Abstract
Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.
Highlights
Microbial evolutionary experiments have received considerable attention in recent years for various reasons
Xylose is an important component of lignocellulose hydrolysates used for the production of bioethanol, but the yeast Saccharomyces cerevisiae is unable to utilize xylose
We show that a circular DNA element was spontaneously created by the yeast, encompassing the xylose isomerase gene and an ARS element, present by coincidence adjacent of the inserted xylose isomerase gene
Summary
Microbial evolutionary experiments have received considerable attention in recent years for various reasons. First they allow in depth understanding of the fundamental process of evolution in a rapid and rigorously controlled way [1]. Microbial evolution raises great interest in various fields such as in medicine and industrial applications [2,3,4]. Using nature’s evolutionary principle of variation and selection, microbial evolution has been used for development and optimization of several production host organisms in industrial applications. Genetic changes that occur during evolution include point mutations, gene deletions or amplifications, and often gene rearrangements involving transposable elements, which in turn might generate deletions or amplifications
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