Metabolic Engineering Group, Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007, Salamanca, Spain.
Imperial College Centre for Synthetic Biology and Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK.
Microb Biotechnol. 2019 Nov;12(6):1293-1301. doi: 10.1111/1751-7915.13425. Epub 2019 May 5.
The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner.
丝状真菌棉阿舒囊霉目前被用于维生素 B2 的工业生产。此外，棉阿舒囊霉能够利用低成本的基质进行生长，并且下游处理成本低廉，这使得该真菌成为一种有吸引力的生物技术底盘。事实上，已经有描述在棉阿舒囊霉中生产其他高附加值化合物，如叶酸、核苷和生物脂。因此，开发新的方法来扩展用于棉阿舒囊霉基因组操作的分子工具包是该真菌生物技术的一个重要问题。在这项工作中，我们提出了一种用于棉阿舒囊霉基因组工程的单载体 CRISPR/Cas9 系统。我们证明了该系统作为一种无标记方法的效率，可用于具有可见和不可见表型的核苷酸缺失和替换。特别是，该系统已经针对三种类型的基因组编辑进行了验证：基因失活、loxP 疤痕的基因组擦除和点突变的引入。我们预计，CRISPR/Cas9 系统在棉阿舒囊霉中的使用将极大地有助于以无标记的方式促进该工业真菌的基因组操作。
