Journal Article10.1002/BIT.26253
Establishing a high yielding streptomyces-based cell-free protein synthesis system.
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TL;DR: It is shown that the optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli‐based CFPS system for increasing percent soluble protein expression for four StrePTomyces‐originated high GC‐content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin.
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Abstract: Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.
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A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery
Ashty S. Karim,Michael C. Jewett +1 more
TL;DR: A new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping that will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms is reported.
Cell-free production and streamlined assay of cytosol-penetrating antibodies.
Seung Eui Min,Kyung-Ho Lee,Seong-Wook Park,Tae Hyeon Yoo,Chan Hee Oh,Ji-Ho Park,Sung Yun Yang,Yong Sung Kim,Dong-Myung Kim +8 more
TL;DR: The proposed approach of consolidating cell‐free synthesis and cell‐based assay will substantially expand the capability of discovering and engineering antibodies that can cross the cell membrane and effectively control protein‐mediated cellular functions.
Genome mining of the Streptomyces avermitilis genome and development of genome-minimized hosts for heterologous expression of biosynthetic gene clusters
TL;DR: The genome data of Streptomyces microorganisms provides significant information for not only industrial applications but also understanding the features of this genus, which is a versatile host for heterologous expression of exogenous biosynthetic gene clusters by genetic engineering.