Zymo-Parts: A Golden Gate Modular Cloning Toolbox for Heterologous Gene Expression in Zymomonas mobilis

Zymomonas mobilis is a microorganism with extremely high sugar consumption and ethanol production rates and is generally considered to hold great potential for biotechnological applications. However, its genetic engineering is still difficult, hampering the efficient construction of genetically modified strains. In this work, we present Zymo-Parts, a modular toolbox based on Golden-Gate cloning offering a collection of promoters (including native, inducible, and synthetic constitutive promoters of varying strength), an array of terminators and several synthetic ribosomal binding sites and reporter genes. All these parts can be combined in an efficient and flexible way to achieve a desired level of gene expression, either from plasmids or via genome integration. Use of the GoldenBraid-based system also enables an assembly of operons consisting of up to five genes. We present the basic structure of the Zymo-Parts cloning system, characterize several constitutive and inducible promoters, and exemplify the construction of an operon and of chromosomal integration of a reporter gene. Finally, we demonstrate the power and utility of the Zymo-Parts toolbox for metabolic engineering applications by overexpressing a heterologous gene encoding for the lactate dehydrogenase of Escherichia coli to achieve different levels of lactate production in Z. mobilis.

Automated summary

Zymo-Parts is a modular toolbox based on Golden-Gate cloning that enables efficient construction of genetically modified strains. It offers a collection of promoters, terminators, ribosomal binding sites, and reporter genes for achieving desired levels of gene expression. The GoldenBraid-based system allows for the assembly of operons with multiple genes. The utility of Zymo-Parts for metabolic engineering applications is demonstrated by overexpressing a heterologous lactate dehydrogenase gene in Z. mobilis.