| Autor: |
Huanyu Zhou, Tian Tian, Jingtong Liu, Hong Lu, Yao Yu, Yongming Wang |
| Jazyk: |
angličtina |
| Rok vydání: |
2024 |
| Předmět: |
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| Zdroj: |
Communications Biology, Vol 7, Iss 1, Pp 1-10 (2024) |
| Druh dokumentu: |
article |
| ISSN: |
2399-3642 |
| DOI: |
10.1038/s42003-024-06487-w |
| Popis: |
Abstract The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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