Regulatory Part Engineering for High-Yield Protein Synthesis in an All-Streptomyces-Based Cell-Free Expression System
- Huiling XuHuiling XuSchool of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Huiling Xu
- ,
- Chen YangChen YangSchool of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Chen Yang
- ,
- Xintong TianXintong TianSchool of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Xintong Tian
- ,
- Yilin ChenYilin ChenSchool of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Yilin Chen
- ,
- Wan-Qiu LiuWan-Qiu LiuSchool of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Wan-Qiu Liu
- , and
- Jian Li*Jian Li*[email protected]School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, ChinaMore by Jian Li
Abstract
Streptomyces-based cell-free expression systems have been developed to meet the demand for synthetic biology applications. However, protein yields from the previous Streptomyces systems are relatively low, and there is a serious limitation of available genetic tools such as plasmids for gene (co)expression. Here, we sought to expand the plasmid toolkit with a focus on the enhancement of protein production. By screening native promoters and ribosome binding sites, we were able to construct a panel of plasmids with different abilities for protein synthesis, which covered a nearly 3-fold range of protein yields. Using the most efficient plasmid, the protein yield reached up to a maximum value of 515.7 ± 25.3 μg/mL. With the plasmid toolkit, we anticipate that our Streptomyces cell-free system will offer great opportunities for cell-free synthetic biology applications such as in vitro biosynthesis of valuable natural products when cell-based systems remain difficult or not amenable.
Cited By
This article is cited by 8 publications.
- Ren Zhang, Wan-Qiu Liu, Shengjie Ling, Jian Li. Combining Cell-Free Expression and Multifactor Optimization for Enhanced Biosynthesis of Cinnamyl Alcohol. Journal of Agricultural and Food Chemistry 2023, 71
(22)
, 8551-8557. https://doi.org/10.1021/acs.jafc.3c02340
- Yushi Liu, Wan-Qiu Liu, Shuhui Huang, Huiling Xu, Haofan Lu, Changzhu Wu, Jian Li. Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals. Metabolic Engineering Communications 2023, 16 , e00217. https://doi.org/10.1016/j.mec.2022.e00217
- Songya Zhang, Yunliang Chen, Jing Zhu, Qiujie Lu, Max J. Cryle, Youming Zhang, Fu Yan. Structural diversity, biosynthesis, and biological functions of lipopeptides from
Streptomyces. Natural Product Reports 2023, 40
(3)
, 557-594. https://doi.org/10.1039/D2NP00044J
- Simon J. Moore, Hung-En Lai, Jian Li, Paul S. Freemont. Streptomyces
cell-free systems for natural product discovery and engineering. Natural Product Reports 2023, 40
(2)
, 228-236. https://doi.org/10.1039/D2NP00057A
- Francesco Del Carratore, Erik KR Hanko, Rainer Breitling, Eriko Takano. Biotechnological application of Streptomyces for the production of clinical drugs and other bioactive molecules. Current Opinion in Biotechnology 2022, 77 , 102762. https://doi.org/10.1016/j.copbio.2022.102762
- Chen Yang, Miaomiao Yang, Wanhua Zhao, Yue Ding, Yu Wang, Jian Li. Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System. Molecules 2022, 27
(15)
, 4684. https://doi.org/10.3390/molecules27154684
- Kara Jew, Philip E. J. Smith, Byungcheol So, Jillian Kasman, Javin P. Oza, Michael W. Black. Characterizing and Improving pET Vectors for Cell-free Expression. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.895069
- Xintong Tian, Wan-Qiu Liu, Huiling Xu, Xiangyang Ji, Yushi Liu, Jian Li. Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan. Synthetic and Systems Biotechnology 2022, 7
(2)
, 775-783. https://doi.org/10.1016/j.synbio.2022.03.006