Interference in molecular regulatory circuits can be used as a powerful tool for redirection of metabolic fluxes in order to optimize industrial use of microorganisms. We have exemplified this by redirection of the fermentative-oxidative carbon balance in baker's yeast in order to reduce aerobic alcoholic fermentation triggered by the presence of glucose. This has been achieved by raising the expression level of Hap4p, the major regulatory protein of the Hap2/3/4/5p complex required for transcriptional induction of respiratory components. Although previously it has been suggested that regulation of the transcript level of HAP4
would be the main determinant for the activity of the Hap complex (3
), we now present the first experimental evidence for this hypothesis. Our results also show that the DNA binding factors of the complex, Hap2p, Hap3p, and Hap5p, are constitutively present at sufficient levels and that posttranslational modifications do not play an important role in glucose control of the Hap complex, in contrast to other transcriptional regulators like Cat8p (23
) and Mig1p (21
). Hence, an artificially elevated expression level of Hap4p is sufficient to increase mRNA levels of respiratory genes on glucose. This resulted in an increased respiratory capacity and in vivo carbon flux through the tricarboxylic acid cycle and respiration. The alleviation of repression of respiration and redirection of respiro-fermentative carbon flux is only partial, which can be explained by the control of other factors and mechanisms on regulation of oxidative function. Nevertheless, this partial effect results in a significant gain in specific growth rate and biomass yield during growth on an excess of glucose due to the large difference in the energetic efficiency between respiration and fermentation.
When grown under anaerobic conditions, Hap4p-overexpressing strains are identical to wild-type cells with respect to growth rate, ethanol production, and biomass yield (data not shown). This implies that overexpression of HAP4 exhibits its effect only during aerobic growth of yeast. Processes depending on anaerobic alcoholic fermentation, like brewing or dough leavening, will be unaffected by HAP4 overexpression. HAP4-modified strains should therefore be suited to optimize biomass yields in the aerobic production phase of industrial yeast strains. Experiments are in progress to test whether HAP4 overexpression has an effect on the rapid triggering of alcoholic fermentation, which occurs during local and transient exposure to an excess of glucose due to imperfect mixing in sugar-limited, aerobic industrial fed-batch cultivations.
The profound effect of Hap4p overproduction on cell physiology during growth on excess of glucose suggest a broad spectrum of effects on different functional groups of genes involved in carbon and energy metabolism. A switch from fermentative to respiratory growth, which normally occurs during the diauxic shift upon depletion of glucose, is correlated with widespread changes in the expression of genes involved in fundamental cellular processes such as carbon metabolism, mitochondrial assembly, stress response, protein synthesis, and carbohydrate storage (4
). Although many genes contain Hap2/3/4/5p consensus binding sites in their promoter regions (10
), only few have been reported to be regulated by the HAP complex (3
). We are currently performing genome-wide transcript profiling studies that will reveal the total spectrum of gene families that are directly or indirectly affected by Hap4p overexpression. Insight into gene families that are affected or unaffected by Hap4p overproduction may provide clues toward modification of other regulatory proteins. The use of transcript-profiling analysis techniques (1
) will be invaluable in the exploration of regulators of metabolic pathways that should be modified for biotechnological applications.
We have presented a clear example of how modification of a transcriptional regulator may serve as a powerful tool for manipulation of the cell's physiology and as an attractive alternative to the unsuccessful alteration of expression levels of single enzymes. It should, however, be realized that overexpression of transcriptional activators can have a deleterious effect on growth which is attributable to squelching of general transcription factors, as observed for GAL4 (13
) and GCN4 (27
), but also in studies with GAL4-HAP4
fusions on 2μm plasmids (J. Stebbins and S. Triezenberg, Abstr. Yeast Genet. Mol. Biol. Meet., abstr. 517, p. 307, 1998). Undesired pleiotropic effects can also occur in case of interference in factors with a central role high in signaling cascades, such as the general repressor complex Tup1/Ssn6 or the Snf1/Snf4 kinase with a central role in the glucose response (reviewed in reference 15
). Our study involving a modest change of a family-specific transcriptional regulator is one of the first successful and promising examples of genetic engineering of metabolic fluxes in yeast.