Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709
Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709
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DOI：
10.1186/s12934-020-01307-2
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发表时间：
2020-02-24
影响因子：
6.4
通讯作者：
Lu, Fuping
中科院分区：
文献类型：
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作者：
Zhou, Cuixia;Zhou, Huiying;Lu, Fuping
背景地衣芽孢杆菌2709是一种广泛应用于高水平生产外源蛋白的宿主菌，但其野生型特性如发酵过程中产生粘性物质和泡沫等，严重影响了其在工业发酵中的应用。如何将其从一个土壤细菌培养成一个具有超强分泌能力、不易驯化的细胞工厂，一直是工业生产中的关键问题。此外，通过比较不同质粒和基因组整合位点在B中的转录效率，还没有优化碱性蛋白酶等诱导酶的最佳表达模式。地衣状。结果通过无标记基因组编辑方法，对地衣芽孢杆菌2709进行了基因改造，成功地将与起泡相关的天然lchAC基因和编码胞外粘多糖的eps簇基因进行了破坏。我们进一步优化了碱性蛋白酶基因（aprE）的表达，通过筛选最有效的表达系统之间的不同模块化质粒和基因组位点。结果表明，aprE的基因组表达上级优于质粒表达，通过宿主优化和染色体整合在复制起点附近，aprE的转录水平提高了1.67倍，酶活比野生型碱性蛋白酶产生菌B提高了62.19%.地衣形的结论成功构建了AprE高产菌株，其发酵特性可通过宿主基因改造和表达优化应用于大规模生产。总之，宿主优化是一种通过消除宿主的有害性状和优化表达模式来提高酶产量的使能技术。我们相信，这些策略可以应用于提高异源蛋白在其他芽孢杆菌属物种中的表达。
Background Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. Result Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. Conclusion We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.