1.
Manipulating the position of DNA expression cassettes using location tags fused to dCas9 (Cas9-Lag) to improve metabolic pathway efficiency.
Xie, Q, Li, S, Zhao, D, Ye, L, Li, Q, Zhang, X, Zhu, L, Bi, C
Microbial cell factories. 2020;(1):229
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 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. 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.
2.
[Research progress in multi-enzyme regulation of genetically engineered bacteria producing lycopene].
Xu, J, Zuo, S, Xie, C, Jiang, L, Li, S, Huang, H, Xu, X
Sheng wu gong cheng xue bao = Chinese journal of biotechnology. 2017;(4):552-564
Abstract
Lycopene plays a crucial role in the biosynthesis pathway of 2-methyl-derythritol-4-phosphate (MEP) and mevalonic acid (MVA). It is a representative product of isoprenoid family, and a typical product of multi-enzyme catalytic reaction in organism. In this paper, we first introduced the general regulation methods in multi-enzyme synthesis reaction, including the construction of multi gene co-expression plasmid, gene order regulation, promoter and ribosome binding site regulation, gene knockout and replacement, aiming at the optimization strategies of multi-enzyme catalytic reaction in lycopene synthesis pathway. Meanwhile, we introduced several new regulation methods in multi-enzyme reaction, including multi-fragment assembly technology, artificial scaffold self-assembly methods and so on. At last, we summarized the application of these multi-enzyme regulation methods in lycopene synthesis. These methods provide a great inspiration and research foundation for the construction of lycopene-producing strains with high yield.