Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Abstract

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste.Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

Modular cell-free expression plasmids to accelerate biological design in cells

“…Standardization of these vector systems allows for new simplified workflows. The pJL1 cell-free vector and variants thereof are routinely used in multiple bacterial cell-free systems (i.e., E. coli , 19 Clostridium , 10 Pseudomonas , 23 Streptomyces , 24,41 Vibrio natriegens 42,43 ). In addition, the pMTL vector system has been demonstrated in several Clostridia species (i.e., autoethanogenum, ljungdahlii, acetobutylicum, beijerinckii, difficile, sporogeneses, perfringens, pasteurianum, tyrobutyricum ) as well as other Gram-negative and Gram-positive model organisms such as E. coli and Bacillus .…”

“…Developments in cell-free synthetic biology have sought to characterize non-model organisms 8-10 and rapidly test metabolic pathways in vitro . 11-14 By using cell-free gene expression (CFE) 15 to produce enzymes directly in vitro , metabolic pathways can be tested without the need to re-engineer organisms or construct new DNA elements between each engineering cycle.…”

“…In this work, we present a modular plasmid system on the basis of standard cell-free vector pJL1 10 ,19,23,24 and universal Clostridium shuttle vector system pMTL80000 22 to rapidly bridge cell-free prototyping efforts and strain engineering in C. autoethanogenum and reduce the overall engineering cycle time. Engineering compatible expression plasmids requires fine tuning to minimize impacts on the genetic context of open reading frames, particularly around the critically important ribosome binding site.…”

Cell-Free Biocatalysis for the Production of Platform Chemicals

“…A recent preprint from the Jewett group (Krüger et al, 2020) describes the use of prototyping for optimisation of protein yield of a non-traditional but industrially relevant Clostridial host as judged by luciferase bioluminescence. The cell free system allowed rapid examination of a number of metabolites, pathways and energy regeneration systems which were shown to vary substantially from the standard E. coli conditions.…”

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

“…We further point such readers to Gregorio et al for an expansive introduction to CFE [ 3 ] and the aforementioned review by Dopp et al on reagent mixes [ 16 ]; in combination with this work, these reviews provide a comprehensive primer on CFE and prokaryotic methodologies. Motivated by the publication of several new methods for E. coli in recent years [ 8 , [12] , [13] , [14] , [15] , [17] , [18] , [19] , [20] , 25 , [33] , [34] , [35] , [36] , [37] ], as well as the creation of extracts from at least 10 new prokaryotic organisms [ [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] ], we trace the development of these methods over the past 20 years. For E. coli extracts, we start by surveying the strains and genetic modifications used in the CFE field, then chronicle efforts to optimize individual steps within the extract preparation process.…”