Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223, Pozuelo de Alarcón, Madrid, Spain.
Present Address: Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CIB-CSIC), 28040, Madrid, Spain.
BMC Biotechnol. 2017 Nov 13;17(1):80. doi: 10.1186/s12896-017-0393-y.
There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.
Here we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.
We present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.
需要开发合成生物学方法和工具，以促进酵母的快速高效工程改造，以满足特定生物技术项目的需求。特别是，由于线粒体蛋白质组的分隔性质，其操纵具有有趣的潜在应用。这些优势之一在于金属化发生在蛋白质导入线粒体之后，线粒体中含有铁、锌、铜和锰离子库，可以用于重组金属蛋白的金属化反应。另一个优势是线粒体是容纳氧敏感蛋白的合适细胞器，因为在细胞呼吸过程中基质内会产生低氧环境。
在这里，我们描述了模块化克隆系统 GoldenBraid2.0 的适应，用于将组装的转录单元整合到酵母基因组的两个不同位置，从而获得高表达水平。我们还生成了一个包含各种启动子、终止子和选择标记的工具包，这使得多基因构建体的生成和在酿酒酵母中重建生物合成途径变得更加容易。为了便于在线粒体基质中特异性表达重组蛋白，我们还在工具包中包含了一系列线粒体靶向信号，并在不同的生长条件下测试了它们的效率。作为概念验证，我们在这里展示了 14 个细菌固氮（nif）基因的整合和表达，其中一些基因已知需要特定的金属簇辅因子来稳定它们，但这些蛋白质对氧气非常敏感。对于其中一个基因 nifU，我们表明通过使用 Su9 线粒体靶向前导序列和甘油作为碳源来维持需氧呼吸，可以实现该蛋白的最佳生产。
我们在这里介绍了一种经过改进的 GoldenBraid2.0 系统，用于酵母中重组蛋白的模块化克隆、基因组整合和表达。我们已经生成了一个工具包，其中包括诱导型和组成型启动子、线粒体靶向信号、终止子和选择标记，以保证重组转录单元设计的多功能性。通过用氮固定酶 Nif 蛋白和不同的线粒体靶向前导序列以及生长条件来测试系统的效率，我们为未来在酵母线粒体中表达异源蛋白的研究铺平了道路。
