Synthetic Biology Engineering Research Articles

Leveraging the dynamics of microalgal CO2 capture to estimate the maximum inherent photosynthetic potential

AbstractBACKGROUNDNatural photosynthesis, utilizing intelligent molecular machinery to harness sunlight, stands as the benchmark for efficient energy generation. Despite its high quantum efficiency compared to synthetic methods, challenges persist due to dynamic nutritional and light requirements in plant, microalgae, and cyanobacteria‐driven processes. Reported microalgae CO2 uptake rates rely on biomass dry cell weight measurement after certain incubation period and systematic study of required CO2 concentrations for optimal photobioreactor operation remaining unexplored. This research is thus aimed at evaluating the critical dissolved CO2 (Ccrit ∙ CO2) concentration and specific CO2 uptake rates (SCUR) of Chlorella minutissima (CM) culture in the presence of surplus light and other nutrients by online monitoring and measuring the dynamic CO2 uptake rates which will help in maintaining the optimal rate of CO2 delivery for continuous cultivation of microalgae while minimizing the escape of CO2.RESULTSDissolved Ccrit ∙ CO2 was determined to be in the range of 16–20 mg/L, and the maximum SCUR in BBM (Bold Basal Medium) was found to be 0.73 mg CO2 (mg dcw ∙ h)−1. Interestingly, this SCUR value is nearly three times greater than that of the most efficient in vitro artificial photosynthetic novel pathway reported so far.CONCLUSIONFrom the calculated SCUR values, it may be noted that 9.6 g of biomass can be obtained from 1 g of microalgal inoculum in a day, thereby setting a benchmark for the scientists working in the areas of bioprocess engineering, synthetic biology, and metabolic engineering to comply. © 2024 Society of Chemical Industry (SCI).

Upcycling C1 gas-derived resources in future food system

Upcycling the one-carbon (C1) gas-derived resources into food-related product offers a dual benefit, alleviating food shortages and reducing greenhouse gas emissions. Powered by synthetic biology and cutting-edge technologies, the conversion of C1 resources into food-related products has become a reality. In this study, we present a state-of-the-art review for the synthesis of C1-based food-related products, broadly categorized as follows: (i) C1-to-carbohydates, (ii) C1-to-proteins, (iii) C1-to-lipids, (iv) C1-to-food additives, and (v) C1-to-food packaging. We assess the technological readiness level of each product category and identify the C1 fixation pathways and C1-based products with industrialization potential. We also address the bottlenecks and explore the opportunities within the less mature product categories. By revealing the existing knowledge gap, we emphasize the imperative of leveraging advanced technologies and collaborative efforts to address these challenges. Taken together, our work highlights the potential of upcycling C1 gas-derived resources in fostering future food system.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

Bacteriophage-Based Bioanalysis.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.

Advances and Challenges in Biomanufacturing of Glycosylation of Natural Products

Glycosylation is one of the most common and important modifications in natural products (NPs), which can alter the biological activities and properties of NPs, effectively increase structural diversity, and improve pharmacological activities. The biosynthesis of glycosylation in natural products involves multiple complex biological processes, which are coordinated by many enzymes. UDP-glycosyltransferases (UGTs) play a crucial role in glycosylation modification, and have attracted long-term and widespread research attention. UGTs can catalyze the O-, C-, S-, and N-glycosylation of different substrates, producing a variety of glycosides with broad biological activity, while improving the solubility, stability, bioavailability, pharmacological activity, and other functions of NPs. In recent years, the rapid development of synthetic biology and advanced manufacturing technologies, especially the widespread application of artificial intelligence in the field of synthetic biology, has led to a series of new discoveries in the biosynthesis of NP glycosides by UGT. This work summarizes the latest progress and challenges in the field of NP glycosylation, covering the research results and potential applications of glycosylated derivatives of terpenes, flavonoids, polyphenols, aromatic compounds, and other compounds in terms of biogenesis. Looking to the future, research may leverage artificial intelligence-driven synthetic biology techniques to decipher genes related to the synthetic pathway, which is expected to further promote the large-scale synthesis and application of glycosylated NPs, and increase the diversity of NPs in the pharmaceutical, functional food, and cosmetic industries.

Towards a Yersinia pestis lipid A recreated in an Escherichia coli scaffold genome.

Synthetic biology and genome engineering capabilities have facilitated the utilization of bacteria for a myriad of applications, ranging from medical treatments to biomanufacturing of complex molecules. The bacterial outer membrane, specifically the lipopolysaccharide (LPS), plays an integral role in the physiology, pathogenesis, and serves as a main target of existing detection assays for Gram-negative bacteria. Here we use CRISPR/Cas9 recombineering to insert Yersinia pestis lipid A biosynthesis genes into the genome of an Escherichia coli strain expressing the lipid IVa subunit. We successfully inserted three genes: kdsD, lpxM, and lpxP into the E. coli genome and demonstrated their expression via reverse transcription PCR (RT-PCR). Despite observing expression of these genes, analytical characterization of the engineered strain's lipid A structure via MALDI-TOF mass spectrometry indicated that the Y. pestis lipid A was not recapitulated in the E. coli background. As synthetic biology and genome engineering technologies advance, novel applications and utilities for the detection and treatments of dangerous pathogens like Yersinia pestis will continue to be developed.

Harnessing the ocean’s pharmacy: marine bioactive compounds as next-generation therapeutics

The ocean’s vast and diverse ecosystem offers a rich reservoir of bioactive compounds with immense clinical potential. Marine organisms produce structurally unique and biologically active compounds, leading to breakthroughs in therapeutic development. Notable examples include anticancer agents like trabectedin and cytarabine, and the analgesic ziconotide. Marine compounds also exhibit potent antimicrobial and antiviral properties, addressing critical challenges like antibiotic resistance and emerging viral infections. Despite the promise, challenges such as sustainable harvesting and complex extraction processes persist. Advances in synthetic biology and metabolic engineering provide solutions for sustainable production, ensuring a stable supply of these valuable compounds. The integration of marine bioactives into modern medicine could revolutionize treatments for cancer, chronic pain, and infectious diseases, underscoring the need for continued investment in marine bioprospecting and biotechnological innovation.

Advances in the biosynthesis and metabolic regulation of terpenoids in Saccharomyces cerevisiae

Terpenoids are the one of most abundant natural products. With diverse varieties and biological activities, they are widely used in the food, medicine, chemical industry, and novel fuels. However, the conventional methods such as plant extraction and chemical synthesis cannot meet the current market demand for terpenoids. Efficient microbial cell factories, especially engineered Saccharomyces cerevisiae strains, have been constructed for the industrial production of terpenoids. In recent years, researchers have constructed multiple S. cerevisiae strains with increased yield and productivity via approaches of synthetic biology and metabolic engineering. This paper reviews the recent progress in the biosynthesis of terpenoids in S. cerevisiae cells and summarizes a variety of metabolic engineering strategies for the production of terpenoids in S. cerevisiae. These strategies include the construction and optimization of metabolic pathways, the mining and modification of key enzymes, the regeneration of cofactors, the engineering of cell localization and cell efflux, and the improvement of cell tolerance. Our review will provide information and strategies for the effective biosynthesis of terpenoids in S. cerevisiae.

Leveraging DNA‐Encoded Cell‐Mimics for Environment‐Adaptive Transmembrane Channel Release‐Induced Cell Death

AbstractThe advancement of cell‐mimic materials, which can forge sophisticated physicochemical dialogues with living cells, has unlocked a realm of intriguing prospects within the fields of synthetic biology and biomedical engineering. Inspired by the evolutionarily acquired ability of T lymphocytes to release perforin and generate transmembrane channels on targeted cells for killing, herein we present a pioneering DNA‐encoded artificial T cell mimic model (ARTC) that accurately mimics T‐cell‐like behavior. ARTC responds to acidic conditions similar to those found in the tumor microenvironment and then selectively releases a G‐rich DNA strand (LG4) embedded with C12 lipid and cholesterol molecules. Once released, LG4 effectively integrates into the membranes of neighboring live cells, behaving as an artificial transmembrane channel that selectively transports K+ ions and disrupts cellular homeostasis, ultimately inducing apoptosis. We hope that the emergence of ARTC will usher in new perspectives for revolutionizing future disease treatment and catalyzing the development of advanced biomedical technologies.

Leveraging DNA-Encoded Cell-Mimics for Environment-Adaptive Transmembrane Channel Release-Induced Cell Death.

The advancement of cell-mimic materials, which can forge sophisticated physicochemical dialogues with living cells, has unlocked a realm of intriguing prospects within the fields of synthetic biology and biomedical engineering. Inspired by the evolutionarily acquired ability of T lymphocytes to release perforin and generate transmembrane channels on targeted cells for killing, herein we present a pioneering DNA-encoded artificial T cell mimic model (ARTC) that accurately mimics T-cell-like behavior. ARTC responds to acidic conditions similar to those found in the tumor microenvironment and then selectively releases a G-rich DNA strand (LG4) embedded with C12 lipid and cholesterol molecules. Once released, LG4 effectively integrates into the membranes of neighboring live cells, behaving as an artificial transmembrane channel that selectively transports K+ ions and disrupts cellular homeostasis, ultimately inducing apoptosis. We hope that the emergence of ARTC will usher in new perspectives for revolutionizing future disease treatment and catalyzing the development of advanced biomedical technologies.

Cyanamide-inducible expression of homing nuclease I−SceI for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae

In synthetic biology, microbial chassis including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped the cyanamide-induced I−SceI expression, which triggered double-strand DNA breaks (DSBs) for selectable marker removal. We further combined cyanamide-induced I−SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution, which simplified the molecular cloning procedure for promoter characterisation. We then characterised three tetracycline-inducible promoters showing differential strength, a non-leaky β-estradiol-inducible promoter, cyanamide-inducible DDI2 promoter, bidirectional MAL32/MAL31 promoters, and five pairs of bidirectional GAL1/GAL10 promoters. Overall, alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.

An Effective DNA-Based File Storage System for Practical Archiving and Retrieval of Medical MRI Data.

DNA-based data storage is a new technology in computational and synthetic biology, that offers a solution for long-term, high-density data archiving. Given the critical importance of medical data in advancing human health, there is a growing interest in developing an effective medical data storage system based on DNA. Data integrity, accuracy, reliability, and efficient retrieval are all significant concerns. Therefore, this study proposes an Effective DNA Storage (EDS) approach for archiving medical MRI data. The EDS approach incorporates three key components (i) a novel fraction strategy to address the critical issue of rotating encoding, which often leads to data loss due to single base error propagation; (ii) a novel rule-based quaternary transcoding method that satisfies bio-constraints and ensure reliable mapping; and (iii) an indexing technique designed to simplify random search and access. The effectiveness of this approach is validated through computer simulations and biological experiments, confirming its practicality. The EDS approach outperforms existing methods, providing superior control over bio-constraints and reducing computational time. The results and code provided in this study open new avenues for practical DNA storage of medical MRI data, offering promising prospects for the future of medical data archiving and retrieval.

Comparative transcriptomic analysis revealed important processes underlying the static magnetic field effects on Arabidopsis.

Static magnetic field (SMF) plays important roles in various biological processes of many organisms including plants, though the molecular mechanism remains largely unclear. Here in this study, we evaluated different magnetic setups to test their effects on growth and development on Arabidopsis (Arabidopsis thaliana), and discovered that plant growth was significantly enhanced by inhomogeneous SMF generated by a regular triangular prism magnet perpendicular to the direction of gravity. Comparative transcriptomic analysis revealed that auxin synthesis and signal transduction genes were upregulated by SMF exposure. SMF also facilitated plants to maintain the iron homeostasis. The expression of iron metabolism-related genes was downregulated by SMF, however, the iron content in plant tissues remains relatively unchanged. Furthermore, SMF exposure also helped the plants to reduce ROS level and synergistically maintain the oxidant balance by enhanced activity of antioxidant enzymes and accumulation of nicotinamide. Taken together, our data suggested that SMF is involved in regulating the growth and development of Arabidopsis thaliana through maintaining iron homeostasis and balancing oxidative stress, which could be beneficial for plant survival and growth. The work presented here would extend our understanding of the mechanism and the regulatory network of how magnetic field affects the plant growth, which would provide insights into the development of novel plant synthetic biology technologies to engineer stress-resistant and high-yielding crops.

CHALLENGES AND OPPORTUNITIES TO DESIGN FUTURE CROPS: GATEWAY TO SUSTAINABLE AGRICULTURE IN 21ST CENTURY

The global food production system is facing numerous challenges due to factors such as population growth, climate change, limited resources, and environmental preservation. To address these challenges, various strategies can be employed to develop future crops that are more productive, nutritious, and resilient. One strategy is to improve the yield potential of existing crops by developing new high-yielding cultivars. This can be achieved through the development of new varieties that can produce more grain under optimal conditions. Additionally, improving the nutritional quality of crops is important to address nutrient deficiencies. Synthetic biology and metabolic engineering methods can be used to develop crops with enhanced nutritional value. Efficient utilization of agricultural resources is another important aspect of crop improvement. This includes developing crops that use water and nutrients more efficiently, reducing the need for irrigation and fertilizers, and minimizing environmental impacts. Increasing the resistance of crops to pests, diseases, and extreme weather events can also help reduce the use of pesticides and minimize crop losses. The domestication of wild or semi-wild plants through genetic manipulation offers new opportunities for crop design. These plants may have high nutritional value, stress tolerance, and specialized metabolites that can be incorporated into cultivated crops. Similarly, the domestication of orphan or neglected plants can contribute to crop improvement by incorporating unique traits. Genetic improvement through the transfer of genes from wild relatives or other species can also enhance crop productivity. Advancements in genomics and genetic technologies can aid in the identification and transfer of beneficial alleles. Agronomic improvements, such as maximizing the effectiveness of crop protection agents and fertilizers while minimizing their environmental impact, can also contribute to crop performance. The emerging field of synthetic biology offers opportunities for developing novel biological devices and systems that can further enhance crop productivity and resilience. Overall, these strategies can help address the challenges faced by the global food production system.

Regulatory microRNAs and phasiRNAs of paclitaxel biosynthesis in Taxus chinensis.

Paclitaxel (trade name Taxol) is a rare diterpenoid with anticancer activity isolated from Taxus. At present, paclitaxel is mainly produced by the semi-synthetic method using extract of Taxus tissues as raw materials. The studies of regulatory mechanisms in paclitaxel biosynthesis would promote the production of paclitaxel through tissue/cell culture approaches. Here, we systematically identified 990 transcription factors (TFs), 460 microRNAs (miRNAs), and 160 phased small interfering RNAs (phasiRNAs) in Taxus chinensis to explore their interactions and potential roles in regulation of paclitaxel synthesis. The expression levels of enzyme genes in cone and root were higher than those in leaf and bark. Nearly all enzyme genes in the paclitaxel synthesis pathway were significantly up-regulated after jasmonate treatment, except for GGPPS and CoA Ligase. The expression level of enzyme genes located in the latter steps of the synthesis pathway was significantly higher in female barks than in male. Regulatory TFs were inferred through co-expression network analysis, resulting in the identification of TFs from diverse families including MYB and AP2. Genes with ADP binding and copper ion binding functions were overrepresented in targets of miRNA genes. The miRNA targets were mainly enriched with genes in plant hormone signal transduction, mRNA surveillance pathway, cell cycle and DNA replication. Genes in oxidoreductase activity, protein-disulfide reductase activity were enriched in targets of phasiRNAs. Regulatory networks were further constructed including components of enzyme genes, TFs, miRNAs, and phasiRNAs. The hierarchical regulation of paclitaxel production by miRNAs and phasiRNAs indicates a robust regulation at post-transcriptional level. Our study on transcriptional and posttranscriptional regulation of paclitaxel synthesis provides clues for enhancing paclitaxel production using synthetic biology technology.

Engineering Microorganisms for Cancer Immunotherapy.

Cancer immunotherapy presents a promising approach to fight against cancer by utilizing the immune system. Recently, engineered microorganisms have emerged as a potential strategy in cancer immunotherapy. These microorganisms, including bacteria and viruses, can be designed and modified using synthetic biology and genetic engineering techniques to target cancer cells and modulate the immune system. This review delves into various microorganism-based therapies for cancer immunotherapy, encompassing strategies for enhancing efficacy while ensuring safety and ethical considerations. The development of these therapies holds immense potential in offering innovative personalized treatments for cancer.

Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

Inverse design of three-dimensional multicellular biobots with target functions

Hybrid living biobots consisting of active cells hold promise for significant applications as, for example, intelligent devices in medical engineering and organisms with specific functions in synthetic biology. However, the design and creation of living biobots with various cells remain a challenge. In this paper, we propose a three-dimensional inverse optimization strategy based on the pixel topology optimization method, to design self-propelled living biobots with the function of biomechanical actuations. For illustration, we design several biobots composed of active and passive elements that mimic cardiomyocytes and passive epidermal cells sourced from such as Xenopus laevis, human induced pluripotent stem cells or neonatal rats. Their topologies are optimized by implementing the active constitutive relations of cells into the multicellular topological interpolation model. Effects of nutrient concentrations, elasticity, and anisotropic contraction of cardiomyocytes on the topologies and functionalities of the biobots are examined. In addition, we unveil the living topological interfaces generated by the collective actuations of the optimized biobots. We show a potential of collective biobots for high-throughput drug screening owing to their distinct biomechanical responses under healthy and sick conditions. The proposed inverse optimization method can be extended to design various functional multicellular biological systems, which impacts the studies of organ development, synthetic biology, and medical engineering.

Aspergillus oryzae as a Cell Factory: Research and Applications in Industrial Production.

Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production.

Reflections on the safety regulation of commercialization of synthetic biology products

With the rapid development of synthetic biology, lots of synthetic biology technology achievements in various application fields have been commercialized, generating broad market prospects. The commercialization of products employing synthetic biology technology (hereinafter referred as synthetic biology products) has brought benefits to human beings, but it has also produced potential safety risks. At present, relevant laws and standards for regulation of biotechnology or genetically modified organisms have been adopted to regulate the safety risks of commercialization of synthetic biology products (CSBP). However, due to the complexity and uncertainty of synthetic biology, the safety risks of CSBP cannot be comprehensively regulated by these laws and standards. Therefore, it is of great significance to formulate specific supervision and management measures for regulating the safety risks of CSBP. This paper summarized the situation of CSBP in the fields of food, medical care, agriculture, environment, energy and materials, analyzed the safety risks existing in the CSBP, and sorted out current supervision situation of its safety risks in European countries, United States, as well as in China. We further proposed suggestions on the safety supervision and management measures on the safety risks of CSBP, including classified examination and approval, classified identification of products, and strict screening and approval of market entities before entering the market, and strengthening safety supervision and emergency treatment as well as accident responsibility investigation after entering the market. This whole-process safety regulation might provide support for the safety of CSBP and promote the healthy and long-term development of synthetic biology industry.