Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-phellandrene synthase (β-PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C₁₀H₁₆ β-phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase (GPPS), promoting its expression, while at the same time serving as a selectable marker for the screening of transformants. The work further showed that enhanced GPPS expression increased the yield of β-phellandrene in Synechocystis transformants harboring the β-PHLS gene. Moreover, the research evaluated the expression efficacy of a DNA fragment comprising 87 nucleotides from the 5′ end of the cmR gene in fusion with the GPPS gene. This short fusion construct substantially increased the intracellular geranyl-diphosphate synthase level, suggesting that “short-stretch” cmR leader sequences can be used to drive a higher expression level of heterologous biosynthetic genes, while avoiding undesirable internal recombinations, as these sequences are shorter than the threshold of 200 bp, commonly assumed to be the threshold of high efficiency recombinations.

中文翻译：

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融合结构中的异源前导序列增强蓝细菌（Synechocystis）中的Geranyl Diphosphate合酶的表达和β-Plandlandrene生产的产量。

事实证明，融合构建体作为蛋白质过表达载体对于萜烯合酶在蓝细菌中的异源表达至关重要。该概念最近被应用于植物中β-水芹烯合酶（β- PHLS）的异源过表达，该酶与编码藻蓝蛋白β亚基的高度表达的内源性cpcB基因融合。过表达的CPCB * PHLS融合蛋白增强的C的异源产率₁₀ ħ ₁₆ β水芹烯烃生产的集胞藻。这项工作通过显示高表达的异源基因还可以充当蓝细菌中蛋白质过表达的前导序列，扩展了融合构建物作为蛋白质过表达载体的概念。检查的是卡那霉素nptI和氯霉素cmR抗性盒，它们均在集胞藻中过表达。证据表明，nptI基因具有双重目的，即与异源香叶基二磷酸合酶（GPPS）融合的前导序列，可促进其表达，同时可作为筛选转化子的选择标记。这项工作进一步表明，增强了GPPS在具有β- PHLS基因的集胞藻转化子中，表达增加了β-芹菜烯的产量。此外，研究评估包括从所述的5'端87个核苷酸的DNA片段的表达效力CMR基因融合与GPPS基因。这种短融合构建体显着提高了细胞内香叶基二磷酸香叶基二磷酸合酶的水平，表明“短拉伸” cmR前导序列可用于驱动异源生物合成基因的更高表达水平，同时避免了不良的内部重组，因为这些序列比200 bp的阈值，通常被认为是高效重组的阈值。