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.     

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.     

High-level cell-free expression and functional characterization of a novel aquaporin from Photobactetrium profundum SS9

AqpSS9 is a novel aquaporin derived from the deep sea bacterium Photobactetrium profundum SS9 and has attracted many attentions in developing water filtration biomimetic membranes. Functional characterization of AqpSS9 was carried out first by its expression in E. coli MM1211 (aqpZ⁻). Results showed that it was similar to bacterial aquaporin Z (AqpZ) and functioned as a real aquaporin. In-vitro expression of AqpSS9 were systematically investigated using three different modes: precipitate-based cell-free (P-CF) mode, the detergent-based cell-free (D-CF) mode and lipid-based cell-free expression mode (L-CF). D-CF mode showed more superiority than P-CF and L-CF mode, and the highest expression level of 571 mg/l was achieved by adding 0.7% Brij-78. Then AqpSS9 was purified by affinity chromatograph and incorporated into DOPC liposomes. Osmotic water permeability values (P_f) of reconstituted AqpSS9 proteoliposomes was measured as 310.7 ± 3.2 μm/s, which was about 3.5 times of empty control liposomes and comparable to reported Aqps. The AqpSS9 embedded layer-by-layer (LbL) membrane was fabricated and tested, which showed enhanced water permeability and salt rejection in comparison with the control membrane. This work demonstrated the good performance of AqpSS9 as a water channel protein, which may become an alternative candidate for biomimetic membrane construction for water filtration.     

Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation

Cell-free protein synthesis has become one of the standard methods for protein expression. One of the major advantages of this method is that PCR-amplified linear DNA fragments can be directly used as templates for protein synthesis. The productivity of cell-free protein synthesis using linear DNA templates is generally lower than that from plasmid DNA templates, especially when using an Escherichia coli cell extract. In the present study, we found that a simple modification of the protocol for cell extract preparation from E. coli, just by altering the cultivation temperature (37 °C) of the cells to a moderately lower range (20-34 °C), dramatically reduced the linear DNA degradation activity in the cell extract. This modification greatly improved the productivity of cell-free protein synthesis from linear DNA templates. The removal of the RecD protein, one of the components of exonuclease V, from the extract had almost the same effect, indicating that the linear DNA degradation activity in the extract was mainly due to the RecD protein and that its expression level was decreased at the lower cultivation temperature.     

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.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Cell-free production and characterisation of human uncoupling protein 1–3

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer.Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.     

Cell-free platforms for flexible expression and screening of enzymes

As was witnessed from PCR technology, in vitro applications of biosynthetic machinery can expand the horizon of biotechnology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocess. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables instant creation of protein molecules from diverse sources of genetic information. Enzyme discovery and engineering is the field of particular interest among the possible applications of cell-free protein synthesis since many of the intrinsic limitations associated with traditional cell-based expression screening of enzymes can be effectively addressed. Cell-free synthesis not only offers excellent throughput in the generation of enzymes, it allows facile integration of expression and analysis of enzymes, greatly accelerating the process of enzyme discovery. This review article is thus intended to survey recent progress in cell-free protein synthesis technology focused on its applications in enzyme expression and screening.     

Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway

An efficient production process is important for industrial microorganisms. The cellular efficiency of solventogenic clostridia, a group of anaerobes capable of producing a wealth of bulk chemicals and biofuels, must be improved for competitive commercialization. Here, using Clostridium acetobutylicum, a species of solventogenic clostridia, we revealed that the insufficient biosynthesis of biotin, a pivotal coenzyme for many important biological processes, is a major limiting bottleneck in this anaerobe’s performance. To address this problem, we strengthened the biotin synthesis of C. acetobutylicum by overexpressing four relevant genes involved in biotin transport and biosynthesis. This strategy led to faster growth and improved the titer and productivity of acetone, butanol and ethanol (ABE solvents) of C. acetobutylicum in both biotin-containing and biotin-free media. Expressionally modulating these four genes by modifying the ribosome binding site further promoted cellular performance, achieving ABE solvent titer and productivity as high as 21.9 g/L and 0.30 g/L/h, respectively, in biotin-free medium; these values exceeded those of the wild-type strain by over 30%. More importantly, biotin synthesis reinforcement also conferred improved ability of C. acetobutylicum to use hexose and pentose sugars, further demonstrating the potential of this metabolic-engineering strategy in solventogenic clostridia.     

Extracellular biosynthesis of cadmium sulphide quantum dot using cell-free extract of Pseudomonas chlororaphis CHR05 and its antibacterial activity

For the first time a novel aquatic bacterium belonging to strain of Pseudomonas chlororaphis was developed for the synthesis of cadmium sulfide quantum dots. The cadmium sulfate solution incubated with cell-free extract (CFE) of P. chlororaphis strain CHR05 to generate cadmium sulfide nanoparticles (CdSNps) which were characterized with combined spectroscopy and microscopy analyses. The preliminary confirmation on the formation of CdSNps was done by UV–vis and fluorescence analyses with the absorption and emission spectra at 435 and 475 nm, respectively. EDX pattern shows that the CdSNps are composed of the elementals Cd and S. Also, XRD analysis of the purified nanoparticles confirmed the formation of CdSNps. FE-SEM, TEM, and DLS analyzed the size and morphology of the CdSNps. The biosynthesis of CdSNps with the strategy of cell free extract was investigated under optimum conditions. After 24 h of incubation, the results showed that the novel isolated strain can produce spherical CdSNps with an average size of 6.7 ± 2.4 nm, after exposure to CdSO₄ solution (2.5 mM) at pH 7.5 and 30 °C. The antibacterial activity CdSNps against some Gram-positive and -negative bacteria were determined using agar well diffusion method and microplate method which has growth-inhibitory effect all on tested bacteria.     

Antioxidant and Antiradical Properties of Probiotic Strains Bacillus amyloliquefaciens ssp. plantarum

The aim of the present study was to investigate the in vitro antioxidant potential of the cell-free extracts (CFE) of two probiotic bacteria Bacillus amyloliquefaciens ssp. plantarum IMV B-7142 and Bacillus amyloliquefaciens ssp. plantarum IMV B-7143 and their hepatoprotective effects. These strains are the main components of the veterinary probiotic preparation endosporyn. The CFE of probiotic bacteria were able to stabilize the 2.2-diphenyl-1-picrylhydrazyl radical to its neutral form at their cultivation during 24–48 h. But this index was more pronounced for the IMV B-7142 strain and amounted to 44.4–51.2%. The hydroxyl radical scavenging activity of the CFE of probiotic bacteria increased more than 70–80% regardless of the cultivation period (24–48 h). The antioxidant potential of probiotic strains is associated with the synthesis of the multiple biologically active molecules. The phenolic and benzoic acids-antioxidants (gallic, 4-hydroxyphenylacetic, caffeic, syringic, p-coumaric, trans-ferulic, and trans-cinnamic acids) were identified among metabolites of B. amyloliquefaciens ssp. plantarum strains. The CFE of probiotic strains were able to protect of rat hepatocytes from the toxic effects of the carbon tetrachloride (CCl₄). Post-treatment of stress-induced rat hepatocytes by CFE of the IMV B-7042 was accompanied by an increase of the catalase activity of cells by 485.2 mM/min × mg of protein, compared to stress-damaged sample. In doing so, the content of the main markers of oxidative stress: lipid hydroperoxides and malondialdehyde decreased significantly. The results suggested that CFE of both probiotic strains have potent antioxidant properties and effectively protect of stress-damaged rat hepatocytes.

     

Wheat germ systems for cell-free protein expression

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.     

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.     

Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×10²⁴ photons/s/mol, the highest reported activity for any characterized luciferase.