A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181
A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181
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hmgA 的单点突变导致苏云金芽孢杆菌 BMB181 中黑色素积累。
DOI：
10.1016/j.enzmictec.2018.10.007
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发表时间：
2019-01-01
影响因子：
3.4
通讯作者：
Cai, Jun
中科院分区：
文献类型：
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作者：
Tan, Tong-tong;Zhang, Xu-dong;Cai, Jun
苏云金芽孢杆菌BMB 181（Bt BMB 181）是一株高产黑色素的菌株，是光稳定杀虫剂工业化生产的理想候选菌株。然而，其黑素生成途径仍不清楚。在本研究中，我们证明了Bt BMB 181在用甲基磺草酮处理后不能产生黑色素，甲基磺草酮是黑色素合成的尿黑酸途径中4-羟基苯丙酮酸双加氧酶的抑制剂。异源表达实验表明，BtBMB 171中的尿黑酸-1，2-双加氧酶（HmgA）功能正常，而BtBMB 181中的HmgA至少部分丧失了活性。利用CRISPR-Cas9系统敲除Bt BMB 171中的hmgA基因，突变菌株获得了产生黑色素的能力。此外，补充的菌株恢复到野生型表型。此外，Bt BMB 181自身的hmgA基因的过量表达也导致不能产生色素。BLAST分析结果表明，BtBMB 181的HmgA基因的G272 E突变是由815 G → A的单点突变引起的。纯化的HmgA 171的酶活性比HmgA 181高10倍以上。最后，我们确定hmgA中的突变是Bt BMB 181中黑色素积累的原因。本研究结果为苏云金B.thuringiensis黑色素的合成与调控提供了新的认识，并将促进其在工业上的应用。
Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815G -> A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new in-sights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.