过表达基因elo1和ole1增强光滑球拟酵母Δmed15B菌株的低pH耐受能力

Enhancing low pH tolerance of Candida glabrata Δmed15B by overexpressing genes elo1 and ole1

摘要:

[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）（IC₅₀）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。

Abstract:

[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 and Δ9 desaturase, in strain Δmed15B we obtained an overexpressed strain Δmed15B/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, strain Δmed15B and overexpression strain Δmed15B/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 strain Δmed15B/elo1-ole1 was increased by 4.1-fold and 3.3-fold compared to those of strain Δmed15B, 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 strain Δmed15B/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 strain Δmed15B/elo1-ole1 was decreased by 60.8% and 37.7%, respectively, compared to that of strain Δmed15B and parent strain ATCC55. Similarly, overexpression strain Δmed15B/elo1-ole1 exhibited more tolerance to low pH and the half-maximal inhibition (IC₅₀) values for pH stress was reached pH 2.3, which was pH 3.7 of strain Δmed15B and pH 3.1 of parent strain ATCC55. [Conclusion] These results indicated that overexpression genes elo1 and ole1 in strain Δmed15B can enhance cell tolerance to low pH by increasing the content of long chain and unsaturated fatty acids.