Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16
Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16
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DOI：
10.1007/s00253-011-3100-2
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发表时间：
2011-03-01
影响因子：
5
通讯作者：
Nakamura, Satoshi
中科院分区：
文献类型：
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作者：
Fukui, Toshiaki;Ohsawa, Kei;Nakamura, Satoshi
评价了5种启动子作为PHA产生菌Cupravidus Necator中基因表达调控工具的作用。构建了报告基因GFP的广宿主域表达载体，分别由来自大肠杆菌的P(Lac)、P(Tac)或P(BAD)或来自链球菌的phaC1(P(Phac))或phaP1(P(Phap))的启动子区域调控。然后，在果糖和大豆油上生长时，测定了含有所构建载体的链球藻菌株的GFP表达谱。P(Lac)、P(Tac)、P(Phac)和P(Phap)介导构成基因的表达，其中P(Tac)是最强的启动子。Laci-P(Tac)即使在加入异丙基-β-d-硫代半乳糖苷(IPTG)后也不能完全发挥作用，这可能是由于弧菌不能吸收IPTG所致。不同浓度的L-阿拉伯糖可以调节Arac-P(BAD)的基因表达，但重组菌的P(3Hb)产量略有下降。Phar-P(Phap)表现出与P(3Hb)积累紧密耦合的表达谱，这表明携带Phar-P(Phap)的载体可用于在PHA生物合成阶段特异表达基因。这些启动子的性质有望为高效工程合成PHA奠定基础。
Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P (lac) , P (tac) , or P (BAD) derived from Escherichia coli, or promoter regions of phaC1 (P (phaC) ) or phaP1 (P (phaP) ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P (lac) , P (tac) , P (phaC) , and P (phaP) mediated constitutive gene expression, among which P (tac) was the strongest promoter. lacI-P (tac) was not thoroughly functional even after addition of isopropyl-beta-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P (BAD) could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P (phaP) exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P (phaP) for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.