Comprehensive in silico analysis is carried out to establish rational guidelines for further genetic modification of recombinant yeast. The basic strategy is to identify the effective target mode for the genetic change. To this end, we examine the effect of overexpressing enzymes (catalyzing the throughput flux of EMs) on the ethanol productivity (Peth) which is computed as follows:

PETH — [xETH (t f ) “ xETH (0)] / tf (6)

where xETH is the (molar or mass) concentration of ethanol, and tf is the batch fermentation time.

For realistic simulations, incorporation of metabolic burden is critical. Metabolic burden is ascribed to the lower availability of internal resources for host cells because the same resources are competitively used by plasmids for their replication and more importantly, the synthesis of exogenous proteins. While several empirical correlations are available to consider the change of growth rate with the plasmid content (e. g., Lee et al., 1985; Satyagal & Agrawal, 1989), cybernetic models are able to directly take into account of the reduction of internal resources (b), for example, as follows:

b0

1 + ф

where ф is the parameter depending on the overexpressed level of heterologous proteins as well as the plasmid copy number, and b0 denotes the fraction of internal resources when no genetic modification is made (i. e., ф = 0). We simulate enzyme overexpression by increasing the constitutive enzyme synthesis rate (aMj rs) in Eq. (3) and relate ф to the ratio of "the total incremental of aM, j’s due to plasmids" to "the summation of inducible enzyme synthesis rates."