Geobacillus spp. are moderate thermophiles that have great potential for use in diverse applications. For effective utilization of the species, genetic tools have been extensively studied; however, an overexpression vector remains to be developed. Here we constructed a plasmid vector that can shuttle between Escherichia coli and Geobacillus spp., and which contained a maltose-inducible promoter from Geobacillus kaustophilus HTA426. Although the vector (termed pGKE119) was originally designed for basal gene expression, it surprisingly directed robust protein production in G. kaustophilus. Protein production essentially occurred in an auto-inducible manner without maltose; however, some proteins were produced more efficiently in the presence of maltose. Although the productivity was affected by culture conditions, three proteins were successfully produced with abundance ratios of 12–27% (on a total protein basis) and yields of 77–170 mg (per L culture). pGKE119 directed substantial protein production even in Geobacillus subterraneus, Geobacillus thermoglucosidasius, and Geobacillus thermoleovorans. This suggests that pGKE119 can use a range of Geobacillus spp. as hosts and widely expand their genetic toolbox. Because Geobacillus spp. are highly proliferative bacteria that are distinct from organisms used as protein production hosts, pGKE119 may also provide a novel platform for hyperproduction of recombinant proteins.

中文翻译：

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指导嗜热芽孢杆菌属物种中重组蛋白超量生产的质粒载体。

嗜菌。是中等温和的嗜热剂，具有广泛的应用前景。为了有效利用该物种，已经广泛研究了遗传工具。然而，过表达载体仍有待开发。在这里，我们构建了一个质粒载体，该质粒载体可在大肠杆菌和Geobacillus spp。之间穿梭，并包含来自嗜碱芽孢杆菌HTA426的麦芽糖诱导型启动子。尽管该载体（称为pGKE119）最初是为基础基因表达而设计的，但它却出人意料地指导了嗜碱性链球菌中强大的蛋白质生产。蛋白质的产生基本上是在没有麦芽糖的情况下以自诱导方式发生的；但是，在麦芽糖的存在下，某些蛋白质的生产效率更高。尽管生产力受到培养条件的影响，但成功生产了三种蛋白，其丰度比为12–27％（以总蛋白为基准），产量为77–170 mg（每升培养物）。pGKE119涉及大量的蛋白生产，即使在土芽孢subterraneus，土芽孢杆菌属thermoglucosidasius，和土芽孢杆菌属thermoleovorans。这表明，pGKE119可以使用一系列的嗜菌。作为主机并广泛扩展其遗传工具箱。因为地杆菌spp。pGKE119是高度增殖的细菌，与用作蛋白质生产宿主的生物不同，pGKE119还可为重组蛋白的超量生产提供新的平台。