English: Construction of a combinatorial library relies on iterative engineering cycles of one-pot assembly reactions, and amplification of assembled products in microbial cells. At the level of the assembly reaction, the reaction cocktail contains libraries of genetic elements such as promoters (blue arrow), genes (green arrow), and terminators (orange “T”). Combinatorial assembly allows assembly of all standard elements (e.g. promoters, genes, and terminators) in different combination in a single cloning step. To do this, homology sequences (for homology-based cloning method) or sequences that consist of a restriction enzyme cleavage site (for classical digestion/ligation method) at the ends of the fragments to assemble are required: X0 and X1 are segments upstream (left) and downstream (right) of the cloning in plasmid 1, respectively; segment Z0 represents the 3′ region of the promoter and overlaps with the sequence upstream (left) of the gene; segment Y0 represents the 3′ region of the gene and overlaps with the sequence upstream (left) of the terminator. Thereafter, the multiple groups of gene modules of may be integrated into multi-locus of the host genome. A first combinatorial reaction cocktail is used for assembly of gene module, while a second reaction is used for generation of two-gene module from individual gene module in plasmid 2. X2 and X3 are segments upstream (left) and downstream (right) of the cloning in plasmid 2, respectively; and segment Z1 represents the 3′ region of the first gene module and overlaps with the sequence upstream (left) of the second gene module. After establishing a plasmid library containing the entire pathway gene modules, the plasmid library can be directly transformed into the host or can be integrated into the genome of the host to generate stable combinatorial library variants.^[1]