A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations.
A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations.
复制标题
DOI：
10.1186/s13068-016-0583-1
复制
发表时间：
2016
影响因子：
6.3
通讯作者：
van Maris AJ
中科院分区：
文献类型：
--
作者：
González-Ramos D;Gorter de Vries AR;Grijseels SS;van Berkum MC;Swinnen S;van den Broek M;Nevoigt E;Daran JM;Pronk JT;van Maris AJ
在用于第二代生物乙醇生产的木质纤维素原料的水解过程中释放的乙酸抑制酵母生长和酒精发酵。因此，在乙醇生产之前的繁殖步骤中产生的酵母生物质在引入木质纤维素水解产物之前应表达高的组成型水平的乙酸耐受性。然而，早期的实验室进化策略，增加酿酒酵母的乙酸耐受性，在乙酸的存在下，选择诱导型，而不是组成性的耐受性，这种抑制剂的基础上长期培养。在乙酸存在下的预适应显示出强烈增加酵母细胞的分数，可以在这种抑制剂的存在下启动生长。连续微氧分批培养，交替转移到新鲜培养基与乙酸和不产生进化的S。酿酒酵母培养物具有组成型乙酸耐受性。在50-55次转移后，从五个这样的进化实验中分离的单细胞系被选择用于进一步研究。紫外诱变后，选择了一个额外的组成型耐乙酸突变体。所有六个突变体显示出从非应激条件转移到含有乙酸的培养基后生长细胞的分数增加。全基因组测序确定了6个基因，其中包含（不同的）突变在多个耐乙酸突变体。单倍体分离研究和未进化祖先菌株中突变等位基因的表达确定了四个基因（ASG 1，ADH 3，SKS 1和GIS 4）中获得乙酸耐受性的因果突变。ASG 1、ADH 3和SKS 1基因突变对乙酸耐性的影响是加性的。一种新的实验室进化策略的基础上交替培养周期的存在和不存在的乙酸赋予了选择性的优势，组成型耐乙酸突变体，并可能适用于选择组成型耐其他压力。4个基因（ASG 1、ADH 3、SKS 1和GIS 4）的突变被鉴定为引起乙酸耐受性的原因。实验室进化策略以及鉴定的突变可以有助于提高工业酵母菌株的乙酸耐受性。本文的在线版本（doi：10.1186/s13068-016-0583-1）包含补充材料，可供授权用户使用。
Acetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor. Preadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50–55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive. A novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains. The online version of this article (doi:10.1186/s13068-016-0583-1) contains supplementary material, which is available to authorized users.