Table 2.

Comparison between Episomal Expression and Site-Specific Chromosomal Integration Expression in A. nidulans

expression system	transforming DNA	advantages	disadvantages
Episomal	E. coli-yeast-fungal shuttle vectors containing expression cassettes Assembled in yeast	High yield of transforming DNA easily obtained by propagation in E. coli Efficient assembly of large clusters in yeast No disruption to the host chromosome Multiple copies of the vectors in the nucleus might increase expression of heterologous genes With 4 heterologous genes per vector and 3 selection markers available, theoretically insertion of 12 genes in one transformation is possible	Possible loss of vector if selection pressure is not maintained Only markers that allow for counter-selection can be recycled (pyrG) Longer time of transforming DNA preparation
Site-specific chromosomal integration	PCR products of expression cassettes flanked by homologous regions Assembled by fusion PCR or Gibson assembly	Genetic stability of expression cassettes for long-term bioproduction Ease of rational engineering Unlimited selection marker recycling Rapid generation of integration DNA by fusion PCR and Gibson assembly Visual selection of correct transformants (yellow or white colonies)	Yield of transforming DNA dependent on PCR Assembly of BGCs limited by fusion PCR (this limitation is circumvented by Gibson assembly) Unknown effects of chromosomal disruption (albeit in a defined locus)