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In search of sustainable approaches to plastics production, many efforts have been made to engineer microbial conversions of renewable feedstock to short-chain (C2-C8) bifunctional polymer precursors (e.g., succinic acid, cadaverine, 1,4-butanediol). Less attention has been given to medium-chain (C12-C14) monomers such as ω-hydroxy fatty acids (ω-OHFAs) and α,ω-dicarboxylic acids (α,ω-DCAs), which are precursors to high performance polyesters and polyamides. Here we engineer a complete microbial conversion of glucose to C12 and C14 ω-OHFAs and α,ω-DCAs, with precise control of product chain length. Using an expanded bioinformatics approach, we screen a wide range of enzymes across phyla to identify combinations that yield complete conversion of intermediates to product α,ω-DCAs. Finally, through optimization of culture conditions, we enhance production titer of C12 α,ω-DCA to nearly 600 mg/L. Our results indicate potential for this microbial factory to enable commercially relevant, renewable production of C12 α,ω-DCA-a valuable precursor to the high-performance plastic, nylon-6,12.

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.

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