Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic pathway efficiency

Qianwen Xie,Changhao Bi,Dongdong Zhao,Lijun Ye,Li Zhu,Qingyan Li,Xueli Zhang,Siwei Li

doi:10.1186/s12934-020-01496-w

Abstract

BackgroundDeactivated 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.ResultsCarotenoids 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 GlpF-Cas9 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.ConclusionThis 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.

Highlights

The RNA-guided Cas9 nuclease has been developed into a powerful genome editing tool in recent years [1,2,3]
We developed a novel molecular device to manipulate the cellular position of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to Deactivated Cas9 (dCas9) (Cas9-Lag) and applied the technique for synthetic biology tasks (Fig. 1a)
The GlpF‐dCas9/gRNA complex binds to the target plasmid To construct a molecular device to manipulate the cellular localization of a specific DNA cassette, GlpF was fused to the amino terminus of dCas9 with a short flexible linker (GGGS)

Summary

Introduction

The RNA-guided Cas nuclease has been developed into a powerful genome editing tool in recent years [1,2,3]. We developed a novel molecular device to manipulate the cellular position of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas (Cas9-Lag) and applied the technique for synthetic biology tasks (Fig. 1a). Deactivated Cas (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. 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 dCas (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

Methods

Results

Discussion

Conclusion