Queensland University of Technology, Brisbane, QLD, Australia.
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, Australia.
Appl Environ Microbiol. 2018 Mar 1;84(6). doi: 10.1128/AEM.02712-17. Print 2018 Mar 15.
(syn. ) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to , where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast , expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as (syn. ). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.
（同义名）是真核生物中外源蛋白生产最常用的表达系统之一。表达盒通常整合到基因组中以获得稳定表达的菌株。与 不同，短的突出端足以靶向高度特异性的整合，而在 中，长的突出端更有效，并且外源 DNA 可以异位整合。在这里，我们通过高通量筛选超过 700 个转化体和对 27 个转化体进行全基因组测序，旨在阐明异位整合的影响。使用不同的载体设计和线性化方法来模拟 中靶向的最常见整合事件 荧光增强型绿色荧光蛋白（eGFP）报告蛋白的荧光在表达盒正确整合到靶标基因座时在转化体中高度均匀。令人惊讶的是，大多数非特异性整合的转化体表现出高度均匀的表达，与特异性整合相当，这表明非特异性整合不一定会影响表达。然而，少数含有异位整合盒的克隆（<10%）表现出更大的变化，跨越 25 倍的范围，高达特异性整合的参考菌株的 6 倍。高表达菌株表现出基因拷贝数增加与报告蛋白荧光水平升高之间的相关性。我们的结果表明，只要比较足够数量的转化菌株，就可以忽略整合基因座来比较菌株之间的表达水平。对于高度可表达蛋白的表达优化，增加拷贝数似乎是主要的积极影响，而不是整合基因座、基因组重排、缺失或单核苷酸多态性（SNP）。酵母通常被用作蛋白质和代谢物的生物技术生产宿主。在酵母 中，携带外源基因的表达盒在基因组的靶位点高度特异性地整合。相比之下，在非传统酵母如 （同义名）中，盒通常随机整合到基因组的位置。因此，来自同一转化事件的细胞通常表现不同，存在显著的克隆变异，需要筛选大量的菌株。这项研究的重要性在于，我们系统地研究了 700 多个菌株中的整合事件的影响。我们的发现为 中的克隆变异提供了新的见解，因此如何避免陷阱并获得可靠的结果。潜在的机制也可能在其他酵母中起作用，因此可能与重组酵母蛋白生产菌株普遍相关。
