,the percentage of propidium iodide(PI)-stained cell in overexpression strainΔmed15B/elo1-ole1 was decreased by 60.8%and 37.7%,respectively,compared to that of strainΔmed15B and parent strain ATCC55.Similarly,overexpression strainΔmed15B/elo1-ole1 exhibited more tolerance to low pH and the half-maximal inhibition(IC_(50))values for pH stress was reached pH 2.3,which was pH 3.7 of strainΔmed15B and pH 3.1 of parent strain ATCC55.[Conclusion]These results indicated that overexpression genes elo1 and ole1 in strainΔmed15B can enhance cell tolerance to low pH by increasing the content of long chain and unsaturated fatty acids.