Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating
Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating
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DOI：
10.1007/s00284-020-01902-x
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发表时间：
2020-01-30
影响因子：
2.6
通讯作者：
Qian, Guoliang
中科院分区：
文献类型：
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作者：
Ren, Xuexiang;Ren, Shuangshuang;Qian, Guoliang
热稳定抗真菌因子 (HSAF) 是一种广谱抗真菌抗生素，由生物防治剂溶酶杆菌产生。在我们早期的工作中，我们应用HSAF来有效控制小麦和梨的真菌病害。然而，其实际应用的一个主要瓶颈是HSAF生产水平较低；因此，提高其产量对于其广泛应用至关重要。过去，我们发现c-di-GMP（一种通用的细菌第二信使）对HSAF的产生具有抑制作用。在这项工作中，我们通过生物信息学和遗传分析进一步鉴定了溶杆菌酶基因中负责 c-di-GMP 合成的八种活性二鸟苷酸环化酶 (DGC)。我们生成了缺乏七个活性 DGC 基因的菌株，并发现这种 DGC 修饰菌株 OH11LC 以 c-di-GMP 浓度依赖性方式产生更高的 HSAF 量。随后，通过使用 OH11LC 作为宿主发酵菌株，我们甚至可以生产更高的 HSAF 量（> 200 倍）。在改进 HSAF 生产后，我们进一步开发了 HSAF 种子包衣方法技术，该技术可有效对抗玉米种子传播的丝状病原体禾谷腐霉 (Pythium gramineacola)。总的来说，通过结合菌株修饰和发酵优化，我们展示了将细菌c-di-GMP信号传导的基础知识转化为生物防治应用的一个很好的例子，其中我们通过删除一堆潜在活性的产酶乳杆菌DGC基因来减轻c-di-GMP对HSAF生物合成的抑制作用，以提高HSAF产量并扩大其在抗真菌种衣中的用途。
Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.