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Rapid evolution of recombinant Saccharomyces cerevisiae for Xylose fermentation through formation of extra-chromosomal circular DNA.

Demeke MM, Foulquié-Moreno MR, Dumortier F, Thevelein JM - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, Leuven-Heverlee, Flanders, Belgium; Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders, Belgium.

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.

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Comparison of the xylose cassette sequence coverage between the parent strain HDY.GUF5 and evolved strain GS1.11–26.Log2 ratio of sequence coverage between the evolved and parent strain in the locus of chromosome XV, where the D-xylose and arabinose gene cassette has been integrated, is plotted over the length of the chromosomal fragment. Annotations present in the locus are indicated by bars at the top of the figure. Bars shaded in blue correspond to the heterologous genes that were inserted into the chromosome, while the unshaded bars represent genes present in part of the original yeast chromosome. The coverage was computed for averaged sliding windows of 500 nucleotide positions.
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pgen.1005010.g002: Comparison of the xylose cassette sequence coverage between the parent strain HDY.GUF5 and evolved strain GS1.11–26.Log2 ratio of sequence coverage between the evolved and parent strain in the locus of chromosome XV, where the D-xylose and arabinose gene cassette has been integrated, is plotted over the length of the chromosomal fragment. Annotations present in the locus are indicated by bars at the top of the figure. Bars shaded in blue correspond to the heterologous genes that were inserted into the chromosome, while the unshaded bars represent genes present in part of the original yeast chromosome. The coverage was computed for averaged sliding windows of 500 nucleotide positions.

Mentions: The most prominent CNV occurred in a region at the right arm of chromosome XV. This was the region where the D-xylose and L-arabinose metabolism gene cassette had been integrated in the genome of the parent strain HDY.GUF5 [19]. Part of the integrated gene cassette, containing the XylA gene, and a sequence upstream of the integrated cassette, that includes the gene REV1, the tRNA gene tP(UGG)O3 and the autonomously replicating sequence ARS1529, were amplified about 9 times (estimated from the log2 ratio) in the evolved strain compared to the parent strain (Fig. 2). XylA encodes xylose isomerase that converts D-xylose to D-xylulose, the rate-limiting step in D-xylose fermentation. We previously showed that the evolved strain GS1.11–26 displayed significantly higher (about 17 times) XI activity than the parent strain, which displayed only moderate activity [19]. The high copy number of XylA in the evolved strain is therefore consistent with its high XI activity, though the fold increase in the XI activity was higher than that of the copy number.


Rapid evolution of recombinant Saccharomyces cerevisiae for Xylose fermentation through formation of extra-chromosomal circular DNA.

Demeke MM, Foulquié-Moreno MR, Dumortier F, Thevelein JM - PLoS Genet. (2015)

Comparison of the xylose cassette sequence coverage between the parent strain HDY.GUF5 and evolved strain GS1.11–26.Log2 ratio of sequence coverage between the evolved and parent strain in the locus of chromosome XV, where the D-xylose and arabinose gene cassette has been integrated, is plotted over the length of the chromosomal fragment. Annotations present in the locus are indicated by bars at the top of the figure. Bars shaded in blue correspond to the heterologous genes that were inserted into the chromosome, while the unshaded bars represent genes present in part of the original yeast chromosome. The coverage was computed for averaged sliding windows of 500 nucleotide positions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352087&req=5

pgen.1005010.g002: Comparison of the xylose cassette sequence coverage between the parent strain HDY.GUF5 and evolved strain GS1.11–26.Log2 ratio of sequence coverage between the evolved and parent strain in the locus of chromosome XV, where the D-xylose and arabinose gene cassette has been integrated, is plotted over the length of the chromosomal fragment. Annotations present in the locus are indicated by bars at the top of the figure. Bars shaded in blue correspond to the heterologous genes that were inserted into the chromosome, while the unshaded bars represent genes present in part of the original yeast chromosome. The coverage was computed for averaged sliding windows of 500 nucleotide positions.
Mentions: The most prominent CNV occurred in a region at the right arm of chromosome XV. This was the region where the D-xylose and L-arabinose metabolism gene cassette had been integrated in the genome of the parent strain HDY.GUF5 [19]. Part of the integrated gene cassette, containing the XylA gene, and a sequence upstream of the integrated cassette, that includes the gene REV1, the tRNA gene tP(UGG)O3 and the autonomously replicating sequence ARS1529, were amplified about 9 times (estimated from the log2 ratio) in the evolved strain compared to the parent strain (Fig. 2). XylA encodes xylose isomerase that converts D-xylose to D-xylulose, the rate-limiting step in D-xylose fermentation. We previously showed that the evolved strain GS1.11–26 displayed significantly higher (about 17 times) XI activity than the parent strain, which displayed only moderate activity [19]. The high copy number of XylA in the evolved strain is therefore consistent with its high XI activity, though the fold increase in the XI activity was higher than that of the copy number.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, Leuven-Heverlee, Flanders, Belgium; Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders, Belgium.

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.

Show MeSH
Related in: MedlinePlus