ACS Synthetic Biology最新文献

Efficient methods for diversifying genes of interest (GOIs) are essential in protein engineering. For example, OrthoRep, a yeast-based orthogonal DNA replication system that achieves the rapid in vivo diversification of GOIs encoded on a cytosolic plasmid (p1), has been successfully used to drive numerous protein engineering campaigns. However, OrthoRep-based GOI evolution has almost always started from single GOI sequences, limiting the number of locations on a fitness landscape from where evolutionary search begins. Here, we present a simple approach for the high-efficiency integration of GOI libraries onto OrthoRep. By leveraging integrases, we demonstrate recombination of donor DNA onto the cytosolic p1 plasmid at exceptionally high transformation efficiencies, even surpassing the transformation efficiency of standard circular plasmids and linearized plasmid fragments into yeast. We demonstrate our method’s utility through the straightforward construction of mock nanobody libraries encoded on OrthoRep, from which rare binders were reliably enriched. Overall, integrase-assisted manipulation of yeast cytosolic plasmids should enhance the versatility of OrthoRep in continuous evolution experiments and support the routine construction of large GOI libraries in yeast, in general.

We have developed a rapid, simple, and high-throughput screening system for recombinant enzymes using disulfide-bonded hydrogel beads (HBs) produced via a microfluidic method. These redox-responsive HBs were compatible with the biosynthesis of enzyme mutants via cell-free protein synthesis, fluorescent staining through an enzymatic reaction, and genetic information recovery after fluorescence-activated droplet sorting (FADS). The expression of microbial transglutaminase zymogen (MTGz) using cell-free protein synthesis and the cross-linking-reactivity-based staining of HBs with a fluorescent product were validated. Next-generation sequencing (NGS) analysis of the genes recovered from highly fluorescent HBs identified novel mutation sites (N25 and N27) in the propeptide domain. The introduction of these mutations allowed for the design of an engineered active MTGz, demonstrating the potential of HBs as artificial compartments for the FADS-based selection of enzymes that catalyze peptide and protein cross-linking reactions.

Escherichia coli surface display technology, which facilitates the stable display of target peptides and proteins on the bacterial surface through fusion with anchor proteins, has become a potent and versatile tool in biotechnology and biomedicine. The E. coli surface display strategy presents a unique alternative to classic intracellular and extracellular expression systems, facilitating the anchorage of target peptides and proteins on the cell surface for functional execution. This distinctive attribute also introduces a novel paradigm in the realm of biocatalysis, harnessing cells with surface-displayed enzymes to catalyze the conversion of substrates. This strategy effectively eliminates the requirement for enzyme purification, overcomes the limitations related to substrate transmembrane transport, improves enzyme activity and stability, and greatly reduces the cost of downstream product purification, thus making it widely used in biocatalysis. Here, we review recent advances in various surface display systems and surface display technology for biocatalytic applications. Additionally, we discuss the current limitations of this technology and several promising alternative display methods.

Colorants are widely used in our daily lives to give colors to diverse chemicals and materials, including clothes, food, drugs, cosmetics, and paints. Although synthetic colorants derived from fossil fuels have been predominantly used due to their low cost, there is a growing need to replace them with natural alternatives. This shift is driven by increasing concerns over the climate crisis caused by excessive fossil fuel use, as well as health issues associated with the consumption of foods, beverages, and cosmetics containing petroleum-derived chemicals. In addition, many natural colorants show health-promoting properties such as antioxidant and antimicrobial activities. Despite such advantages, natural colorants could not be readily commercialized and distributed in the market due to their low stability, limited color spectrum, and low yields from natural resources. To this end, synthetic biology approaches have been developed to efficiently produce natural colorants from renewable resources with high yields. Strategies to diversify natural colorants to produce non-natural derivatives with enhanced properties and an expanded color spectrum have been also developed. In this Review, we discuss the recent synthetic biology strategies developed for enhancing the production of natural colorants and their non-natural derivatives, together with accompanying examples. Challenges ahead and future perspectives are also discussed.

The wine strains of Saccharomyces cerevisiae transform glucose into ethanol and other byproducts such as glycerol and acetate. The balance of these metabolites is important during the fermentation process, which impacts the organoleptic properties of wines. Ethanol and glycerol productions are mainly controlled by the ADH1 and GPD1 genes, which encode for the alcohol dehydrogenase and glycerol-3-phosphate-dehydrogenase enzymes, respectively. Genetic modification of these genes can thus be used to alter the levels of the corresponding metabolites and to reroute fermentation. In this work, we used an optogenetic system named FUN-LOV (FUNgal-Light Oxygen Voltage) to regulate the expression of ADH1 and GPD1 in a wine yeast strain using light. Initially, we confirmed the light-controlled expression of GPD1 and ADH1 in the engineered strains via RT-qPCR and a translational reporter, respectively. To characterize the generated yeast strains, we performed growth curve assays and laboratory-scale fermentations, observing phenotypic differences between illumination conditions that confirm the optogenetic control of the target genes. We also monitored glucose consumption and ethanol and glycerol productions during a fermentation time course, observing that the optogenetic control of GPD1 increased glycerol production under constant illumination without affecting ethanol production. Interestingly, the optogenetic control of ADH1 showed an inverted phenotype, where glycerol production increased under constant darkness conditions. Altogether, our results highlight the feasibility of using optogenetic tools to control yeast fermentation in a wine yeast strain, which allows changing the balance of metabolic products of interest in a light-dependent manner.

Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harboring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.

Synthetic riboswitches are attracting increasing interest for a diverse range of applications, including synthetic biology, functional genomics, and prospective therapeutic strategies. This study demonstrates that controlling alternative splicing with synthetic riboswitches represents a promising approach to effectively regulating transgene expression in mammalian cells. However, the function of synthetic riboswitches in the eukaryotic system in controlling gene expression is often limited to certain genes or cell types. So far, strategies to increase the dynamic range of regulation have been focused on adapting and modifying the riboswitch sequence itself without taking into account the context in which the riboswitch was inserted. In the present study, the tetracycline riboswitch was chosen to investigate the effects of the context and insertion site of a cassette exon within the gene to control the expression of an artificial arachidonate 5-lipoxygenase gene (ALOX5) in HEK293 cells. We demonstrate here that the use of riboswitch-controlled cassette exons for the control of gene expression via alternative splicing can be easily transferred to another gene through the process of contextual sequence adaptation. This was achieved through the introduction of gene-specific intronic and exonic sequences with different intron lengths and positions being tested. In contrast, the introduction of nonadapted constructs resulted in an unanticipated functionality outcome of the gene switch. Furthermore, we demonstrate that the combination of two cassette exons into a single gene resulted in a notable enhancement in the dynamic range. Finally, we generated a novel riboswitch-controlled splicing concept that enabled us to switch 5-LO wild-type to expression of an ALOX5 isoform that lacks exon 13 (5-LOΔ13). Taken together, this study demonstrates that synthetic riboswitches that control alternative splicing are a powerful tool to regulate gene expression when applied in combination with gene-specific intronic and exonic sequences.

Cell-free systems, which can express an easily detectable output (protein) with a DNA or mRNA template, are promising as foundations of biosensors devoid of cellular constraints. Moreover, by encasing them in membranes such as natural cells to create artificial cells, these systems can avoid the adverse effects of environmental inhibitory molecules. However, the bacterial systems generally used for this purpose do not function well at ambient temperatures. We here encapsulated a eukaryotic cell-free system consisting of wheat germ extract (WGE) and a DNA template encoding an analyte-responsive regulatory RNA (called a riboswitch) into giant unilamellar vesicles (GUVs) to create eukaryotic artificial cell-based sensors that function well at ambient temperature. First, we improved our previously reported eukaryotic synthetic riboswitches and WGE for use in GUVs by chimerizing two internal ribosome entry sites and optimizing magnesium concentrations, respectively, both of which increased the expression efficiency in GUVs several fold. Then, a DNA template encoding one of these riboswitches followed by a reporter protein was encapsulated with the optimized GUV-friendly WGE. Importantly, our previously established versatile method allowed for the rational design of highly efficient eukaryotic riboswitches that are responsive to a user-defined analyte. In fact, we utilized this method to successfully create three types of artificial cells, each of which responded to a specific, membrane-permeable analyte with wide-range, analyte-dose dependency and high sensitivity at ambient temperature. Finally, due to their orthogonality and robustness, we were able to mix a cocktail of these artificial cells to achieve simultaneous detection of the three analytes without significant barriers.

Triterpenoids widely exist in nature with diverse structures and possess various functional properties and biological effects. However, research on triterpenoids biosynthesis in Corynebacterium glutamicum is still limited to squalene, which restricts the development of C. glutamicum to produce high-value triterpenoids. In this study, C. glutamicum was developed as an efficient and flexible platform for the biosynthesis of different types of triterpenoids. Squalene was synthesized and the titer was improved to 400.1 mg/L in flask combining strategies of metabolic engineering and fermentation optimization. Particularly, intracellular squalene accounted for more than 97%, addressing the problem of leaking squalene in C. glutamicum, which may restrict the subsequent synthesis of other triterpenoids derived from squalene. Furthermore, 201.9 mg/L (3S)-2,3-oxidosqualene (SQO) and 264.9 mg/L (3S,22S)-2,3,22,23-dioxidosqualene (SDO) were successfully synthesized in strains harboring heterogeneous squalene epoxidase from Arabidopsis thaliana with different expression strengths. Therefore, a platform for de novo triterpenoids synthesis based on SQO or SDO was constructed in C. glutamicum. For instance, biosynthesis of α-amyrin and α-onocerin was achieved for the first time by introducing oxidosqualene cyclases in SQO- and SDO-producing C. glutamicum strains, respectively. After optimization, the titer of α-amyrin and α-onocerin was improved to 65.3 and 136.85 mg/L, respectively. Furthermore, ursolic acid, derived from α-amyrin, was synthesized after expressing cytochrome P450 enzyme and its compatible cytochrome P450 reductases with a titer of 486 μg/L. For the first time, reactions of epoxidation, cyclization, and oxidation from squalene were achieved in C. glutamicum, leading to the production of different types of triterpenoids. Our study provides a new platform for the production of triterpenoids, which will be helpful for the large-scale production of triterpenoids employing C. glutamicum as a chassis strain.

One of the most pressing challenges in cell-free synthetic biology is to assemble well-controlled genetic circuits. However, no complex circuits have been reported in eukaryotic cell-free systems, unlike the case in bacterial ones, despite several unique advantages of the former. We here developed protein-responsive upregulating riboswitches (ON-riboswitches) that function in wheat germ extract to create multistep gene regulatory cascades. Although the initial two types of ON-riboswitches we first designed were less efficient than desired, we improved one of them by incorporating hybridization switches to successfully construct a pair of highly efficient, protein-responsive ON-riboswitches. Both upregulated expression up to 20-fold through self-cleavage by a hammerhead ribozyme (HHR) in response to the corresponding protein ligands expressed in situ. We then combined them with similar types of HHR-based, small-molecule-responsive ON-riboswitches regulating protein ligand expression, to create four kinds of two-step regulatory cascades. Due to the high orthogonality of all the riboswitches used, we also succeeded in regulating two-step cascades concurrently and even in creating three-step cascades. Interestingly, the switching efficiency of each multistep cascade constructed was equivalent to that of the worst step within it. Therefore, more complex cascades with additional steps could be constructed using other efficient and orthogonal, protein-responsive ON-riboswitches with minimal loss of total switching efficiency, although the reaction conditions must be optimized to prevent a reduction of expression efficiencies. Riboswitch-based cascades fashioned through our proposed strategy would aid in the construction of eukaryotic genetic circuits for programmed cell-free systems or artificial cells with functionalities surpassing those of natural cells.