Microbial Sciences, Fungal Genetics and Biotechnology, Institute of Biology Leiden, Leiden University, The Netherlands.
Molecular Enzymology, University of Groningen, The Netherlands.
FEBS J. 2023 Nov;290(21):5127-5140. doi: 10.1111/febs.16891. Epub 2023 Jun 27.
The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL PatE protein in the culture medium with a purity just under 90%.
丝状真菌黑曲霉以其高蛋白质分泌能力而闻名，是同源和异源蛋白质生产的首选宿主。为了进一步提高黑曲霉的蛋白质生产能力，我们构建了一组专用的蛋白质生产菌株，这些菌株在基因组的预定位置含有多达 10 个糖化酶着陆位点 (GLS)。这些 GLS 取代了编码大量存在的酶或编码不需要的功能的基因。每个 GLS 都包含糖化酶基因 (glaA) 的启动子和终止子区域，glaA 是黑曲霉中表达量最高的基因之一。众所周知，整合多个基因拷贝（通常通过随机整合实现）可以提高蛋白质的生产产量。在我们的方法中，GLS 允许使用 CRISPR/Cas9 介导的基因组编辑快速靶向基因替换。通过在每个 GLS 中引入相同或不同的独特 DNA 序列（称为 KORE 序列）并设计 Cas9 兼容的单指导 RNA，可以选择目标基因在哪个 GLS 处进行整合。通过这种方式，可以轻松快速地构建一组具有不同目的基因拷贝数的相同菌株，以比较蛋白质的生产水平。作为其潜力的例证，我们成功地使用该表达平台生成了多拷贝黑曲霉菌株，该菌株表达催化棒曲霉素生物合成最后一步的展青霉素 PatE::6xHis 蛋白。表达 10 个 patE::6xHis 表达盒的黑曲霉菌株在培养基中产生了约 70μg·mL 的 PatE 蛋白，纯度略低于 90%。
