Fig. 3.

Overview of innovative methods for pathway assembly. A Assembly of multiple genes into one large plasmid or one genomic locus. Restriction digestion/ligation-based methods with usage of type II and type IIs restriction enzymes are employed for in vitro gene assembly. Sequence homology-based methods are adopted to join multiple DNA fragments within one step in vivo or in vitro via short overlap. B Multi-locus integration of long pathway by splitting the whole pathway into several segments and then inserting them into multiple sites within one step or by multi-round integration, utilizing multiple markers, recycling markers or adopting marker-free systems. C Combination of multi-locus integration approach and in vivo assembly method to facilitate long-pathway construction