Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic
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Microbial Cell Factories Open Access
RESEARCH
Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9‑Lag) to improve metabolic pathway efficiency Qianwen Xie1,3†, Siwei Li3,4†, Dongdong Zhao3,4, Lijun Ye3,4, Qingyan Li3,4, Xueli Zhang3,4*, Li Zhu2* and Changhao Bi3,4*
Abstract Background: Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment. Results: Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpFCas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components. Conclusion: This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location. Keywords: dCas9, Complex localization, Astaxanthin, Carotenoids, Escherichia coli
*Correspondence: [email protected]; [email protected]; [email protected] † Qianwen Xie and Siwei Li have contributed equally to this work 2 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing 100071, China 3 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China Full list of author information is available at the end of the article
Introduction The RNA-guided Cas9 nuclease has been developed into a powerful genome editing tool in recent years [1–3]. The Clustered Regularly Interspersed Short Palindromic Repeats CRISPR/C
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