Amplification of the xylose isomerase gene XylA through an extra-chromosomal ARS-carrying circular DNA during evolutionary engineering for rapid xylose fermentation in yeast

Evolutionary engineering experiments have been used to improve the performance of industrial microorganisms, for instance to enhance xylose fermentation capacity in yeast strains intended for conversion of lignocellulose biomass into ethanol. In this case chromosomal amplification of the growth-limiting xylose isomerase gene is a general outcome. The mechanism responsible for the amplification, however, has remained unclear, especially in the absence of flanking repeats. We noticed that the amplified XylA fragment in our previously developed xylose-fermenting industrial yeast strain included an adjacent ARS element. We now show that XylA amplification involved the generation of an extrachromosomal circular DNA element (eccDNA), which was used for amplification of the XylA locus consecutively in the two chromosomal loci of the diploid strain. We have been able to isolate this plasmid from an intermediate strain in the evolutionary engineering process, sequenced it completely and showed that it confers high xylose fermentation capacity. In this way, we have provided the first clear evidence that gene amplification can occur through generation of an eccDNA without the presence of flanking repeats. We noted that many yeast genes previously reported to undergo amplification under growth-limiting conditions, have an ARS element immediately adjacent to the gene. We suggest that there may be a mechanistic link between the relief of the growth limitation and the DNA replication initiated at the ARS element adjacent to the growth-limiting gene. This may cause eccDNA formation to be initiated preferentially at the locus of the growth-limiting gene, rather than randomly throughout the genome.