The Gram-negative beta-proteobacterium Cupriavidus necator is a chemolithotroph that can convert CO2 into biomass. C. necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon-optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for gfp. The effect of codon-optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

中文翻译：

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扩展 Cupriavidus necator 的合成生物学工具箱以建立脂肪酸生产

革兰氏阴性β-变形菌 Cupriavidus necator 是一种化能营养生物，可以将二氧化碳转化为生物质。过去，C. necator 已被设计用于生产各种高价值化学品。然而，基因表达和基因组工程仍然缺乏特征良好的工具箱。代谢工程微生物中生物合成途径的开发和优化需要通过功能性遗传元件（例如启动子、核糖体结合位点 (RBS) 和密码子优化）来控制基因表达。在这项工作中，在 C. necator 中验证和表征了一组诱导型和组成型启动子，并设计和测试了 RBS 文库，以显示 gfp 的 50 倍表达范围。通过表达具有不同密码子适应指数的 gfp 和 mCherry 基因来研究密码子优化对 C. necator 基因表达的影响，并通过表达 C12 特异性脂肪酸硫酯酶的密码子优化变体以产生十二烷酸来验证。我们讨论了 C. necator 广泛用于生物合成过程需要克服的进一步障碍。