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.

中文翻译：

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转基因地衣芽孢杆菌2709可优化碱性蛋白酶的表达并提高其产量。

背景技术地衣芽孢杆菌2709被广泛用作高水平生产异源蛋白的宿主，但芽孢杆菌细胞通常具有不利的野生型特性，例如发酵过程中产生粘性物质和泡沫，这严重影响了工业发酵的应用。 。如何将其从土壤细菌发展为具有较少未驯化性质的超分泌细胞工厂，始终在工业生产中起着至关重要的作用。此外，还没有通过比较地衣芽孢杆菌中不同质粒的转录效率和基因组整合位点来优化诱导酶（如碱性蛋白酶）的最佳表达方式。结果地衣芽孢杆菌2709通过无标记基因组编辑方法破坏了与起泡有关的天然lchAC基因和编码胞外粘多糖的eps簇而进行了遗传修饰。通过筛选不同模块质粒和基因组位点之间最有效的表达系统，我们进一步优化了碱性蛋白酶基因（aprE）的表达。结果表明，aprE的基因组表达优于质粒表达，并且通过宿主优化和复制起点附近的染色体整合，aprE的转录水平最终大大提高了1.67倍，而酶活性与p53相比显着提高了62.19％。野生型产生碱性蛋白酶的菌株地衣芽孢杆菌。结论我们成功地设计了无不良特性的AprE高产菌株，其发酵特性可通过宿主遗传修饰和表达优化应用于批量生产。总之，宿主优化是通过消除宿主的有害性状和优化表达模式来提高酶生产的一项使能技术。我们认为，这些策略可用于改善其他芽孢杆菌属物种中的异源蛋白表达。