BACKGROUND & AIMS:
:Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.
背景与目标:
:生物素原养是酿酒酵母菌株的一种罕见,尚未完全理解并且在工业上具有相关性的特征。单倍体实验室菌株CEN.PK113-7D的基因组包含完整的生物素生物合成基因,但在不含生物素的合成培养基中的生长极为缓慢(比生长率[μ]≈0.01 h-1)。在重复的分批培养和加速器中进行的四个独立进化实验得出的菌株在无生物素和补充生物素的培养基中的生长速率(μ≤0.36 h-1)相似。这些进化菌株的全基因组重测序揭示了BIO1的40倍扩增,该BIO1编码庚二酰辅酶A(CoA)合成酶。在不同的染色体上发现了BIO1的其他副本,其扩增与大量的染色体重排相符。 BIO1在菌株CEN.PK113-7D中的过表达证实了该基因扩增的关键作用,该菌株能够在不含生物素的培养基中生长(μ= 0.15 h-1)。在一些进化的菌株中发现了膜转运蛋白基因TPO1和/或PDR12的突变。在过表达BIO1的菌株中TPO1和PDR12的缺失将其比生长速率提高到0.25 h-1。这些基因中无效突变的作用是非可累加的,这些突变以前未与生物素代谢相关。这项研究表明,具有生物素合成基本遗传信息的酿酒酵母菌株可以进化为完整的生物素原营养型,并为将生物素原营养型工程化到该酵母的实验室和工业菌株中确定了新的目标。在所有生物体中发挥作用,并非所有生物体都能合成这种维生素。面包酵母的许多菌株是工业生物技术中的重要微生物,至少含有一些生物素合成所需的基因。然而,这些菌株中的大多数不能完全合成生物素,或者不能以足以维持快速生长和产物形成的速率合成生物素。因此,通常将这种昂贵的维生素添加到面包师的酵母培养物中。在这项研究中,实验室在无生物素生长培养基中的进化产生了新菌株,该菌株在不存在生物素的情况下的生长速度与在存在生物素的情况下一样快。通过分析进化的不依赖生物素的菌株的DNA序列,鉴定出有助于此能力的突变。这项工作证明了与工业相关的酵母具有完全的生物素独立性,并鉴定了突变,将其引入其他酵母菌株可以降低或消除其对生物素的需求。