A new function of a putative UDP-glucose 4-epimerase on the expression of glycoside hydrolase genes in Aspergillus aculeatus
A new function of a putative UDP-glucose 4-epimerase on the expression of glycoside hydrolase genes in Aspergillus aculeatus
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DOI：
10.1007/s00253-022-12337-8
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发表时间：
2023-01
影响因子：
5
通讯作者：
M. Kuga;Hidetoshi Shiroyanagi;T. Kawaguchi;S. Tani
中科院分区：
文献类型：
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作者：
M. Kuga;Hidetoshi Shiroyanagi;T. Kawaguchi;S. Tani
为了弄清棘孢曲霉糖苷水解酶基因的诱导机制，我们筛选了约9,000个插入转移DNA（T-DNA）的突变体，寻找参与诱导的正调控因子。由于突变体具有乳清酸核苷5′-单磷酸脱羧酶基因作为报告基因来监测纤维二糖水解酶I基因（cbhI）的纤维素响应性表达，因此通过针对5-氟乳清酸（5-FOA）的反选择来分离候选菌株。一个在uge 5位点携带T-DNA的5-FOA抗性突变体显示出降低的纤维素利用和cbhi I表达。aculeatusUge 5与烟曲霉uge 5（AspergillusfumigatusUge 5）同源（Afu 5g 10780;E-value，0.0; identities，93%），其催化尿苷二磷酸（UDP）-葡萄糖转化为UDP-吡喃半乳糖。A. aculeatus（Δ uge 5）在补充有半乳糖、刺槐豆胶（LBG）和瓜尔豆胶作为碳源的基本培养基上显示出减少的分生孢子形成。在含LBG的液体培养基中，β-1，4-内切葡聚糖酶和β-1，4-甘露聚糖酶的产量在第5天分别降低到对照菌株的10%和6%，而在含麦麸的培养基中没有观察到差异。在Δ uge 5中存在甘露二糖的情况下，主要纤维素分解基因和甘露糖分解基因的表达降低至对照菌株的15%以下，而纤维二糖响应性表达仅在早期诱导时间点适度降低。由于所有测试基因都由转录因子ManR控制，这些数据表明Uge 5参与了通过ManR控制的基因的诱导物依赖性选择性表达。关键点·UDP-葡萄糖4-差向异构酶（Uge 5）调节糖基水解酶基因的表达。·ManR调节纤维二糖和甘露二糖响应性表达。·Uge 5在甘露二糖应答表达中起着关键作用。
In order to figure out the induction mechanisms of glycoside hydrolase genes inAspergillus aculeatus, we screened approximately 9,000 transfer DNA (T-DNA)-inserted mutants for positive regulators involved in the induction. Since the mutants possess the orotidine 5′-monophosphate decarboxylase gene as a reporter gene to monitor the cellulose-responsive expression of the cellobiohydrolase I gene (cbhI), candidate strains were isolated by counterselection against 5-fluoroorotic acid (5-FOA). One 5-FOA-resistant mutant harboring the T-DNA at theuge5locus showed reduced cellulose utilization andcbhIexpression.A. aculeatusUge5 is homologous toAspergillus fumigatus uge5(Afu5g10780;E-value, 0.0; identities, 93%), which catalyzes the conversion of uridine diphosphate (UDP)-glucose to UDP-galactopyranose. Theuge5deletion mutant inA. aculeatus(Δuge5) showed reduced conidium formation on minimal media supplemented with galactose, locust bean gum (LBG), and guar gum as a carbon source. β-1,4-Endoglucanase and β-1,4-mannanase production in submerged culture containing LBG was reduced to 10% and 6% of the control strain at day 5, respectively, but no difference was observed in cultures containing wheat bran. The expression of major cellulolytic and mannolytic genes in the presence of mannobiose in Δuge5was reduced to less than 15% of the control strain, while cellobiose-responsive expression was only modestly reduced at early inducing time points. Since all test genes were controlled by a transcription factor ManR, these data demonstrate that Uge5 is involved in inducer-dependent selective expression of genes controlled via ManR.Key points•UDP-glucose 4-epimerase (Uge5) regulates expression of glycosyl hydrolase genes.•ManR regulates both cellobiose- and mannobiose-responsive expression.•Uge5 plays a key role in mannobiose-responsive expression.Graphical Abstract