Tianjin R&D Center for Petrochemical Technology, Tianjin University, Tianjin, People's Republic of China.
School of Chemical Engineering and Technology, Tianjin University, Tianjin, People's Republic of China.
Biotechnol Appl Biochem. 2021 Oct;68(5):953-963. doi: 10.1002/bab.1984. Epub 2020 Aug 24.
Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167 bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77 g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.
工业酵母菌株的遗传改造通常比实验室菌株面临更多的困难。因此，仍然需要新的方法。在这项研究中，通过URA3 回收，对安琪酵母来源的单倍体菌株 Kα 进行了多轮δ-整合遗传修饰。首先构建了三个带有两个 167bpδ序列和重复 URA3-重复片段的δ整合质粒 pGδRU、pGδRU-BGL 和 pGδRU-EG。然后，通过线性化 pGδRU-BGL 或 pGδRU-EG 片段的一次转化，分别成功获得了含有 bgl1 或 egl2 基因的δ整合菌株。通过选择在 5´-氟乳清酸平板上生长，也很容易分离出URA3 基因环出的对应菌株，尽管随着相应整合纤维素酶编码基因拷贝数的增加，缺乏 URA3 的菌落与总菌落数的比例降低。在第二轮δ-整合中也观察到了类似的结果，第一轮获得的来自 Kα(δ::bgl1-repeat)的δ-整合菌株用线性化的 pGδRU-EG 片段转化。经过 10 轮细胞生长和转移到新鲜培养基后，Kα(δ::bgl1-repeat)、Kα(δ::egl2-repeat)和 Kα(δ::bgl1-repeat)(δ::egl2-repeat)的倍增时间和酶活性均无显著变化且稳定。此外，它们在预处理玉米芯同步糖化发酵的 7 天内的最大乙醇浓度分别为 46.35、33.13 和 51.77g/L，均显著高于亲本 Kα 菌株。因此，带有 URA3 回收的重复δ-整合可以成为安琪酵母遗传工程的一种可行且有价值的方法。这些结果还为一些与δ-整合相关的重要问题提供了线索，例如δ-整合基因的结构稳定性和单个整合位点位置对基因表达的影响。进一步阐明这些问题应有助于充分实现基于δ-整合方法在工业酵母育种中的潜力。
