Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System |
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| Authors: | Chen Yang Miaomiao Yang Wanhua Zhao Yue Ding Yu Wang Jian Li |
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| Affiliation: | 1.School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China;2.Clinical Pathology Center, The Fourth Affiliated Hospital of Anhui Medical University, Hefei 230012, China;3.Department of Biological Physics, University of Science and Technology of China, Hefei 230026, China;4.College of Life Sciences, Jiangxi Agricultural University, Nanchang 330045, China; (W.Z.); (Y.D.) |
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| Abstract: | Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 μg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals. |
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| Keywords: | Klebsiella pneumoniae cell-free protein synthesis genome editing cell-free synthetic biology |
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