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.

中文翻译：

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通过 URA3 循环将纤维素酶编码基因重复 δ 整合到工业天使酵母衍生菌株的染色体中

工业酵母菌株的基因改造往往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，天使酵母衍生的单倍体菌株 Kα 通过多轮 δ 整合进行基因改造，这是通过URA 3 回收实现的。三个 δ 整合质粒，pGδRU、pGδRU-BGL 和 pGδRU-EG，首先用两个 167 bp δ 序列和一个重复URA 3 重复片段构建。然后，分别对线性化的pGδRU-BGL或pGδRU-EG片段进行一次转化，成功获得了含有bgl 1或egl 2基因的δ整合菌株。市建局所在的同行环出的 3 基因也很容易通过选择在 5´-氟乳清酸板上生长来分离，尽管缺乏URA3的菌落与菌落总数的比例随着相应整合的纤维素酶编码基因拷贝数的增加而降低。在第二轮 δ 整合期间观察到类似的结果，其中从第一轮获得的 δ 整合菌株 Kα(δ:: bgl 1-repeat) 被转化为线性化的 pGδRU-EG 片段。经过 10 轮细胞生长并转移至新鲜培养基后，Kα(δ:: bgl 1-repeat)、Kα(δ:: egl 2-repeat) 和 Kα(δ:: bgl 1-重复)(δ:: egl2-repeat) 显示没有显着变化并且是稳定的。此外，它们在 7 天的时间内同时糖化和发酵预处理玉米芯的最大乙醇浓度分别为 46.35、33.13 和 51.77 g/L，均显着高于亲本 Kα 菌株。因此，重复 δ 整合与URA 3 回收可能是一种可行且有价值的天使酵母基因工程方法。这些结果还提供了一些与 δ 整合相关的重要问题的线索，例如 δ 整合基因的结构稳定性和个体整合位点位置对基因表达的影响。进一步阐明这些问题将有助于充分发挥基于 δ 整合的方法在工业酵母育种中的潜力。