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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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Sequence analysis at the borders of the amplified XylA-locus, and verification of the presence of circular or tandem repeats.(A) Illumina sequence reads mapped to the reference sequence at both ends of the amplified XylA-locus. Part of the reads that match to the reference sequence are shown in dark red, while sub-reads that do not match to the reference sequence are indicated in faded red color. The reference sequence is shown in black at the top of the reads. Microhomologies located on either ends of the amplified locus are shown in black rectangular boxes. (B) Chromatogram from Sanger sequencing of the amplified locus, showing continuous reads through the break point of the Illumina sequence reads alignment. The Sanger sequencing was performed using a PCR product obtained by amplification with primers that anneal outwards from both ends of the locus. The same result was obtained when the plasmid isolated from strain GS1.2–6 was sequenced. (C) Schematic representation of the amplified XylA-locus and PCR primer sets used. Horizontal arrows stand for annealing sites and direction of the PCR primers used to verify circular or tandem repeat formation (primer set P1), the presence of the XylA locus at the right position (primer set P2) and the possibility of deletion or single copy of the XylA-locus (P3). The size of the expected PCR product is given in kb. (D) Agarose gel electrophoresis picture of the PCR products obtained using the three sets of primers (P1, P2 and P3) with DNA samples from different strains. For primer set P3, the result obtained with a long extension time (8 min) is shown. ER (Ethanol Red) is the original industrial strain in which the xylose/arabinose gene cassette has been inserted into the genome.
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pgen.1005010.g003: Sequence analysis at the borders of the amplified XylA-locus, and verification of the presence of circular or tandem repeats.(A) Illumina sequence reads mapped to the reference sequence at both ends of the amplified XylA-locus. Part of the reads that match to the reference sequence are shown in dark red, while sub-reads that do not match to the reference sequence are indicated in faded red color. The reference sequence is shown in black at the top of the reads. Microhomologies located on either ends of the amplified locus are shown in black rectangular boxes. (B) Chromatogram from Sanger sequencing of the amplified locus, showing continuous reads through the break point of the Illumina sequence reads alignment. The Sanger sequencing was performed using a PCR product obtained by amplification with primers that anneal outwards from both ends of the locus. The same result was obtained when the plasmid isolated from strain GS1.2–6 was sequenced. (C) Schematic representation of the amplified XylA-locus and PCR primer sets used. Horizontal arrows stand for annealing sites and direction of the PCR primers used to verify circular or tandem repeat formation (primer set P1), the presence of the XylA locus at the right position (primer set P2) and the possibility of deletion or single copy of the XylA-locus (P3). The size of the expected PCR product is given in kb. (D) Agarose gel electrophoresis picture of the PCR products obtained using the three sets of primers (P1, P2 and P3) with DNA samples from different strains. For primer set P3, the result obtained with a long extension time (8 min) is shown. ER (Ethanol Red) is the original industrial strain in which the xylose/arabinose gene cassette has been inserted into the genome.

Mentions: We sought to understand the structural arrangement of the amplification of the XylA-locus. The presence of the autonomous replication sequence ARS1529 in the amplified XylA-locus made us consider the possibility that this region got amplified as a self-replicating eccDNA. This idea was supported by the observation of break points on either end of the amplified region when the Illumina sequence reads were mapped to the reference genome (Fig. 3A). The sequence reads at one end of the break point contained partially unmatched sequences that matched with the sequence of the opposite end. This condition implies either circular DNA or tandem repeat formation.


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)

Sequence analysis at the borders of the amplified XylA-locus, and verification of the presence of circular or tandem repeats.(A) Illumina sequence reads mapped to the reference sequence at both ends of the amplified XylA-locus. Part of the reads that match to the reference sequence are shown in dark red, while sub-reads that do not match to the reference sequence are indicated in faded red color. The reference sequence is shown in black at the top of the reads. Microhomologies located on either ends of the amplified locus are shown in black rectangular boxes. (B) Chromatogram from Sanger sequencing of the amplified locus, showing continuous reads through the break point of the Illumina sequence reads alignment. The Sanger sequencing was performed using a PCR product obtained by amplification with primers that anneal outwards from both ends of the locus. The same result was obtained when the plasmid isolated from strain GS1.2–6 was sequenced. (C) Schematic representation of the amplified XylA-locus and PCR primer sets used. Horizontal arrows stand for annealing sites and direction of the PCR primers used to verify circular or tandem repeat formation (primer set P1), the presence of the XylA locus at the right position (primer set P2) and the possibility of deletion or single copy of the XylA-locus (P3). The size of the expected PCR product is given in kb. (D) Agarose gel electrophoresis picture of the PCR products obtained using the three sets of primers (P1, P2 and P3) with DNA samples from different strains. For primer set P3, the result obtained with a long extension time (8 min) is shown. ER (Ethanol Red) is the original industrial strain in which the xylose/arabinose gene cassette has been inserted into the genome.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352087&req=5

pgen.1005010.g003: Sequence analysis at the borders of the amplified XylA-locus, and verification of the presence of circular or tandem repeats.(A) Illumina sequence reads mapped to the reference sequence at both ends of the amplified XylA-locus. Part of the reads that match to the reference sequence are shown in dark red, while sub-reads that do not match to the reference sequence are indicated in faded red color. The reference sequence is shown in black at the top of the reads. Microhomologies located on either ends of the amplified locus are shown in black rectangular boxes. (B) Chromatogram from Sanger sequencing of the amplified locus, showing continuous reads through the break point of the Illumina sequence reads alignment. The Sanger sequencing was performed using a PCR product obtained by amplification with primers that anneal outwards from both ends of the locus. The same result was obtained when the plasmid isolated from strain GS1.2–6 was sequenced. (C) Schematic representation of the amplified XylA-locus and PCR primer sets used. Horizontal arrows stand for annealing sites and direction of the PCR primers used to verify circular or tandem repeat formation (primer set P1), the presence of the XylA locus at the right position (primer set P2) and the possibility of deletion or single copy of the XylA-locus (P3). The size of the expected PCR product is given in kb. (D) Agarose gel electrophoresis picture of the PCR products obtained using the three sets of primers (P1, P2 and P3) with DNA samples from different strains. For primer set P3, the result obtained with a long extension time (8 min) is shown. ER (Ethanol Red) is the original industrial strain in which the xylose/arabinose gene cassette has been inserted into the genome.
Mentions: We sought to understand the structural arrangement of the amplification of the XylA-locus. The presence of the autonomous replication sequence ARS1529 in the amplified XylA-locus made us consider the possibility that this region got amplified as a self-replicating eccDNA. This idea was supported by the observation of break points on either end of the amplified region when the Illumina sequence reads were mapped to the reference genome (Fig. 3A). The sequence reads at one end of the break point contained partially unmatched sequences that matched with the sequence of the opposite end. This condition implies either circular DNA or tandem repeat formation.

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