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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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Evaluation of the XylA-locus in cultures and single cell clones obtained from the various stages of the evolutionary adaptation process.Agarose gel electrophoresis of PCR products obtained using primer sets P1 and P3 are shown (see Fig. 3C for the primer sets). DNA was isolated from a sample of the whole culture obtained just after the genome shuffling step (GS1.0) and after each batch of the first 5 cultures of the evolutionary adaptation process (GS1.1 to GS1.5). GS1.2–2 and GS1.2–6 are single cell isolates from the culture GS1.2, while GS1.4–14 and GS1.4–17 are single cell isolates from the culture GS1.4. The strains M315, HDY.GUF5, ER, (Ethanol Red) and GS1.11–26 have been included for comparison.
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pgen.1005010.g005: Evaluation of the XylA-locus in cultures and single cell clones obtained from the various stages of the evolutionary adaptation process.Agarose gel electrophoresis of PCR products obtained using primer sets P1 and P3 are shown (see Fig. 3C for the primer sets). DNA was isolated from a sample of the whole culture obtained just after the genome shuffling step (GS1.0) and after each batch of the first 5 cultures of the evolutionary adaptation process (GS1.1 to GS1.5). GS1.2–2 and GS1.2–6 are single cell isolates from the culture GS1.2, while GS1.4–14 and GS1.4–17 are single cell isolates from the culture GS1.4. The strains M315, HDY.GUF5, ER, (Ethanol Red) and GS1.11–26 have been included for comparison.

Mentions: As described in our previous report [19], the evolutionary adaptation step used to obtain the strain GS1.11–26 involved a series of 11 sequential batch cultures in D-xylose medium. To verify at which stage of the evolutionary adaptation process the amplification of the XylA-locus had occurred, a sample from the culture before the evolutionary adaption (GS1.0), and samples at the end of the first 5 serial transfers during the evolutionary adaptation (GS1.1, GS1.2, GS1.3, GS1.4 and GS1.5) were tested by PCR for the presence of the tandem amplification or circular DNA formation using PCR primer set P1. Interestingly, a positive PCR result was obtained in all the samples derived from the second culture (GS1.2) onwards, whereas isolates from GS1.0, GS1.1, as well as the original strains used for the genome shuffling step (HDY.GUF5 and M315) did not give rise to the PCR product (Fig. 5). Southern blot analysis of the same samples after HindIII digestion also confirmed the presence of either circular or multiple copies of the locus in the samples obtained from GS1.2 onwards (Fig. 4B). This strongly suggests that amplification of the XylA-locus had occurred at the second step of the evolutionary adaptation process (GS1.2). As we anticipated, the sharp rise in the rate of D-xylose fermentation in the second culture [19], correlated with amplification of the XylA-locus. Although XylA was expressed from a strong promoter in the parent strain HDY.GUF5, the level of expression was not high enough to confer strong D-xylose fermentation capacity. Amplification of the gene likely increased the expression of XI, which in turn alleviated the rate limiting bottleneck for fermentation of D-xylose.


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)

Evaluation of the XylA-locus in cultures and single cell clones obtained from the various stages of the evolutionary adaptation process.Agarose gel electrophoresis of PCR products obtained using primer sets P1 and P3 are shown (see Fig. 3C for the primer sets). DNA was isolated from a sample of the whole culture obtained just after the genome shuffling step (GS1.0) and after each batch of the first 5 cultures of the evolutionary adaptation process (GS1.1 to GS1.5). GS1.2–2 and GS1.2–6 are single cell isolates from the culture GS1.2, while GS1.4–14 and GS1.4–17 are single cell isolates from the culture GS1.4. The strains M315, HDY.GUF5, ER, (Ethanol Red) and GS1.11–26 have been included for comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005010.g005: Evaluation of the XylA-locus in cultures and single cell clones obtained from the various stages of the evolutionary adaptation process.Agarose gel electrophoresis of PCR products obtained using primer sets P1 and P3 are shown (see Fig. 3C for the primer sets). DNA was isolated from a sample of the whole culture obtained just after the genome shuffling step (GS1.0) and after each batch of the first 5 cultures of the evolutionary adaptation process (GS1.1 to GS1.5). GS1.2–2 and GS1.2–6 are single cell isolates from the culture GS1.2, while GS1.4–14 and GS1.4–17 are single cell isolates from the culture GS1.4. The strains M315, HDY.GUF5, ER, (Ethanol Red) and GS1.11–26 have been included for comparison.
Mentions: As described in our previous report [19], the evolutionary adaptation step used to obtain the strain GS1.11–26 involved a series of 11 sequential batch cultures in D-xylose medium. To verify at which stage of the evolutionary adaptation process the amplification of the XylA-locus had occurred, a sample from the culture before the evolutionary adaption (GS1.0), and samples at the end of the first 5 serial transfers during the evolutionary adaptation (GS1.1, GS1.2, GS1.3, GS1.4 and GS1.5) were tested by PCR for the presence of the tandem amplification or circular DNA formation using PCR primer set P1. Interestingly, a positive PCR result was obtained in all the samples derived from the second culture (GS1.2) onwards, whereas isolates from GS1.0, GS1.1, as well as the original strains used for the genome shuffling step (HDY.GUF5 and M315) did not give rise to the PCR product (Fig. 5). Southern blot analysis of the same samples after HindIII digestion also confirmed the presence of either circular or multiple copies of the locus in the samples obtained from GS1.2 onwards (Fig. 4B). This strongly suggests that amplification of the XylA-locus had occurred at the second step of the evolutionary adaptation process (GS1.2). As we anticipated, the sharp rise in the rate of D-xylose fermentation in the second culture [19], correlated with amplification of the XylA-locus. Although XylA was expressed from a strong promoter in the parent strain HDY.GUF5, the level of expression was not high enough to confer strong D-xylose fermentation capacity. Amplification of the gene likely increased the expression of XI, which in turn alleviated the rate limiting bottleneck for fermentation of D-xylose.

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