Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

Abstract

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

“…Cell‐free systems, made up of either individually reconstituted cellular components (Shimizu et al , ; Wang et al , ; Villarreal et al , ) or cell lysates (Jewett et al , ; Garamella et al , ), can support a variety of catalytic reactions in vitro when supplied with energy sources, cofactors, and ions (Calhoun & Swartz, , ; Jewett et al , ). These cell‐free approaches have facilitated the development of therapeutically useful natural products (Dudley et al , ; Maini et al , ) and biologics with non‐standard amino acids (Martin et al , ) or chemical moieties otherwise challenging to synthesize (Jaroentomeechai et al , ). They have also been used to generate viable phage particles directly from purified phage DNA (Shin et al , ; Garamella et al , ; Rustad et al , ) and detect pathogens in emerging diagnostic applications (Pardee et al , ; Takahashi et al , ).…”

Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

“…We sought to leverage the advantages of cell-free biosensing platforms to create a new approach for monitoring for the presence of fluoride in water using a fluoride-responsive riboswitch that regulates the expression of the crcB fluoride efflux pump in Bacillus cereus (7,9,10). By configuring the B. cereus crcB fluoride riboswitch to control the transcription of downstream reporter genes (11), we show that a cell-free gene expression system can activate both protein and RNA reporter expression in the presence of fluoride.…”

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

“…Preparation of cell extracts for CFPS. CFPS of glycosylation enzymes and target proteins was performed using crude E. coli lysate from a recently described, high-yielding MG1655-derived E. coli strain C321.∆A.759 32 prepared using well-established methods 26,32 . Briefly, 1-liter cultures of E. coli cells were grown from a starting OD600 = 0.08 in 2xYTPG media (yeast extract 10 g/l, tryptone 16 g/l, NaCl 5 g/l, K2HPO4 7 g/l, KH2PO4 3 g/l, and glucose 18 g/l, pH 7.2) in 2.5-liter Tunair flasks at 34°C with shaking at 250 r.p.m.…”

“…Quantification of CFPS yields. CFPS yields of glycosylation targets and GTs for GlycoPRIME were determined by supplementation of standard CFPS reactions with 10 µM [ 14 C]-leucine using established protocols 26,32 . Briefly, proteins produced in CFPS were precipitated and washed three times using 5% trichloroacetic acid (TCA) followed by quantification of incorporated radioactivity by a Microbeta2 liquid scintillation counter.…”

“…One promising alternative to bypass current technical bottlenecks is the use of cell-free systems in which the production of proteins and metabolites occurs in vitro without using intact, living cells 30,31 . Complementing efforts in cellular engineering, E. coli-based cell-free protein synthesis (CFPS) systems can achieve gram per liter titers of diverse proteins in hours 30,32 and have been shown to enable the rapid discovery, prototyping, and optimization of biosynthetic pathways without the need to reengineer an organism for each pathway iteration 31,33,34 . By shifting the design-build-test unit from a genetic construct to a lysate, this approach allows for the construction of glycosylation pathways in hours [18][19][20]26 .…”