Abstract:
An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, 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 specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration 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 E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.
摘要:
酿酒酵母中从头合成生物素的氧需求排除了生物素原养型菌株在使用无生物素培养基的缺氧过程中的应用。为了克服这个问题,本研究探讨了在酿酒酵母中引入不依赖氧的大肠杆菌生物素生物合成途径。该途径的实施需要表达参与脂肪酸合成的七个大肠杆菌基因和形成庚二酸硫酯所必需的三个大肠杆菌基因。生物素合成的关键前体。表达这些基因的酵母菌株容易在无生物素培养基中生长,不管氧气的存在。然而,工程菌株在无生物素培养基中的比生长速率比在补充生物素的培养基中低25%。在缺氧培养的适应性实验室进化之后,进化的细胞系在受控的生物反应器中不再显示这种生长差异,通过基因组测序和蛋白质组分析进行表征。进化的分离株表现出全基因组重复,并伴随着生物合成途径基因的相对基因剂量的改变。这些改变导致催化大肠杆菌生物素途径的前三个步骤的酶的丰度降低。进化的途径构型在二倍体工业酿酒酵母菌株乙醇红中被逆向工程改造。在厌氧室中进行的补充生物素和无生物素的非对照培养基批次中,所得菌株以几乎相同的速率生长。这项研究建立了一种独特的基因工程策略,以使酿酒酵母不依赖生物素的缺氧生长,并证明了其在工业菌株背景下的便携性。