Combinatorial Assembly of Multigene Pathways by Combining Single-Strand Assembly with Golden Gate Assembly

: Biotechnological production routes for fine and bulk chemicals are progressively explored and developed. Yet this development is hampered by the many constraints determining the metabolic sweet spot, such as optimal expression levels, metabolic stress, feedback regulation, etc.In this regard, we introduce a novel, highly reliable, and rapid single-strand assembly (SSA) methods for combinatorial pathway engineering. In this contribution, SSA is elucidated which enables one to modulate the expression via promoter and/or RBS randomization. Moreover, a new combinatorial multigene pathway assembly scheme based on single-strand assembly (SSA) methods and Golden Gate Assembly is introduced, exploiting the strengths of both assembly techniques.