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。

关键词:光滑球拟酵母;Med15B;长链脂肪酸;不饱和脂肪酸;低pH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.

Key words:Candida glabrata;Med15B;long chain fatty acids;unsaturated fatty acids;low pH tolerance