Synthetic Biology Approaches Research Articles

Extremophilic Cellulases: A Comprehensive Review

Microbial cellulases are an important industrial enzyme having diverse applications in biotechnology, environmental challenges, industrial products and processes. Extremophiles like thermophillic bacteria are a good source of industrially important cellulases as these can withstand industrially rigorous procedures like paper deinking, fabric material softening, bio stoning, paper and pulp, biopolishing cloth material, animal feed and juice. Identification of novel cellulases or improving them through biotechnological interventions has remained a challenge for researchers. Genetic manipulation of thermophilic bacteria for increased cellulase production or synthetic biology approaches for cellulase gene/gene cluster extraction from thermophilic bacteria and expression in appropriate hosts for improved cellulase synthesis. The classic and high-throughput technologies like genomics, metagenomics and bioinformatics could be exploited to isolate cellulase genes from a variety of thermophilic bacteria and further processing. Keeping in view the ultimate requirement of extremophilic cellulases in industries, present study is a compilation of various aspects related to extremophilic cellulases their sources, production, biotechnological interventions and challenges.

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-d-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Renewable biosynthesis of isoprene from wastewater through a synthetic biology approach: the role of individual organic compounds

Renewable biosynthesis of isoprene from wastewater through a synthetic biology approach: the role of individual organic compounds

Biosynthesis of Phomactin Platelet Activating Factor AntagonistRequires a Two-Enzyme Cascade.

Phomactin diterpenoids possess a unique bicyclo[9.3.1]-pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from amarine fungus,Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate-analogue FGGPP and elucidated the novel metal ion binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This work provides the first insights into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Biosynthesis of Phomactin Platelet Activating Factor Antagonist Requires a Two‐Enzyme Cascade

AbstractPhomactin diterpenoids possess a unique bicyclo[9.3.1]pentadecane skeleton with multiple oxidative modifications, and are good platelet‐activating factor (PAF) antagonists that can inhibit PAF‐induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from a marine fungus, Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate analogue FGGPP and elucidated the novel metal‐ion‐binding mode and enzymatic mechanism of PhmA through site‐directed mutagenesis. This study provides the first insight into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Cellulose Degradation Enzymes in Filamentous Fungi, A Bioprocessing Approach Towards Biorefinery.

The economic exploration of renewable energy resources has hot fundamentals among the countries besides dwindling energy resources and increasing public pressure. Cellulose accumulation is a major bio-natural resource from agricultural waste. Cellulases are the most potential enzymes that systematically degrade cellulosic biomass into monomers which could be further processed into several efficient value-added products via chemical and biological reactions including useful biomaterial for human benefits. This could lower the environmental risks problems followed by an energy crisis. Cellulases are mainly synthesized by special fungal genotypes. The strain Trichoderma orientalis could highly express cellulases and was regarded as an ideal strain for further research, as the genetic tools have found compatibility for cellulose breakdown by producing effective cellulose-degrading enzymes. This strain has found a cellulase production of about 35g/L that needs further studies for advancement. The enzyme activity of strain Trichoderma orientalis needed to be further improved from a molecular level which is one of the important methods. Considering synthetic biological approaches to unveil the genetic tools will boost the knowledge about commercial cellulases bioproduction. Several genetic transformation methods were significantly cited in this study. The transformation approaches that are currently researchers are exploring is transcription regulatory factors that are deeply explained in this study, that are considered essential regulators of gene expression.

Technological Insights on Glycerol Valorization into Propanediol through Thermocatalytic and Synthetic Biology Approaches

The adverse effects of climate change, predominantly propelled by greenhouse gas emissions from fossil fuels, underscore the urgency of seeking sustainable alternatives to fossil fuel use. Amid growing concerns about climate change caused by fossil fuels and petrochemicals, this review focuses on sustainable solutions through the conversion of glycerol into value-added biochemicals. Glycerol, as the main byproduct of biodiesel production, is a particularly attractive chemical due to its potential to be upgraded into value-added building blocks and biochemicals. This review provides a detailed analysis of different thermochemical (catalytic) and synthetic biology (fermentative) pathways for the conversion of glycerol into 1,2-propanediol and 1,3-propanediol, which have proven industrial and commercial applications globally. The synthesis of propanediol from glycerol hydrogenolysis and other catalytic processes using different active metals and acidic oxides is reviewed. The reaction mechanism involved in hydrogenolysis reactions concerning the surface reaction mechanism is systematically discussed. The metabolic activities of promising microorganisms in fermenting glycerol, as the carbon source used to produce propanediol, are illustrated and elaborated. Combining these insights, this review is a comprehensive resource that can foster a better understanding of glycerol transformation into propanediol and its implications for sustainable chemistry and industrial practices. This exploration of alternative methods emphasizes the potential of sustainable approaches to reshape production practices and contribute to climate change mitigation.

Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization.

Recombinant adeno-associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale-up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity. To identify potential factors that restrained the productivity, we systematically characterized virus production kinetics through targeted quantitative proteomics and various physical assays of viral components. We demonstrated that reducing the excessive expression of gene of interest by its conditional expression greatly increased the productivity of these synthetic cell lines. Further enhancement was gained by optimizing induction profiles and alleviating proteasomal degradation of viral capsid protein by the addition of proteasome inhibitors. Altogether, these enhancements brought the productivity close to traditional multiple plasmid transfection. The rAAV produced had comparable full particle contents as those produced by conventional transient plasmid transfection. The present work exemplified the versatility of our synthetic biology-based viral vector production platform and its potential for plasmid- and virus-free rAAV manufacturing.

Multilayered safety framework for living diagnostics in the colon

Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed “Colourectal”, a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy.Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered.Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in mosquitoes.

Transcriptome analysis provides insights into coumarin biosynthesis in the medicinal plant Angelica dahurica cv. Yubaizhi

Angelica dahurica roots have a long history of use in traditional Chinese medicine due to their high coumarin content. To address the increasing demand for these roots, a synthetic biology approach has been proposed. Nevertheless, our comprehension of coumarin biosynthesis and its regulation remains limited. In this study, we utilized Hiseq2500 sequencing to analyze the transcriptomes of A. dahurica at different growth stages while concurrently quantifying coumarin content. Differentially expressed gene (DEG) analysis was employed to identify key genes involved in coumarin and terpenoid backbone biosynthesis. Weighted gene co-expression network analysis (WGCNA) was applied to identify gene modules strongly associated with coumarin content, elucidating the regulatory relationships between transcription factors (TFs) and pathway genes. Furthermore, KEGG enrichment analysis was used to explore essential pathways governing coumarin biosynthesis, with the identification of hub genes. Our results indicated that total coumarin content was highest in the roots, followed by leaves and stems, across all three developmental stages. Transcriptome analysis identified a total of 92,478 genes, among which 215 and 30 genes were implicated in coumarin and terpenoid backbone biosynthesis, respectively. Within the 73 identified gene modules by WGCNA, three modules—namely aquamarine1 (comprising two OMTs, one CSE, one AACT, one HDS, two PSs, one 2OGO, four UGTs, and seven CYP450s), darkmagenta (containing one UGT and one HDR), and navajowhite2 (consisting of one HCT, three UGTs, one CYP71A25, one OMT, one CSE, one HDS, and one PT)—were strongly associated with imperatorin, oxypeucedanin, and isoimperatorin content, respectively. KEGG enrichment analysis highlighted significant enrichment of cytochrome P450, transporter, and ubiquitin system pathways. Moreover, TF-gene regulatory analysis unveiled the complexity of coumarin biosynthesis, with 17 TF families regulating 17 genes in the aquamarine1 module, 8 TF families regulating 2 genes in the darkmagenta module, and 8 TF families regulating 7 genes in the navajowhite2 module. These comprehensive findings provide valuable insights into coumarin biosynthesis in A. dahurica, facilitating future research and potential applications in traditional Chinese medicine and synthetic biology strategies.

Transcription factor module NLP-NIGT1 fine-tunes NITRATE TRANSPORTER2.1 expression.

Arabidopsis (Arabidopsis thaliana) high-affinity NITRATE TRANSPORTER2.1 (NRT2.1) plays a dominant role in the uptake of nitrate, the most important nitrogen (N) source for most terrestrial plants. The nitrate-inducible expression of NRT2.1 is regulated by NIN-LIKE PROTEIN (NLP) family transcriptional activators and NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) family transcriptional repressors. Phosphorus (P) availability also affects the expression of NRT2.1 because the PHOSPHATE STARVATION RESPONSE1 transcriptional activator activates NIGT1 genes in P-deficient environments. Here, we show a biology-based mathematical understanding of the complex regulation of NRT2.1 expression by multiple transcription factors using 2 different approaches: a microplate-based assay for the real-time measurement of temporal changes in NRT2.1 promoter activity under different nutritional conditions, and an ordinary differential equation (ODE)-based mathematical modeling of the NLP- and NIGT1-regulated expression patterns of NRT2.1. Both approaches consistently reveal that NIGT1 stabilizes the amplitude of NRT2.1 expression under a wide range of nitrate concentrations. Furthermore, the ODE model suggests that parameters such as the synthesis rate of NIGT1 mRNA and NIGT1 proteins and the affinity of NIGT1 proteins for the NRT2.1 promoter substantially influence the temporal expression patterns of NRT2.1 in response to nitrate. These results suggest that the NLP-NIGT1 feedforward loop allows a precise control of nitrate uptake. Hence, this study paves the way for understanding the complex regulation of nutrient acquisition in plants, thus facilitating engineered nutrient uptake and plant response patterns using synthetic biology approaches.

Cytokine signaling in chimeric antigen receptor T-cell therapy.

Adoptive immunotherapy using chimeric antigen-receptor (CAR)-engineered T cells can induce robust antitumor responses against hematologic malignancies. However, its efficacy is not durable in the majority of the patients, warranting further improvement of T-cell functions. Cytokine signaling is one of the key cascades regulating T-cell survival and effector functions. In addition to cytokines that use the common γ chain as a receptor subunit, multiple cytokines regulate T-cell functions directly or indirectly. Modulating cytokine signaling in CAR-T cells by genetic engineering is one promising strategy to augment their therapeutic efficacy. These strategies include ectopic expression of cytokines, cytokine receptors, and synthetic molecules that mimic endogenous cytokine signaling. Alternatively, autocrine IL-2 signaling can be augmented through reprogramming of CAR-T cell properties through transcriptional and epigenetic modification. On the other hand, cytokine production by CAR-T cells triggers systemic inflammatory responses, which mainly manifest as adverse events such as cytokine-release syndrome (CRS) and neurotoxicity. In addition to inhibiting direct inflammatory mediators such as IL-6 and IL-1 released from activated macrophages, suppression of T-cell-derived cytokines associated with the priming of macrophages can be accomplished through genetic modification of CAR-T cells. In this review, I will outline recently developed synthetic biology approaches to exploit cytokine signaling to enhance CAR-T cell functions. I will also discuss therapeutic target molecules to prevent or alleviate CAR-T cell-related toxicities.

Synthetic Biology Toolkit for a New Species of Pseudomonas Promissory for Electricity Generation in Microbial Fuel Cells.

Microbial fuel cells (MFCs) offer sustainable solutions for various biotechnological applications and are a crucial area of research in biotechnology. MFCs can effectively treat various refuse, such as wastewater and biodiesel waste by decomposing organic matter and generating electricity. Certain Pseudomonas species possess extracellular electron transfer (EET) pathways, enabling them to transfer electrons from organic compounds to the MFC's anode. Moreover, Pseudomonas species can grow under low-oxygen conditions, which is advantageous considering that the electron transfer process in an MFC typically leads to reduced oxygen levels at the anode. This study focuses on evaluating MFCs inoculated with a new Pseudomonas species grown with 1 g.L-1 glycerol, a common byproduct of biodiesel production. Pseudomonas sp. BJa5 exhibited a maximum power density of 39 mW.m-2. Also, the observed voltammograms and genome analysis indicate the potential production of novel redox mediators by BJa5. Additionally, we investigated the bacterium's potential as a synthetic biology non-model chassis. Through testing various genetic parts, including constitutive promoters, replication origins and cargos using pSEVA vectors as a scaffold, we assessed the bacterium's suitability. Overall, our findings offer valuable insights into utilizing Pseudomonas spp. BJa5 as a novel chassis for MFCs. Synthetic biology approaches can further enhance the performance of this bacterium in MFCs, providing avenues for improvement.

Establishment of a halotolerant bioremediation platform from Halomonas cupida using synthetic biology approaches

At present, the vast majority of organic pollutants-degrading microbes are unable to grow and lose their degradation capability under high-salt conditions owing to lack of resistance to high osmotic pressure imposed by salt stress. To overcome the bottleneck, in this study, a halotolerant bioremediation platform is established from a halophilic chassis Halomonas cupida J9. As a proof of concept, we chose a typical environmental pollutant p-nitrophenol (PNP) to validate the feasibility of engineering H. cupida J9 for efficient mineralization of environmental pollutants in high saline environments. Firstly, four strong promoters including P3, P15, P16 and P22 were screened from H. cupida J9 by transcriptome analysis and promoter strength characterization. Subsequently, the constructed strain J9U-P15-AB and J9U-P8KT-AB with a chromosome-borne P15- and P8KT-pnpAB expression cassette could rapidly degrade and tolerate 200 mg/L PNP. Furthermore, a pathway for PNP biodegradation was functionally assembled in the genome of H. cupida J9 and optimized by enhanced expression of all PNP degradation genes with P15 and P8KT promoter, resulting in two stable genetically engineered strains J9U-P15PNP and J9U-P8KTPNP capable of metabolizing the only carbon source PNP to support cell growth in high-salinity media. Eventually, stable isotope analysis for elucidating the degradation pathway of 13C6-PNP indicated that the finally constructed strains were capable of converting PNP into CO2 in high saline media. These results suggest that promoter engineering may serve as a useful strategy for significantly improving the mineralization efficiency of organic pollutants. The engineered strains could degrade 25 mg/L PNP in seawater within 6 h, suggesting good potential of these degraders for in situ bioremediation. The gfp-labeled degraders also can be tracked during bioremediation. More importantly, this study underscores the value of H. cupida J9 as a promising chassis for the establishment of a halotolerant bioremediation platform using synthetic biology approaches.

The Impact of Viral Infection on the Chemistries of the Earth's Most Abundant Photosynthesizes: Metabolically Talented Aquatic Cyanobacteria.

Cyanobacteria are the most abundant photosynthesizers on earth, and as such, they play a central role in marine metabolite generation, ocean nutrient cycling, and the control of planetary oxygen generation. Cyanobacteriophage infection exerts control on all of these critical processes of the planet, with the phage-ported homologs of genes linked to photosynthesis, catabolism, and secondary metabolism (marine metabolite generation). Here, we analyze the 153 fully sequenced cyanophages from the National Center for Biotechnology Information (NCBI) database and the 45 auxiliary metabolic genes (AMGs) that they deliver into their hosts. Most of these AMGs are homologs of those found within cyanobacteria and play a key role in cyanobacterial metabolism-encoding proteins involved in photosynthesis, central carbon metabolism, phosphate metabolism, methylation, and cellular regulation. A greater understanding of cyanobacteriophage infection will pave the way to a better understanding of carbon fixation and nutrient cycling, as well as provide new tools for synthetic biology and alternative approaches for the use of cyanobacteria in biotechnology and sustainable manufacturing.

Beyond natural biology: rewiring cellular networks to study innate immunity.

Within immune cells, microbial and self-ligands trigger pattern recognition receptors (PRRs) to nucleate and activate the signaling organelles of the immune system. Much work in this area has derived from observational biology of natural innate immune signaling. More recently, synthetic biology approaches have been used to rewire and study innate immune networks. By utilizing controllable chemical or optogenetic inputs, rearranging protein building blocks, or engineering signal recording circuits, synthetic biology-based techniques complement and inform studies of natural immune pathway operation. In this review, we describe recent synthetic biology-based approaches that have uncovered new insights into PRR signaling, virus-host interactions, and systemic cytokine responses.

Exploring the landscape of synthetic IL-6-type cytokines.

Interleukin-6 (IL-6)-type cytokines not only have key immunomodulatory functions that affect the pathogenesis of diseases such as autoimmune diseases, chronic inflammatory conditions, and cancer, but also fulfill important homeostatic tasks. Even though the pro-inflammatory arm has hindered the development of therapeutics based on natural-like IL-6-type cytokines to date, current synthetic trends might pave the way to overcome these limitations and eventually lead to immune-inert designer cytokines to aid type 2 diabetes and brain injuries. Those synthetic biology approaches include mutations, fusion proteins, and inter-cytokine swapping, and resulted in IL-6-type cytokines with altered receptor affinities, extended target cell profiles, and targeting of non-natural cytokine receptor complexes. Here, we survey synthetic cytokine developments within the IL-6-type cytokine family and discuss potential clinical applications.

Designing plant flavonoids: harnessing transcriptional regulation and enzyme variation to enhance yield and diversity.

Plant synthetic biology has emerged as a powerful and promising approach to enhance the production of value-added metabolites in plants. Flavonoids, a class of plant secondary metabolites, offer numerous health benefits and have attracted attention for their potential use in plant-based products. However, achieving high yields of specific flavonoids remains challenging due to the complex and diverse metabolic pathways involved in their biosynthesis. In recent years, synthetic biology approaches leveraging transcription factors and enzyme diversity have demonstrated promise in enhancing flavonoid yields and expanding their production repertoire. This review delves into the latest research progress in flavonoid metabolic engineering, encompassing the identification and manipulation of transcription factors and enzymes involved in flavonoid biosynthesis, as well as the deployment of synthetic biology tools for designing metabolic pathways. This review underscores the importance of employing carefully-selected transcription factors to boost plant flavonoid production and harnessing enzyme promiscuity to broaden flavonoid diversity or streamline the biosynthetic steps required for effective metabolic engineering. By harnessing the power of synthetic biology and a deeper understanding of flavonoid biosynthesis, future researchers can potentially transform the landscape of plant-based product development across the food and beverage, pharmaceutical, and cosmetic industries, ultimately benefiting consumers worldwide.

Advances in Metabolic Engineering of Plant Monoterpene Indole Alkaloids.

Monoterpene indole alkaloids (MIAs) encompass a diverse family of over 3000 plant natural products with a wide range of medical applications. Further utilizations of these compounds, however, are hampered due to low levels of abundance in their natural sources, causing difficult isolation and complex multi-steps in uneconomical chemical syntheses. Metabolic engineering of MIA biosynthesis in heterologous hosts is attractive, particularly for increasing the yield of natural products of interest and expanding their chemical diversity. Here, we review recent advances and strategies which have been adopted to engineer microbial and plant systems for the purpose of generating MIAs and discuss the current issues and future developments of manufacturing MIAs by synthetic biology approaches.