Department of Biochemistry, Campus B2.2, 66123, Saarland University, Saarbrücken, Germany.
OakLabs GmbH, Neuendorfstraβe 16b, 16761 Berlin/Hennigsdorf, Germany.
J Biotechnol. 2019 Mar 20;294:38-48. doi: 10.1016/j.jbiotec.2019.01.018. Epub 2019 Feb 13.
Over the past decades, Bacillus megaterium has gained significant interest in the biotechnological industry due to its high capacity for protein production. Although many proteins have been expressed efficiently using the optimized xylose inducible system so far, there is a considerable demand for novel promoters with varying activities, particularly for the adjustment of protein levels in multi-enzyme cascades. Genome-wide microarray analyses of the industrially important B. megaterium strain MS941 were applied to identify constitutive and growth phase dependent promoters for the expression of heterologous proteins from the early exponential to the early stationary phase of bacterial growth. Fifteen putative promoter elements were selected based on differential gene expression profiles and signal intensities of the generated microarray data. The corresponding promoter activities were evaluated in B. megaterium via β-galactosidase screening. β-Galactosidase expression levels ranged from 15% to 130% compared to the optimized xylose inducible promoter. Apart from these constitutive promoters we also identified and characterized novel inducible promoters, which were regulated by the addition of arabinose, galactose and the commonly used allolactose analog IPTG. The potential application of the identified promoters for biotechnologically relevant processes was demonstrated by overexpression of the cholesterol oxidase II from Brevibacterium sterolicum, thus obtaining product yields of up to 1.13 g/l/d. The provided toolbox of novel promoters offers versatile promoter strengths and will significantly contribute to harmonize protein expression in synthetic metabolic pathways, thereby pushing forward the engineering of B. megaterium as microbial cell factory for the biosynthesis and conversion of valuable compounds.
在过去的几十年中，巨大芽孢杆菌因其具有较高的蛋白质生产能力而在生物技术行业中引起了广泛关注。尽管迄今为止，许多蛋白质已通过优化的木糖诱导系统得到了高效表达，但仍需要具有不同活性的新型启动子，特别是需要用于多酶级联中蛋白质水平的调节。对工业上重要的巨大芽孢杆菌 MS941 菌株进行了全基因组微阵列分析，以鉴定从细菌生长的早期指数期到早期稳定期表达异源蛋白质的组成型和生长阶段依赖性启动子。根据差异基因表达谱和生成的微阵列数据的信号强度，选择了十五个假定的启动子元件。通过β-半乳糖苷酶筛选在巨大芽孢杆菌中评估了相应的启动子活性。与优化的木糖诱导启动子相比，β-半乳糖苷酶表达水平的范围为 15%至 130%。除了这些组成型启动子之外，我们还鉴定并表征了新型诱导型启动子，这些启动子受阿拉伯糖、半乳糖和常用的别乳糖类似物 IPTG 的添加调节。通过过表达 Brevibacterium sterolicum 的胆固醇氧化酶 II 证明了所鉴定启动子在生物技术相关过程中的潜在应用，从而获得了高达 1.13 g/l/d 的产物产量。所提供的新型启动子工具包提供了多种启动子强度，将极大地有助于协调合成代谢途径中的蛋白质表达，从而推动巨大芽孢杆菌作为微生物细胞工厂用于有价值化合物的生物合成和转化的工程化。
