Table 2.

Strategies for assembling BGCs.

Strategy (Max size assembled)	Basic principles	Advantages	Limitations
MoClo243,245 (50 kb)	Type IIs restriction enzymes, Golden Gate technology	Scarless cloning, single-step, multi-gene assembly High cloning efficiency: 95–100% for 10 fragments Suitable for construction of large and complex gene clusters Automation friendly	Limited construct size for E. coli transformation Limited selection of type IIs restriction enzymes requires additional modification of higher-level modules for continued assembly Not suitable for genes containing type IIs restriction sites
MASTER ligation244 (29 kb)	MspJI and Golden Gate cloning	Scarless cloning Specific digestion based on incorporated 5-methylcytosine Suitable for assembly of large and complex constructs High throughput and automation friendly	Unreported efficiency
DNA assembler246,247 (50 kb)	Yeast HR	Scarless cloning, single-step, multi-gene assembly High efficiency for less fragments Automation friendly	Instability of homologous regions in yeast Additional plasmid propagation step in E. coli is required
Reiterative recombinalion241	Endonuclease-induced HR paired with recyclable markers	High yielding to build large mock libraries >104 biosynthetic pathways High efficiency User friendly Suitable for very large DNA constructs Automation friendly	Sequential assembly of multiple genes Additional plasmid propagation step in E. coli is required
LCR-DNA assembler248 (44 kb)	Ligase cycling reaction followed by in vivo yeast assembly	High throughput and automation friendly Perfect efficiency for small clusters (<13 kb, 10 genes)	Additional work needed to build cassettes containing gene of interests Additional plasmid propagation step in E. coli is required