Direct Combinatorial Pathway Optimization.

Combinatorial engineering approaches are becoming increasingly popular, yet they are hindered by the lack of specialized techniques for both efficient introduction of sequence variability and assembly of numerous DNA parts, required for the construction of lengthy multigene pathways. In this contribution, we introduce a new combinatorial multigene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly, exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accompanying set of well-characterized and ready-to-use genetic parts, the developed workflow allows effective introduction of various libraries and efficient assembly of multigene pathways. It was put to the test by optimizing the lycopene pathway as proof-of-principle. The here constructed libraries yield ample variation in lycopene production. In addition, good-performing transformants with a significantly higher lycopene production were obtained as compared to previously published reference strains. The best selected producer yielded 3-fold improvement in lycopene titers up to 448 mg lycopene/g CDW. The proposed workflow in combination with the accompanying sets of ready-to-use expression and carrier plasmids, will allow the combinatorial assembly of increasingly lengthy product pathways with minimal effort.