Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways
Introduction
Microbes can be engineered to manufacture biofuels and high-value compounds such as chemicals, materials, and therapeutics (Keasling, 2012; Nielsen and Keasling, 2016). This biomanufacturing capability promises to help address rapid population growth, an increase in energy demand, and waste generation (Nielsen et al., 2014). However, even the most advanced design-build-test cycles for optimizing a given compound's biosynthetic pathway in model organisms such as Escherichia coli and yeast are still on the order of weeks to months. In addition, process-based challenges associated with these organisms remain (e.g., limited substrate range, reduced yields through CO2 losses, and susceptibility to contamination, among others) (Keasling, 2012; Nielsen and Keasling, 2016). These challenges have prevented a more rapid commercialization of new bioproduct manufacturing processes, with only a handful successfully commercialized to date apart from ethanol fermentation (Meadows et al., 2016; Nakamura and Whited, 2003; Nielsen et al., 2014; Yim et al., 2011). As such, most industrial bioprocesses (e.g., synthesis of amino acids (Leuchtenberger et al., 2005), acetone-butanol-ethanol (ABE) (Jiang et al., 2015; Jones, 2005), organic acids (Ghaffar et al., 2014; Rodriguez et al., 2014; Wee et al., 2006)) rely on other “non-model” organisms.
Clostridia are one such group of organisms, which are industrially proven and have exceptional substrate and metabolite diversity, as well as tolerance to metabolic end-products and contaminants (Tracy et al., 2012). Industrial, large-scale fermentations with clostridia have been carried out for over 100 years with the ABE fermentation being the second largest industrial fermentation process only behind ethanol fermentation (Jones, 2005). In addition to ABE clostridia (solventogenic), there are also clostridia species that are able to degrade lignocellulosic biomass (cellulolytic) and species that are capable of autotrophic growth on C1 substrates, such as carbon monoxide (CO) and CO2 (acetogenic) (Tracy et al., 2012). Gas fermentation with acetogenic clostridia offers an attractive route for conversion of syngas that can be generated from any biomass resource (e.g., agricultural waste or unsorted and non-recyclable municipal solid waste) and industrial waste resources (e.g., off-gases from steel mills, processing plants or refineries) to fuels and chemicals Köpke and Simpson, 2020. However, the current state-of-the-art strain engineering for clostridia remains a low-throughput, labor-intensive endeavor. Specific challenges include organism-specific genetic constraints (Daniell et al., 2015; Joseph et al., 2018; Liew et al., 2017, 2016; Nagaraju et al., 2016), the requirement of an anaerobic environment, and, in case of acetogens, handling of gases. As a result, developments in clostridia biotechnology and basic knowledge of clostridia biology have lagged behind achievements in aerobic prokaryotic and eukaryotic biology. New robust tools are needed to study clostridia and speed up the designing, building, and testing of biological processes in these organisms.
Extract-based cell-free systems are emerging as powerful platforms for synthetic biology applications such as metabolic engineering (Bujara et al., 2011; Carlson et al., 2012; Dudley et al., 2020, 2019; Grubbe et al., 2020; Hodgman and Jewett, 2012; Karim et al., 2020; Karim and Jewett, 2016; Kelwick et al., 2018; Kightlinger et al., 2019; Morgado et al., 2018; Silverman et al., 2019a). Assembling metabolic pathways in the cell-free environment has been done traditionally by assembling purified enzymes and substrates, enabling identification of key rate-limiting information, gaining insights into fundamental biochemistry and improving in vivo engineering approaches for increasing the titer of targeted products (Karim et al., 2020; Liu et al., 2017; Yu et al., 2011; Zhu et al., 2014).
However, the development of cell-free gene expression (CFE) systems has transformed the way pathways can be built and tested (Silverman et al., 2019a). These systems consist of crude cell extracts, energy substrates, co-factors and genetic instructions in the form of DNA, and facilitate the activation, manipulation and usage of cellular processes in a test tube. While cell-free systems have historically been used to study fundamental biology (e.g., the genetic code) (Nirenberg and Matthaei, 1961), recent development of cell-free protein synthesis capabilities (Caschera and Noireaux, 2014; Des Soye et al., 2019; Jewett et al., 2008; Jewett and Swartz, 2004) has expanded the application space to include prototyping of genetic parts (Chappell et al., 2013; Moore et al., 2018; Siegal-Gaskins et al., 2014; Takahashi et al., 2015a; Takahashi et al., 2015b; Yim et al., 2019) and studying whole metabolic pathways (Bujara et al., 2011; Dudley et al., 2019; Karim et al., 2020; Karim and Jewett, 2016; Kelwick et al., 2018). As compared to in vivo approaches, cell-free systems have several key advantages: First, these systems lack a cell wall, and thereby allow active monitoring, rapid sampling and direct manipulation. Second, because genetic instructions can be simply added to CFE reactions in form of plasmid DNA or linear PCR products, they circumvent laborious cloning and transformation steps, and can thereby facilitate testing of genetic designs within a few hours instead of several days or weeks. Third, this approach does not rely on time-consuming enzyme purification procedures but rapidly builds and tests metabolic pathways directly in cell extracts by synthesizing required enzymes in vitro (Karim et al., 2018; Karim and Jewett, 2016; Liu et al., 2019). Given these advantages, cell-free systems have emerged as an important approach for accelerating biological design, especially with the advent of new extract based systems from non-model organisms: Bacillus (Moore et al., 2018), Streptomyces (Li et al., 2018, 2017), Vibrio (Des Soye et al., 2018; Failmezger et al., 2018; Wiegand et al., 2018), and Pseudomonas (Wang et al., 2018) among others. However, no clostridia cell-free system exists that produces protein yields sufficient for prototyping genetic parts and metabolic pathways.
Here, we present the first, to our knowledge, easy-to-use, robust and high-yielding clostridia CFE platform derived from an industrially relevant strain, Clostridium autoethanogenum (C. autoethanogenum), which promises to facilitate metabolic engineering applications. Specifically, the goal was to enable cell-free protein synthesis yields of more than 100 μg/ml by optimizing process parameters. To achieve this goal, we first streamline and optimize the extract preparation and processing procedure. Second, we carry out a systematic optimization of CFE reaction conditions to tune the physicochemical environment for stimulating highly active combined transcription and translation from linear DNA templates. We observed a ~100,000-fold increase in protein synthesis yields relative to the original unoptimized case, resulting in a final titer of approximately 240 μg/mL in 3-h batch reaction. This yield could be further improved in a semi-continuous reaction to more than 300 μg/mL. Finally, with the clostridia CFE system at hand, we demonstrate the capability of our system for clostridia-specific prototyping: clostridia genetic parts by expressing luciferase from constructs under the control of endogenous promoters and 5′UTRs derived from clostridia metabolic enzymes or by utilizing different gene coding sequences, as well as activity of clostridia metabolic pathways in the extracts (Fig. 1). We anticipate that this platform, the first high-yielding CFE system from an obligate anaerobe, will speed-up metabolic engineering efforts for bioprocess development in clostridia.
Section snippets
Results
Developing a system capable of CFE from a new organism requires optimization at several levels. The choice of organism, fermentation conditions, extract preparation and processing, and cell-free reaction conditions each play an important role. In this work, we aimed to develop a high-yielding CFE system using an industrially relevant clostridia strain as our source organism, C. autoethanogenum. Based on extensive optimization that has gone into establishing anaerobic fermentation conditions for
Discussion
In this work, we describe the development of a robust, high-yielding, and easy to use CFE system derived from the non-model and anaerobic bacterium C. autoethanogenum. To do so, we optimized the extract lysis preparation procedure, streamlined the extract processing steps, and optimized cell-free reaction conditions. We found C. autoethanogenum-derived CFE requires different conditions than E. coli-derived CFE. Surprisingly, C. autoethanogenum CFE requires unusually high magnesium
Materials & methods
Strains and plasmid constructs. Clostridium autoethanogenum DSM 19630, a derivate of type strain DSM10061 was used in this study (Heijstra et al., 2016). The gene sequences and oligonucleotides used in this study are listed in the Supplemental Materials and Table S1, respectively.
Codon-adapted luciferase genes for CFE were synthesized by IDT, cloned into the pJL1 plasmid using Gibson assembly and confirmed by Sanger sequencing by ACGT, Inc. Kanamycin (50 μg/mL) was used to maintain pJL1-based
Author contributions
A.K. and M.C.J. designed the experiments. A.P.M. and M.K. generated C. autoethanogenum cells. A.K., G.A.R., N.L.E., Z.K.Y., T.J.T. conducted and analyzed experiments. A.K. and M.C.J. wrote the manuscript. M.C.J. supervised the research.
Declaration of competing interest
A.P.M., S.D.S and M.K. are employees of LanzaTech, which has commercial interest in gas fermentation with C. autoethanogenum. A.K., A.P.M., M.K. and M.C.J. are co-inventors on the U.S. Patent Application Serial No. 62/810,014 that incorporates discoveries described in this manuscript. All other authors declare no conflicts.
Acknowledgements
The authors thank Ashty Karim (ORCID # 0000-0002-5789-7715), Lauren Clark, and Ben De-Soye for helpful comments on the manuscript and Ava Rhule-Smith, Jim Daleiden, Monica MacDonald, and Steven Glasker for their help growing the C. autoethanogenum cultures and Robert Nogle for help with subcloning the promoter regions. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research in the DOE Office of Science under Award Number DE-SC0018249 and FWP
References (85)
- et al.
Cell-free protein synthesis: applications come of age
Biotechnol. Adv.
(2012) - et al.
Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription–translation system
Biochimie
(2014) - et al.
Effective approaches for the production of heterologous proteins using the Thermococcus kodakaraensis-based translation system
J. Biotechnol.
(2008) - et al.
Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile
J. Biotechnol.
(2006) - et al.
Recent trends in lactic acid biotechnology: a brief review on production to purification
J. Radiat. Res. Appl. Sci.
(2014) Wheat germ systems for cell-free protein expression
FEBS Lett.
(2014)- et al.
Cell-free synthetic biology: thinking outside the cell
Metab. Eng.
(2012) - et al.
Recent advances in producing and selecting functional proteins by using cell-free translation
Curr. Opin. Biotechnol.
(1998) - et al.
Current status and prospects of industrial bio-production of n-butanol in China
Biotechnol. Adv.
(2015) - et al.
Controlling cell-free metabolism through physiochemical perturbations
Metab. Eng.
(2018)