Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase.

During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.

Membrane protein synthesis: no cells required.

Despite advances in membrane protein (MP) structural biology and a growing interest in their applications, these proteins remain challenging to study. Progress has been hindered by the complex nature of MPs and innovative methods will be required to circumvent technical hurdles. Cell-free protein synthesis (CFPS) is a burgeoning technique for synthesizing MPs directly into a membrane environment using reconstituted components of the cellular transcription and translation machinery in vitro. We provide an overview of CFPS and how this technique can be applied to the synthesis and study of MPs. We highlight numerous strategies including synthesis methods and folding environments, each with advantages and limitations, to provide a survey of how CFPS techniques can advance the study of MPs.

A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

Establishing a Klebsiella pneumoniae-Based Cell-Free Protein Synthesis System.

Cell-free protein synthesis (CFPS) systems are emerging as powerful platforms for in vitro protein production, which leads to the development of new CFPS systems for different applications. To expand the current CFPS toolkit, here we develop a novel CFPS system derived from a chassis microorganism Klebsiella pneumoniae, an important industrial host for heterologous protein expression and the production of many useful chemicals. First, we engineered the K. pneumoniae strain by deleting a capsule formation-associated wzy gene. This capsule-deficient strain enabled easy collection of the cell biomass for preparing cell extracts. Then, we optimized the procedure of cell extract preparation and the reaction conditions for CFPS. Finally, the optimized CFPS system was able to synthesize a reporter protein (superfolder green fluorescent protein, sfGFP) with a maximum yield of 253 ± 15.79 µg/mL. Looking forward, our K. pneumoniae-based CFPS system will not only expand the toolkit for protein synthesis, but also provide a new platform for constructing in vitro metabolic pathways for the synthesis of high-value chemicals.

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins.

Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Human blood contains circulating cell-free mitochondria, but are they really functional?

Dache et al. (FASEB J 34: 3616-3630, 2020) recently reported the presence of respiratory-competent cell-free mitochondria in human blood (up to 3.7 × 106 per mL of blood), providing exciting perspectives on the potential role of these extracellular mitochondria. Although their evidence for the presence of cell-free mitochondria in human blood is compelling, their conclusion that these cell-free mitochondria are respiratory competent or functional has to be reevaluated. To this end, we evaluated the functionality of cell-free mitochondria in human blood using high-resolution respirometry and mitochondria extracted from platelets of the same blood samples as positive controls. Although cell-free mitochondria were present in human plasma (i.e., significant MitoTracker Green fluorescence and complex IV activity), there was no evidence suggesting that their mitochondrial electron transport system (ETS) was functional (i.e., respiration rate not significantly different from 0; no significant responses to ADP, uncoupler, or mitochondrial inhibitors oligomycin and antimycin A). Yet, in vitro complex IV activity was detectable and even slightly higher than levels found in mitochondria extracted from platelets, suggesting that cell-free mitochondria in human blood are likely to only retain a nonfunctional part of the ETS. Despite being unlikely to be fully functional in the narrow sense (i.e., capable of oxidative phosphorylation), circulating cell-free mitochondria may have significant physiological roles that remain to be elucidated.NEW & NOTEWORTHY The recently reported cell-free mitochondria in human blood have been thought to be respiratory competent, giving rise to speculation about their biological function(s). By characterizing their bioenergetics in vitro, we show that circulating cell-free mitochondria are unlikely to be functional in vivo since they display no potential for oxidative phosphorylation.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Reprogramming extracellular vesicles with engineered proteins.

Extracellular vesicles (EVs) have been emerging as a new class of cell-free therapy for the treatment of a variety of diseases, including cancer, tissue injuries, and inflammatory diseases. Reprograming native EVs by genetic engineering and other approaches offers an attractive prospect of extending therapeutic capabilities of EVs beyond their natural functions and properties. In this review article, we survey the state-of-the-art methods of EVs engineering and summarize major therapeutic applications of the reprogrammed EVs.

Inosine induces context-dependent recoding and translational stalling.

RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.

Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

Decoding on the ribosome depends on the structure of the mRNA phosphodiester backbone.

During translation, the ribosome plays an active role in ensuring that mRNA is decoded accurately and rapidly. Recently, biochemical studies have also implicated certain accessory factors in maintaining decoding accuracy. However, it is currently unclear whether the mRNA itself plays an active role in the process beyond its ability to base pair with the tRNA. Structural studies revealed that the mRNA kinks at the interface of the P and A sites. A magnesium ion appears to stabilize this structure through electrostatic interactions with the phosphodiester backbone of the mRNA. Here we examined the role of the kink structure on decoding using a well-defined in vitro translation system. Disruption of the kink structure through site-specific phosphorothioate modification resulted in an acute hyperaccurate phenotype. We measured rates of peptidyl transfer for near-cognate tRNAs that were severely diminished and in some instances were almost 100-fold slower than unmodified mRNAs. In contrast to peptidyl transfer, the modifications had little effect on GTP hydrolysis by elongation factor thermal unstable (EF-Tu), suggesting that only the proofreading phase of tRNA selection depends critically on the kink structure. Although the modifications appear to have no effect on typical cognate interactions, peptidyl transfer for a tRNA that uses atypical base pairing is compromised. These observations suggest that the kink structure is important for decoding in the absence of Watson-Crick or G-U wobble base pairing at the third position. Our findings provide evidence for a previously unappreciated role for the mRNA backbone in ensuring uniform decoding of the genetic code.

ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation.

ZFAND5/ZNF216, a member of the zinc finger AN1-type domain family, is abundant in heart and brain, but is induced in skeletal muscle during atrophy (although not in proteotoxic stress). Because mice lacking ZFAND5 exhibit decreased atrophy, a role in stimulating protein breakdown seemed likely. Addition of recombinant ZFAND5 to purified 26S proteasomes stimulated hydrolysis of ubiquitinated proteins, short peptides, and ATP. Mutating its C-terminal AN1 domain abolished the stimulation of proteasomal peptidase activity. Mutating its N-terminal zinc finger A20 domain, which binds ubiquitin chains, prevented the enhanced degradation of ubiquitinated proteins without affecting peptidase activity. Mouse embryonic fibroblast (MEF) cells lacking ZFAND5 had lower rates of protein degradation and proteasomal activity than WT MEFs. ZFAND5 addition to cell lysates stimulated proteasomal activity and protein degradation. Unlike other proteasome regulators, ZFAND5 enhances multiple 26S activities and overall cellular protein breakdown.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays.

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, ß-D-apio-, and ß-D-ribofuranosides. T and HOT ß-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT ß-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T ß-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT ß-D-fructofuranoside and ß-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.

Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca2+/Mn2+ Pump.

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

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.

Determination of enrichment factors for modified RNA in MeRIP experiments.

In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

Flexibility and structure of flanking DNA impact transcription factor affinity for its core motif.

Spatial and temporal expression of genes is essential for maintaining phenotype integrity. Transcription factors (TFs) modulate expression patterns by binding to specific DNA sequences in the genome. Along with the core binding motif, the flanking sequence context can play a role in DNA-TF recognition. Here, we employ high-throughput in vitro and in silico analyses to understand the influence of sequences flanking the cognate sites in binding of three most prevalent eukaryotic TF families (zinc finger, homeodomain and bZIP). In vitro binding preferences of each TF toward the entire DNA sequence space were correlated with a wide range of DNA structural parameters, including DNA flexibility. Results demonstrate that conformational plasticity of flanking regions modulates binding affinity of certain TF families. DNA duplex stability and minor groove width also play an important role in DNA-TF recognition but differ in how exactly they influence the binding in each specific case. Our analyses further reveal that the structural features of preferred flanking sequences are not universal, as similar DNA-binding folds can employ distinct DNA recognition modes.

The deoxyribose phosphate lyase of DNA polymerase ß suppresses a processive DNA synthesis to prevent trinucleotide repeat instability.

Trinucleotide repeat (TNR) instability is associated with over 42 neurodegenerative diseases and cancer, for which the molecular mechanisms remain to be elucidated. We have shown that the DNA base excision repair (BER) pathway and its central component, DNA polymerase ß (pol ß), in particular, its polymerase activity plays an active role in regulating somatic TNR instability. Herein, we revealed a unique role of the pol ß dRP lyase in preventing somatic TNR instability. We found that deficiency of pol ß deoxyribose phosphate (dRP) lyase activity locked the pol ß dRP lyase domain to a dRP group, and this 'tethered' pol ß to its template forcing the polymerase to perform a processive DNA synthesis. This subsequently promoted DNA strand slippage allowing pol ß to skip over a template loop and causing TNR deletion. We showed that the effects were eliminated by complementation of the dRP lyase deficiency with wild-type pol ß protein. The results indicate that pol ß dRP lyase activity restrained the pol ß-dRP interaction to suppress a pol ß processive DNA synthesis, thereby preventing TNR deletion. This further implicates a potential of pol ß dRP lyase inhibition as a novel treatment of TNR-expansion diseases.