Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, 300071, China.
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, 300071, China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin, 300071, China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin, 300071, China.
Enzyme Microb Technol. 2019 Jan;120:91-97. doi: 10.1016/j.enzmictec.2018.10.007. Epub 2018 Oct 19.
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 insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.
苏云金芽胞杆菌 BMB181（Bt BMB181）具有较高的黑色素产量，是工业规模生产光稳定杀虫剂的理想候选菌株。然而，其黑色素生物合成途径尚不清楚。在本研究中，我们发现 Bt BMB181 在霍格尼斯酸途径中 4-羟基苯丙酮酸双加氧酶抑制剂 mesotrione 的作用下无法产生黑色素。异源表达实验表明，Bt BMB171 中的 4-羟基苯丙酮酸双加氧酶（HmgA）正常发挥作用，但 Bt BMB181 中的 HmgA 至少部分丧失了活性。利用 CRISPR-Cas9 系统敲除了 Bt BMB171 中的 hmgA 基因，该突变株获得了产生黑色素的能力。此外，互补菌株恢复为野生型表型。另外，在 Bt BMB181 中过表达其自身的 hmgA 基因也导致色素无法产生。BLAST 结果表明，Bt BMB181 中 HmgA 的氨基酸改变（G272E）是由单个点突变（815G→A）引起的。纯化的 HmgA171 的酶活性比 HmgA181 高 10 多倍。最后，我们确定 hmgA 的突变是导致 Bt BMB181 黑色素积累的原因。我们的研究结果为苏云金芽胞杆菌黑色素生物合成和调控提供了新的见解，并将促进其未来的工业应用。
