Data underlying research on engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae

An oxygen requirement for <i>de novo</i> biotin synthesis in <i>Saccharomyces cerevisiae</i> precludes the application of biotin-prototrophic strains in anaerobic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent <i>Escherichia coli</i> biotin-biosynthesis pathway in <i>S. cerevisiae</i>. Implementation of this pathway required expression of seven <i>E. coli </i>genes involved in fatty-acid synthesis and three <i>E. coli </i>genes essential for the formation of a pimelate thioester, a key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited lower specific growth rates in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anaerobic cultures, evolved cell lines that no longer showed this growth difference were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited several genomic rearrangements, including a whole-genome duplication, which caused alterations in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the <i>E. coli</i> biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial <i>S. cerevisiae </i>strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media. This study established the first genetic engineering strategy to enable biotin-independent anaerobic growth of <i>S. cerevisiae</i> and demonstrated its portability in industrial strain backgrounds.

酿酒酵母（Saccharomyces cerevisiae）的从头生物素合成存在氧气依赖特性，这使得生物素原养型菌株无法应用于无生物素培养基的厌氧培养工艺中。为解决这一难题，本研究探索将不依赖氧气的大肠杆菌（Escherichia coli）生物素合成途径引入酿酒酵母的可行性。该途径的重构需要表达7个参与脂肪酸合成的大肠杆菌基因，以及3个负责合成庚二酸硫酯——生物素合成的关键前体——的大肠杆菌必需基因。表达上述基因的酵母菌株可在无生物素培养基中正常生长，且不受氧气存在与否的影响。但该工程菌株在无生物素培养基中的比生长速率仍低于添加生物素的培养基。通过厌氧环境下的适应性实验室进化，我们获得了不再表现出生长速率差异的进化菌株，并通过基因组测序与蛋白质组分析对其进行了表征。进化菌株出现了多种基因组重排现象，其中包括全基因组复制（whole-genome duplication），该变异改变了生物素合成途径相关基因的相对基因剂量。上述改变使得催化大肠杆菌生物素合成途径前三个步骤的酶丰度降低。我们将该优化后的途径构型反向工程化导入二倍体工业酿酒酵母菌株Ethanol Red中。最终获得的工程菌株在添加生物素与无生物素培养基中的生长速率几乎一致。本研究首次构建了可实现酿酒酵母厌氧生长不依赖生物素的基因工程策略，并验证了该策略在工业菌株背景中的可移植性。