Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Road, Tianjin Economic-Technological Development Area, Tianjin 022, 300457, People's Republic of China.
Microb Cell Fact. 2020 Feb 24;19(1):45. doi: 10.1186/s12934-020-01307-2.
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.
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.
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.
地衣芽孢杆菌 2709 被广泛应用于异源蛋白的高水平生产，但其细胞常具有不良的野生型特性，如在发酵过程中产生粘性物质和泡沫，这严重影响了其在工业发酵中的应用。如何将其从土壤细菌开发成具有较少野生型特性的超分泌细胞工厂，在工业生产中一直起着至关重要的作用。此外，碱性蛋白酶等诱导酶的最佳表达模式尚未通过比较不同质粒和地衣芽孢杆菌基因组整合位点的转录效率来优化。
通过无标记基因组编辑方法，破坏与起泡相关的天然 lchAC 基因和编码细胞外粘多糖的 eps 簇，对地衣芽孢杆菌 2709 进行了遗传修饰。我们进一步通过筛选不同模块质粒和基因组基因座中最有效的表达系统，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，最终通过宿主优化和在复制起始点附近的染色体整合，aprE 的转录水平大大提高了 1.67 倍，而酶活与野生型碱性蛋白酶生产菌株地衣芽孢杆菌相比提高了 62.19%。
我们成功构建了一种无不良特性的 AprE 高产菌株，通过宿主遗传修饰和表达优化，可以将其发酵特性应用于大规模生产。总之，宿主优化是一种通过消除宿主的有害特性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其他芽孢杆菌属中异源蛋白的表达。
