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首页> 外文期刊>Applied Microbiology and Biotechnology >New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii
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New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

机译：用于叶绿体基因工程的新工具允许在绿藻中合成人类生长激素莱茵衣藻

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In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species-Chlamydomonas reinhardtii-and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5'UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient Delta psbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.
机译：近年来，对工业生物技术中微藻的开发越来越感兴趣。这些光养真核生物有可能用于低成本合成有价值的重组产物，例如生物活性代谢产物和治疗性蛋白质。藻类叶绿体特别代表了此类基因工程的诱人靶标，因为它具有主要的代谢途径，并且因为外源基因可以靶向叶绿体基因组内的特定基因座，从而导致高水平，稳定的表达。然而，叶绿体基因工程的常规方法目前仅适用于莱茵衣藻（Chlamydomonas reinhardtii）这样的物种，即使在这里，现有技术也存在局限性，包括需要用于DNA传递的昂贵的生物弹装置，缺乏健壮的表达载体，以及抗生素抗性标记的不良使用。在这里，我们描述了一种新的菌株和载体，用于将转基因靶向插入中性叶绿体基因座，（i）使转基因编码序列与高表达内源基因psaA或atpA的启动子/ 5'UTR元件无疤痕融合。 ，（ii）将内源性基因psbH作为有效但良性的选择标记，并且（iii）确保转基因构建体成功整合到所有转化株中。通过用玻璃珠搅拌DNA /细胞悬浮液的一种简单而廉价的方法即可实现转化，并根据细胞壁缺陷型Delta psbH菌株的光养性进行选择。我们展示了这些工具在产生高水平功能性人类生长激素的转基因品系中的实用性。

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