Rapid evolution of recombinant Saccharomyces cerevisiae for Xylose fermentation through formation of extra-chromosomal circular DNA.
Rapid evolution of recombinant Saccharomyces cerevisiae for Xylose fermentation through formation of extra-chromosomal circular DNA.
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DOI：
10.1371/journal.pgen.1005010
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发表时间：
2015-03
期刊：
影响因子：
4.5
通讯作者：
Thevelein JM
中科院分区：
文献类型：
--
作者：
Demeke MM;Foulquié-Moreno MR;Dumortier F;Thevelein JM
环状DNA元件参与基因组的可塑性，特别是串联重复序列。然而，迄今为止报道的酿酒酵母DNA片段扩增涉及预先存在的重复序列，如核糖体DNA、Ty元件和长末端重复序列（LTRs）。在这里，我们报告了在适应性进化实验中，在没有任何重复序列的区域中产生的eccDNA（染色体外环状DNA元件）。利用代谢和进化工程技术培养的高效d -木糖发酵酵母菌及其亲本工业菌株进行了全基因组序列比较。我们发现，在亲本中插入ARS序列附近的外源基因XylA在进化菌株的染色体位点的两个等位基因中都比亲本扩增了约9倍。对适应进化过程中的扩增过程进行分析，发现在进化适应过程中形成了携带xyla的eccDNA pXI2-6，随后染色体串联整合。在没有任何重复DNA元素的情况下，ecdna的形成可能使用了扩增序列两侧8个核苷酸的微同源序列。我们从一个进化适应过程的中间菌株中分离出pXI2-6 eccDNA，并对其进行了完整的测序，结果表明，当它转移到一个新的菌株时，它具有很高的木糖发酵能力。通过这种方式，我们已经提供了明确的证据，证明基因扩增可以通过产生eccDNA而不存在侧翼重复序列，并且可以作为适应选择压力的快速手段。木糖是用于生产生物乙醇的木质纤维素水解物的重要成分，但酿酒酵母不能利用木糖。细菌木糖异构酶基因的插入和进化适应对木糖生长的改善导致了该基因的扩增和有效的木糖发酵能力。对进化适应过程中最终菌株和中间菌株的进一步分析揭示了基因扩增事件机制的有趣特征，这些事件在自然进化中经常发生。我们现在表明，酵母自发地产生了一个环状DNA元件，包含木糖异构酶基因和一个ARS元件，巧合地出现在插入的木糖异构酶基因附近。ARS元件是DNA聚合酶启动DNA复制的位点。有趣的是，这在酵母中首次揭示了环状DNA质粒可以在没有侧翼重复序列的情况下从基因组DNA中产生。
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. Xylose is an important component of lignocellulose hydrolysates used for the production of bioethanol, but the yeast Saccharomyces cerevisiae is unable to utilize xylose. Insertion of a bacterial xylose isomerase gene and improvement of growth on xylose by evolutionary adaptation resulted in amplification of this gene and efficient xylose fermentation capacity. Further analysis of the final and intermediate strains from the evolutionary adaptation process revealed interesting features about the mechanisms involved in gene amplification events, which have occurred frequently in natural evolution. We now 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. ARS elements are the sites where DNA polymerase initiates duplication of DNA. Interestingly, this has revealed for the first time in yeast that circular DNA plasmids can be created from genomic DNA in the absence of flanking repetitive sequences.
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影响因子：
2.6
作者：
GIETZ, RD;SCHIESTL, RH;WOODS, RA
通讯作者：
WOODS, RA
影响因子：
3.5
作者：
Carginale, V;Trinchella, F;Parisi, E
通讯作者：
Parisi, E
影响因子：
3.7
作者：
Galeote V;Bigey F;Beyne E;Novo M;Legras JL;Casaregola S;Dequin S
通讯作者：
Dequin S
DOI：
10.1073/pnas.1014023107
发表时间：
2010-10-26
影响因子：
11.1
作者：
Gresham, David;Usaite, Renata;Regenberg, Birgitte
通讯作者：
Regenberg, Birgitte
影响因子：
4.4
作者：
Araya CL;Payen C;Dunham MJ;Fields S
通讯作者：
Fields S