In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism. (C) 2000 Elsevier Science Inc. All rights reserved.