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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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Restriction analysis and xylose fermentation conferring capacity of plasmid pXI2–6.(A) Restriction analysis of pXI2–6. Lane 1, undigested plasmid showing two bands that stand for the supercoiled (lower band) and nicked (upper band) structure; lane 2, XhoI digestion which has single restriction site but shows incomplete digestion; lane 3, SphI and SacI double digestion showing the expected 1kb, 3kb and 3.4kb band; lane 4, HindIII which has a single restriction site and produced the expected 7.4 kb band. (B) D-xylose fermentation performance of the mutant strain M315 expressing the plasmid pXI2–6 after deletion of the chromosomal XylA copies. Batch fermentation was performed in YP medium containing 4% xylose using standing fermentation bottles. The CO2 production was estimated from the weight loss due to conversion of xylose to ethanol and CO2. Error bars represent standard deviation from the mean values of triplicate experiments.
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pgen.1005010.g007: Restriction analysis and xylose fermentation conferring capacity of plasmid pXI2–6.(A) Restriction analysis of pXI2–6. Lane 1, undigested plasmid showing two bands that stand for the supercoiled (lower band) and nicked (upper band) structure; lane 2, XhoI digestion which has single restriction site but shows incomplete digestion; lane 3, SphI and SacI double digestion showing the expected 1kb, 3kb and 3.4kb band; lane 4, HindIII which has a single restriction site and produced the expected 7.4 kb band. (B) D-xylose fermentation performance of the mutant strain M315 expressing the plasmid pXI2–6 after deletion of the chromosomal XylA copies. Batch fermentation was performed in YP medium containing 4% xylose using standing fermentation bottles. The CO2 production was estimated from the weight loss due to conversion of xylose to ethanol and CO2. Error bars represent standard deviation from the mean values of triplicate experiments.

Mentions: To further confirm the presence of the eccDNA, plasmid DNA isolation was performed from the strain GS1.2–6, GS1.4–14 and GS1.11–26, using a protocol modified from Singh and Weil [23]. Cells were pre-grown in 100 ml YPX medium for 24 h to enrich the pXI2–6 plasmid content in the cells. The whole 100 ml culture was used for plasmid isolation (see material and methods). As a result, a substantial amount of pXI2–6 plasmid DNA (more than 1 μg) was obtained from GS1.2–6 (Fig. 7A). On the other hand, the amount of pXI2–6 plasmid DNA obtained from GS1.11–26 and GS1.4–14 was too low to be conclusive. This is probably due to the loss of the pXI2–6 plasmid in the later steps of the evolutionary adaptation process, since there was no longer a need for the strain to maintain the plasmid when enough copies of the essential gene XylA had been integrated in the genome sustaining rapid D-xylose utilization. When the pXI2–6 plasmid isolated from GS1.2–6 was sequenced, a 7483 bp circular sequence was obtained, matching the size of the amplified XylA-locus. The complete sequence of the pXI2–6 plasmid has been provided as supplementary information (S1 Dataset). Though there were several polymorphisms compared to the corresponding sequences in the reference S288c genome, the pXI2–6 plasmid sequence was identical to that of the original parent strain obtained by Illumina sequencing. Restriction analysis using two different enzymes also confirmed the correct size of the isolated pXI2–6 plasmid (Fig. 7A).


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)

Restriction analysis and xylose fermentation conferring capacity of plasmid pXI2–6.(A) Restriction analysis of pXI2–6. Lane 1, undigested plasmid showing two bands that stand for the supercoiled (lower band) and nicked (upper band) structure; lane 2, XhoI digestion which has single restriction site but shows incomplete digestion; lane 3, SphI and SacI double digestion showing the expected 1kb, 3kb and 3.4kb band; lane 4, HindIII which has a single restriction site and produced the expected 7.4 kb band. (B) D-xylose fermentation performance of the mutant strain M315 expressing the plasmid pXI2–6 after deletion of the chromosomal XylA copies. Batch fermentation was performed in YP medium containing 4% xylose using standing fermentation bottles. The CO2 production was estimated from the weight loss due to conversion of xylose to ethanol and CO2. Error bars represent standard deviation from the mean values of triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005010.g007: Restriction analysis and xylose fermentation conferring capacity of plasmid pXI2–6.(A) Restriction analysis of pXI2–6. Lane 1, undigested plasmid showing two bands that stand for the supercoiled (lower band) and nicked (upper band) structure; lane 2, XhoI digestion which has single restriction site but shows incomplete digestion; lane 3, SphI and SacI double digestion showing the expected 1kb, 3kb and 3.4kb band; lane 4, HindIII which has a single restriction site and produced the expected 7.4 kb band. (B) D-xylose fermentation performance of the mutant strain M315 expressing the plasmid pXI2–6 after deletion of the chromosomal XylA copies. Batch fermentation was performed in YP medium containing 4% xylose using standing fermentation bottles. The CO2 production was estimated from the weight loss due to conversion of xylose to ethanol and CO2. Error bars represent standard deviation from the mean values of triplicate experiments.
Mentions: To further confirm the presence of the eccDNA, plasmid DNA isolation was performed from the strain GS1.2–6, GS1.4–14 and GS1.11–26, using a protocol modified from Singh and Weil [23]. Cells were pre-grown in 100 ml YPX medium for 24 h to enrich the pXI2–6 plasmid content in the cells. The whole 100 ml culture was used for plasmid isolation (see material and methods). As a result, a substantial amount of pXI2–6 plasmid DNA (more than 1 μg) was obtained from GS1.2–6 (Fig. 7A). On the other hand, the amount of pXI2–6 plasmid DNA obtained from GS1.11–26 and GS1.4–14 was too low to be conclusive. This is probably due to the loss of the pXI2–6 plasmid in the later steps of the evolutionary adaptation process, since there was no longer a need for the strain to maintain the plasmid when enough copies of the essential gene XylA had been integrated in the genome sustaining rapid D-xylose utilization. When the pXI2–6 plasmid isolated from GS1.2–6 was sequenced, a 7483 bp circular sequence was obtained, matching the size of the amplified XylA-locus. The complete sequence of the pXI2–6 plasmid has been provided as supplementary information (S1 Dataset). Though there were several polymorphisms compared to the corresponding sequences in the reference S288c genome, the pXI2–6 plasmid sequence was identical to that of the original parent strain obtained by Illumina sequencing. Restriction analysis using two different enzymes also confirmed the correct size of the isolated pXI2–6 plasmid (Fig. 7A).

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