A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems

Abstract

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

“…Interestingly, the Streptomyces venezuelae system, which was considered in our calculations, showed lower protein concentrations using larger amounts of amino acids resulting in significantly lower fractional yields (Table 2). 36 However, the concentration of the reporter protein could be significantly increased for this system using a novel approach 43 . Instead of a commercial standard, rich media components were added as amino acid sources.…”

“…36 However, the concentration of the reporter protein could be significantly increased for this system using a novel approach. 43 Instead of a commercial standard, rich media components were added as amino acid sources. Especially tryptone and casamino acids gave up to a 2-fold increase in protein concentration for the S. venezuelae system.…”

Streptomyces cell-free systems for natural product discovery and engineering

“…27,30 Alternatively, a range of Streptomyces CFE systems have emerged, compatible with multiple strains, 32 for high-yield protein production. [32][33][34][35][36][37] Currently, Streptomyces venezuelae and Streptomyces lividans CFE systems have been shown to reach up to 0.45 mg mL −1 35,37 and 0.52 mg mL −1 , 33,38 respectively. These protein yields are close to commercial E. coli cell-free systems, which were developed over several decades of research and include many genetic changes to increase recombinant protein yields.…”

“…This is because several other Streptomyces strains have also been used to establish CFE systems using the same preparation procedure. 32 These recent studies have provided user-friendly protocols, [32][33][34][35][36][37][38][39] which will likely lead to the development of more productive Streptomyces CFE systems in the future for natural product research. We will now discuss the current advances for these specialised CFE systems, before summarising the next steps for their continued and wider use.…”

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity

“…This vision of an "astropharmacy" could be realized by onboarding light weight cellfree transcription-translation (TXTL) reagents capable of rapidly synthesizing RNA and proteins from DNA blueprints, just as required. Relying only on isolated biochemical components that can be freeze-dried for storage, TXTL systems combine simplicity and flexibility, while reducing the need for downstream processing [4][5][6] and biocontainment in planetary protection efforts 7 . TXTL technology enables the synthesis of large, self-assembling macromolecular complexes such as bacteriophages 8,9 and virus-like particles 10 .…”