Cell-free gene expression: an expanded repertoire of applications.

Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits.

Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences.

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

A dynamic kinetic model captures cell-free metabolism for improved butanol production.

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 h. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping.

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

Cell-Free Exploration of the Natural Product Chemical Space.

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis.

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23-610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.

Cell-free styrene biosynthesis at high titers.

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin.

Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 µg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Controlling cell-free metabolism through physiochemical perturbations.

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts.

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.

Ribosome Pool Engineering Increases Protein Biosynthesis Yields.

The biosynthetic capability of the bacterial ribosome motivates efforts to understand and harness sequence-optimized versions for synthetic biology. However, functional differences between natively occurring ribosomal RNA (rRNA) operon sequences remain poorly characterized. Here, we use an in vitro ribosome synthesis and translation platform to measure protein production capabilities of ribosomes derived from all unique combinations of 16S and 23S rRNAs from seven distinct Escherichia coli rRNA operon sequences. We observe that polymorphisms that distinguish native E. coli rRNA operons lead to significant functional changes in the resulting ribosomes, ranging from negligible or low gene expression to matching the protein production activity of the standard rRNA operon B sequence. We go on to generate strains expressing single rRNA operons and show that not only do some purified in vivo expressed homogeneous ribosome pools outperform the wild-type, heterogeneous ribosome pool but also that a crude cell lysate made from the strain expressing only operon A ribosomes shows significant yield increases for a panel of medically and industrially relevant proteins. We anticipate that ribosome pool engineering can be applied as a tool to increase yields across many protein biomanufacturing systems, as well as improve basic understanding of ribosome heterogeneity and evolution.

Deconstructing synthetic biology across scales: a conceptual approach for training synthetic biologists.

Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales-molecular, circuit/network, cell/cell-free systems, biological communities, and societal-giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists.

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.

Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.

Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between an expression-enhancing terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing an expression-enhancing terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with an expression-enhancing terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications.

Many metabolic engineering and genetic engineering applications in yeast rely on the use of plasmids. Despite their pervasive use and the diverse collections available, there is a fundamental lack of understanding of how commonly used DNA plasmids affect the cell's ability to grow and how the choice of plasmid components can influence plasmid load and burden. In this study, we characterized the major attributes of the 2 micron and centromeric plasmids typically used in yeast by examining the impact of choice of selection marker, promoter, origin of replication, and strain ploidy on conferred growth rates and plasmid copy number. We conclude that the 'plasmid burden', as demonstrated by a reduced growth rate, is primarily due to the choice of selection marker, especially when auxotrophic markers are utilized. The plasmid burden traditionally attributed to replication and maintenance of plasmid DNA plays only a minor role in haploid yeast yet is much more significant in diploid strains. The selection marker can also significantly change plasmid copy number. In fact, plasmid copy number can be influenced to some extent by all of the parameters tested. The information presented in this study will allow for more rational design and selection of plasmids for engineering applications.

Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing.

Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze-thaw cycles retain consistent metabolic activity. This work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.

A Cell-Free Gene Expression Platform for Discovering and Characterizing Stop Codon Suppressing tRNAs.

Non-canonical amino acids (ncAAs) can be incorporated into peptides and proteins to create new properties and functions. Site-specific ncAA incorporation is typically enabled by orthogonal translation systems comprising a stop codon suppressing tRNA (typically UAG), an aminoacyl-tRNA synthetase, and an ncAA of interest. Unfortunately, methods to discover and characterize suppressor tRNAs are limited because of laborious and time-consuming workflows in living cells. In this work, we develop anEscherichia coli crude extract-based cell-free gene expression system to rapidly express and characterize functional suppressor tRNAs. Our approach co-expresses orthogonal tRNAs using endogenous machinery alongside a stop-codon containing superfolder green fluorescent protein (sfGFP) reporter, which can be used as a simple read-out for suppression. As a model, we evaluate the UAG and UAA suppressing activity of several orthogonal tRNAs. Then, we demonstrate that co-transcription of two mutually orthogonal tRNAs can direct the incorporation of two unique ncAAs within a single modified sfGFP. Finally, we show that the cell-free workflow can be used to discover putative UAG-suppressor tRNAs found in metagenomic data, which are nonspecifically recognized by endogenous aminoacyl-tRNA synthetases. We anticipate that our cell-free system will accelerate the development of orthogonal translation systems for synthetic biology.