Synthetic Biology Engineering Research Articles

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.

Research Progress on the Assembly of Large DNA Fragments.

Synthetic biology, a newly and rapidly developing interdisciplinary field, has demonstrated increasing potential for extensive applications in the wide areas of biomedicine, biofuels, and novel materials. DNA assembly is a key enabling technology of synthetic biology and a central point for realizing fully synthetic artificial life. While the assembly of small DNA fragments has been successfully commercialized, the assembly of large DNA fragments remains a challenge due to their high molecular weight and susceptibility to breakage. This article provides an overview of the development and current state of DNA assembly technology, with a focus on recent advancements in the assembly of large DNA fragments in Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. In particular, the methods and challenges associated with the assembly of large DNA fragment in different hosts are highlighted. The advancements in DNA assembly have the potential to facilitate the construction of customized genomes, giving us the ability to modify cellular functions and even create artificial life. It is also contributing to our ability to understand, predict, and manipulate living organisms.

Genetically Engineered Microorganisms and Their Impact on Human Health.

The emergence of antibiotic-resistant strains, the decreased effectiveness of conventional therapies, and the side effects have led researchers to seek a safer, more cost-effective, patient-friendly, and effective method that does not develop antibiotic resistance. With progress in synthetic biology and genetic engineering, genetically engineered microorganisms effective in treatment, prophylaxis, drug delivery, and diagnosis have been developed. The present study reviews the types of genetically engineered bacteria and phages, their impacts on diseases, cancer, and metabolic and inflammatory disorders, the biosynthesis of these modified strains, the route of administration, and their effects on the environment. We conclude that genetically engineered microorganisms can be considered promising candidates for adjunctive treatment of diseases and cancers.

Mid-Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects.

Mid-to-long-chain dicarboxylic acids (DCAi, i ≥ 6) are organic compounds in which two carboxylic acid functional groups are present at the terminal position of the carbon chain. These acids find important applications as structural components and intermediates across various industrial sectors, including organic compound synthesis, food production, pharmaceutical development, and agricultural manufacturing. However, conventional petroleum-based DCA production methods cause environmental pollution, making sustainable development challenging. Hence, the demand for eco-friendly processes and renewable raw materials for DCA production is rising. Owing to advances in systems metabolic engineering, new tools from systems biology, synthetic biology, and evolutionary engineering can now be used for the sustainable production of energy-dense biofuels. Here, we explore systems metabolic engineering strategies for DCA synthesis in various chassis via the conversion of different raw materials into mid-to-long-chain DCAs. Subsequently, we discuss the future challenges in this field and propose synthetic biology approaches for the efficient production and successful commercialization of these acids.

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

Deep generative design of RNA family sequences.

RNA engineering has immense potential to drive innovation in biotechnology and medicine. Despite its importance, a versatile platform for the automated design of functional RNA is still lacking. Here, we propose RNA family sequence generator (RfamGen), a deep generative model that designs RNA family sequences in a data-efficient manner by explicitly incorporating alignment and consensus secondary structure information. RfamGen can generate novel and functional RNA family sequences by sampling points from a semantically rich and continuous representation. We have experimentally demonstrated the versatility of RfamGen using diverse RNA families. Furthermore, we confirmed the high success rate of RfamGen in designing functional ribozymes through a quantitative massively parallel assay. Notably, RfamGen successfully generates artificial sequences with higher activity than natural sequences. Overall, RfamGen significantly improves our ability to design functional RNA and opens up new potential for generative RNA engineering in synthetic biology.

Bioengineering of the Optimized Biosynthesis of Commercially Vital Carotenoids- Techno-Advanced Applications

Beta-carotene, a carotenoid found in plants, fungi, and algae, is a crucial antioxidant and anti-cancer agent. It is primarily derived from plants, algae, and microbes, but this method has drawbacks like high costs and low productivity. The growing demand for carotenoids has led to large-scale industrial manufacturing. However, extracting and synthesizing these chemicals can be costly and technical. Microbial synthesis offers a cost-effective alternative. Synthetic biology and metabolic engineering technologies have been used in various studies for the optimization of pathways for the overproduction of carotenoids. Four metabolic components are involved in carotenoid biosynthesis, central carbon (C), isoprene supplement, and cofactor metabolism. Metabolic engineering is a potential solution to enhance β-carotene production. This article explores the biochemical routes, methods used by natural microbial species, and metabolic engineering potential of microbial organisms for β-carotenoids production. Currently, Escherichia coli, certain euglena and yeast species are the primary microorganisms used in metabolic engineering, offering minimal environmental impact, cost-effective manufacturing, and high yield.

Long oligodeoxynucleotides: chemical synthesis, isolation via catching-by-polymerization, verification via sequencing, and gene expression demonstration.

Long oligodeoxynucleotides (ODNs) are segments of DNAs having over one hundred nucleotides (nt). They are typically assembled using enzymatic methods such as PCR and ligation from shorter 20 to 60 nt ODNs produced by automated de novo chemical synthesis. While these methods have made many projects in areas such as synthetic biology and protein engineering possible, they have various drawbacks. For example, they cannot produce genes and genomes with long repeats and have difficulty to produce sequences containing stable secondary structures. Here, we report a direct de novo chemical synthesis of 400 nt ODNs, and their isolation from the complex reaction mixture using the catching-by-polymerization (CBP) method. To determine the authenticity of the ODNs, 399 and 401 nt ODNs were synthesized and purified with CBP. The two were joined together using Gibson assembly to give the 800 nt green fluorescent protein (GFP) gene construct. The sequence of the construct was verified via Sanger sequencing. To demonstrate the potential use of the long ODN synthesis method, the GFP gene was expressed in E. coli. The long ODN synthesis and isolation method presented here provides a pathway to the production of genes and genomes containing long repeats or stable secondary structures that cannot be produced or are highly challenging to produce using existing technologies.

The Construction of the Self-Induced Sal System and Its Application in Salicylic Acid Production

The design and construction of more complex and delicate genetic control circuits suffer from poor orthogonality in quorum sensing (QS) systems. The Sal system, which relies on salicylic acid as a signaling molecule, is an artificially engineered regulatory system with a structure that differs significantly from that of natural QS signaling molecules. Salicylic acid is an important drug precursor, mainly used in the production of drugs such as aspirin and anti-HIV drugs. However, there have been no reports on the construction of a self-induced Sal system in single cells. In this study, a high-copy plasmid backbone was used to construct the regulatory proteins and a self-induced promoter of salicylic acid in E. coli by adjusting the precise regulation of key gene expression; the sensitivity and induction range of this system were improved. Subsequently, the exogenous gene pchBA was introduced in E. coli to extend the shikimate pathway and synthesize salicylic acid, resulting in the construction of the first complete self-induced Sal system. Finally, the self-induced Sal System was combined with artificial trans-encoded sRNAs (atsRNAs) to repress the growth-essential gene ppc and accumulate the precursor substance PEP, thereby increasing the titer of salicylic acid by 151%. This construction of a self-induced artificial system introduces a new tool for selecting communication tools and induction systems in synthetic biology and metabolic engineering, but also demonstrates a self-inducible pathway design strategy for salicylic acid biosynthesis.

Accelerating the design of pili-enabled living materials using an integrative technological workflow.

Bacteria can be programmed to create engineered living materials (ELMs) with self-healing and evolvable functionalities. However, further development of ELMs is greatly hampered by the lack of engineerable nonpathogenic chassis and corresponding programmable endogenous biopolymers. Here, we describe a technological workflow for facilitating ELMs design by rationally integrating bioinformatics, structural biology and synthetic biology technologies. We first develop bioinformatics software, termed Bacteria Biopolymer Sniffer (BBSniffer), that allows fast mining of biopolymers and biopolymer-producing bacteria of interest. As a proof-of-principle study, using existing pathogenic pilus as input, we identify the covalently linked pili (CLP) biosynthetic gene cluster in the industrial workhorse Corynebacterium glutamicum. Genetic manipulation and structural characterization reveal the molecular mechanism of the CLP assembly, ultimately enabling a type of programmable pili for ELM design. Finally, engineering of the CLP-enabled living materials transforms cellulosic biomass into lycopene by coupling the extracellular and intracellular bioconversion ability.

Biomanufacturing of bioactive compounds: current situation, challenges, and future perspectives

Biomanufacturing uses biological systems, including cells, microorganisms, and enzymes, to produce natural or synthetic molecules with biological activities for use in various industries, such as pharmaceuticals, cosmetics, and agriculture. These bioactive compounds are expected to play important roles in improving the quality of life and prolonging its length. Fortunately, recent advances in synthetic biology and automation technologies have accelerated the development of biomanufacturing, enabling us to create new products and replace conventional methods in a more sustainable manner. As of now, the role of biomanufacturing in the growth and innovation of bioeconomy is steadily increasing, and this techbology becomes a prevalent technology in global markets. To gain a comprehensive understanding of this field, this article presents a retrospective review of Bloomage Biotechnology's Research and Development and briefly reviews the developments of biomanufacturing and offers insights into the futre prospects. In conclusion, biomanufacturing will continue to be an important, environmentally friendly, and sustainable production mode in the ongoing development of bioeconomy.

Genome Mining Reveals a Surprising Number of Sugar Reductases in Aspergillus niger.

Metabolic engineering of filamentous fungi has received increasing attention in recent years, especially in the context of creating better industrial fungal cell factories to produce a wide range of valuable enzymes and metabolites from plant biomass. Recent studies into the pentose catabolic pathway (PCP) in Aspergillus niger have revealed functional redundancy in most of the pathway steps. In this study, a closer examination of the A. niger genome revealed five additional paralogs for the three original pentose reductases (LarA, XyrA, XyrB). Analysis of these genes using phylogeny, in vitro and in vivo functional analysis of the enzymes, and gene expression revealed that all can functionally replace LarA, XyrA, and XyrB. However, they are also active on several other sugars, suggesting a role for them in other pathways. This study therefore reveals the diversity of primary carbon metabolism in fungi, suggesting an intricate evolutionary process that distinguishes different species. In addition, through this study, the metabolic toolkit for synthetic biology and metabolic engineering of A. niger and other fungal cell factories has been expanded.

Reverse Engineering in Biotechnology: The Role of Genetic Engineering in Synthetic Biology.

Synthetic biology is built on genetic engineering and principles of design engineering, which provides control over the biological functions of interest. This chapter explores the uses, processes, and applications of genetic engineering in synthetic biology. The chapter provides a brief history and course of development of the field of synthetic biology and genetic engineering and their unbreakable association. Next, the chapter delves into materials and methods and the applications of synthetic biology. This includes discussing the generally used components of genetic engineering to design new functions into organisms and even the general steps that are part of any synthetic biology experiment. Lastly, the chapter also explains the use of the materials and methodology discussed in solving a specific problem related to amodel mentioned in the paper titled"Development of Integrase-mediated differentiation circuits to improve evolutionary stability in E. coli." It explains how by using genetic engineering a synthetic biology-related problem was solved efficiently.

Engineering cell-electrode interfacial electron transfer to boost power generation of electroactive biofilm

Electroactive biofilm plays a crucial rule in the electron transfer efficiency of bio-electrochemical systems (BES). However, the slow rate of interfacial electron transfer (IET) restricts practical applications of various BES. Here, a modular engineering strategy was developed to enhance IET rate. Firstly, to accelerate the low transmembrane electron transfer rate caused by insulative cell membrane of Shewanella oneidensis, pili-based artificial conductive nanowires and outer-membrane c-cytochrome OmcF from Geobacter sulfurreducens were heterologously expressed to construct transmembrane electron conduits. Secondly, to improve the low electron transfer rate from S. oneidensis to anode owing to the poor electron collection ability of the anode, N-doped carbon nanotubes and polyriboflavin were used to increase the surface area and active sites of the anode. Thirdly, to further reduce the resistance due to the low conductivity of biofilm, the polydopamine was coated in situ to construct high-speed conductive networks, obtaining an unprecedented power density of 5233.7 ± 364.7 mW/m2, ∼83.1-fold higher than that of the wild-type strain (62.2 ± 5.5 mW/m2), and the maximum coulomb efficiency of ESR1 @PDA was 85.4%, which, to the best of our knowledge, is one of the highest output power densities and coulomb efficiencies that have ever reported in the genetically engineered Shewanella strains. This study demonstrated an integrated biotic-electrode modular engineering strategy to boost power generation of electroactive biofilm via synthetic biology and material engineering.

Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, β-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli.

Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.

Microbial Biofilms: Harnessing the Power of Complex Microbial Communities for Industrial Applications

Microbial biofilms are complex microbial colonies that attach to surfaces and grow inside an extracellular polymeric substance (EPS) matrix. These biofilms have great promise for industrial applications and play crucial roles in several natural and artificial systems. The use of microbial biofilms in industrial settings is examined in this abstract, which also places an emphasis on techniques to improve biofilm development and performance through surface modification and quorum sensing manipulation. Due to their versatility, durability, and cooperative behaviour, biofilms have attracted interest and are useful in a variety of industrial industries. They work in the food business, bioenergy generation, agriculture industry and biosensor, among other fields.Biofilms are more effective in industrial processes because of their intricate interactions, which also lead to higher metabolic capacities, increased stress tolerance, and improved retention of immobilised cells. The production and performance of microbial biofilms need to be improved in order to fully realise their potential. Techniques for surface modification provide a promising way to customise the characteristics of biofilms. It is possible to modify the topography, hydrophobicity, and charge of the substrate to affect how quickly biofilms form after initial microbial attachment. Additionally, functionalization and coatings based on nanomaterials provide novel approaches to improve biofilm adhesion, cohesiveness, and stability. The method of cell-to-cell communication known as quorum sensing (QS) directs the development and behaviour of biofilms. Controlling QS pathways enables fine-grained regulation of biofilm growth. QS may be controlled via genetic engineering and small molecule therapy, which affects the phenotypic and architecture of biofilms. With the use of these techniques, biofilms may be designed with the required properties, including greater thickness, higher resistance to shear pressures, and increased production of desirable chemicals. In conclusion, because of their cooperative nature and adaptable functioning, microbial biofilms show enormous promise for industrial applications. The importance of surface modification and quorum sensing modulation as tactics to improve biofilm performance is highlighted in this abstract. As science advances, a fuller comprehension of the ecology of biofilms, interspecies interactions, and synthetic biology technologies will make it easier to create biofilms that are specifically suited to a given industrial purpose. Understanding the complex principles underlying biofilm production and behaviour will allow for the full realisation of the promise for sustainable and effective industrial processes, ushering in a new age of biofilm-based technology.

Next-generation chimeric antigen receptors for T- and natural killer-cell therapies against cancer.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cells has led to a paradigm shift in the treatment of various hematologic malignancies. However, the broad application of this approach for myeloid malignancies and solid cancers has been limited by the paucity and heterogeneity of target antigen expression, and lack of bona fide tumor-specific antigens that can be targeted without cross-reactivity against normal tissues. This may lead to unwanted on-target off-tumor toxicities that could undermine the desired antitumor effect. Recent advances in synthetic biology and genetic engineering have enabled reprogramming of immune effector cells to enhance their selectivity toward tumors, thus mitigating on-target off-tumor adverse effects. In this review, we outline the current strategies being explored to improve CAR selectivity toward tumor cells with a focus on natural killer (NK) cells, and the progress made in translating these strategies to the clinic.