Advances In Synthetic Biology Research Articles

Removal, detection, and limits of endotoxin in the industry of recombinant proteins

With the advancement of synthetic biology, recombinant proteins are poised to play a significant role in medical applications. The scaled manufacturing is a pillar for the extensive application and development of recombinant proteins across various fields. In the large-scale production process of recombinant proteins, the removal and detection of endotoxins are essential to reduce their levels to safe thresholds in the final products. Currently, establishing stringent endotoxin limits for different recombinant protein products is a crucial aspect of safety assessment. This review begins by shedding light on the pathogenicity of endotoxins and discusses the methods for the removal and determination of endotoxins during the production processes of recombinant proteins. Subsequently, this review summarizes the endotoxin limits in industries such as biologics, medical devices, and human recombinant DNA products, particularly those in recombinant protein injection products. It is highlighted that regardless of whether the hosts for recombinant protein expression are bacteria or not, endotoxin testing is required for the final products of injectable recombinant proteins, and compliance with relevant industry standards is necessary. This review aims to provide a reference for the research on endotoxins in the large-scale production process of recombinant proteins.

Red Light Responsive Cre Recombinase for Bacterial Optogenetics.

Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

Transforming Lignocellulosic Biomass into Biofuels: Recent Innovations in Pretreatment and Bioconversion Techniques

Abstract: This scholarly review investigates contemporary advancements in the conversion of lignocellulosic biomass to biofuels, emphasizing innovative pretreatment and bioconversion technologies that aim to surmount the intrinsic challenges associated with the processing of this complex biomass. Lignocellulosic materials, which are predominantly comprised of cellulose, hemicellulose, and lignin, represent a renewable and plentiful source for biofuel production; however, their structural complexity necessitates sophisticated methodologies for the effective degradation of resistant components. The review scrutinizes a variety of pretreatment methodologies, encompassing physical, chemical, and burgeoning techniques such as plasma-assisted processing, which are engineered to augment cellulose accessibility for enzymatic hydrolysis. Additionally, it elucidates the progress made in bioconversion processes, concentrating on enzymatic hydrolysis, microbial fermentation, and consolidated bioprocessing (CBP), wherein recent endeavors in genetic engineering are refining microbial strains to enhance yield and efficiency. By addressing economic, technological, and environmental challenges, this article emphasizes the role of integrated biorefineries and innovative biotechnologies in facilitating scalable and cost-effective production of lignocellulosic biofuels. Prospective research trajectories include the formulation of sustainable pretreatment techniques and the advancement of synthetic biology to fully harness the potential of lignocellulosic biomass as a renewable energy resource. Ultimately, this review accentuates the significance of lignocellulosic biofuels as a feasible alternative to fossil fuels, thereby contributing to energy sustainability and climate change mitigation through diminished carbon emissions

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model.

The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immunogenic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV. In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine. The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein. The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Enhancing fatty acid and omega-3 production in Schizochytrium sp. using developed safe-harboring expression system

BackgroundSchizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production.ResultsTransgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain.ConclusionThis study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.

Biosynthesis of novel non-proteinogenic amino acids β-hydroxyenduracididine and β-methylphenylalanine in Escherichia coli.

Non-proteinogenic amino acids (npAAs) are valuable building blocks for the development of advanced pharmaceuticals and agrochemicals. The surge in interest in their synthesis is primarily due to the potential to enhance and diversify existing bioactive molecules. This can be achieved by altering these bioactive molecules to improve their effectiveness, reducing resistance compared to their natural counterparts or generating molecules with novel functions. Traditional production of npAAs in native hosts requires specialized conditions and complex cultivation media. Furthermore, these compounds are often found in organisms that challenge genetic manipulation. Thus, the recombinant production of these npAAs in a model organism like Escherichia coli paves the way for groundbreaking advancements in synthetic biology. Two synthetic operons, comprising of five heterologous proteins were genomically integrated into E. coli for the synthesis of npAAs β-methylphenylalanine (BmePhe), β-hydroxyenduracididine (BhEnd), and enduracididine (End). Proteomic and metabolomic analysis confirmed production of these compounds in E. coli for the first time. Interestingly, we discovered that the exogenous addition of pathway precursors to the E. coli system enhanced the yield of BmePhe by 2.5 times, whereas it concurrently attenuated the production of BhEnd and End, signifying a selective precursor-dependent yield enhancement. The synthetic biology landscape is broadened in this study by expanding the repertoire of amino acids beyond the conventional set of 22 standard proteinogenic amino acids. The biosynthesized npAAs, End, BhEnd, and BmePhe hold promise for engineering proteins with modified functions, integrating into novel metabolites and/or enhancing biological stability and activity. Additionally, these amino acids' biological production and subsequent purification present an alternative to traditional chemical synthesis methods, paving a direct pathway for pharmacological evaluation.

Advances in Metabolic Engineering for the Accumulation of Astaxanthin Biosynthesis.

Astaxanthin, a lipophilic carotenoid renowned for itsstrong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application inmedicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longercompared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged asa promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use ofthe natural host Xanthophyllomyces dendrorhous, andthe heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed.

Engineering bacterial theranostics: from logic gates to in vivo applications.

Over the past 2decades, rapid advances in synthetic biology have enabled the design of increasingly intricate and biologically relevant systems with broad applications in healthcare. A growing area of interest is in designing bacteria that sense and respond to endogenous disease-associated signals, creating engineered theranostics that function as disease surveyors for human health. In particular, engineered cells hold potential in facilitating greatly enhanced temporal and spatial control over the release of a range of therapeutics. Such systems are particularly useful for targeting challenging, under-drugged disease targets in a more nuanced manner than is currently possible. This review provides an overview of the recent advances in the design, delivery, and dynamics of bacterial theranostics to enable safe, robust, and genetically tractable therapies to treat disease. It outlines the primary challenges in theranostic clinical translation, proposes strategies to overcome these issues, and explores promising future avenues for the field.

From β-Carotene to Retinoids: A Review of Microbial Production of Vitamin A.

Vitamin A (retinoids) is crucial for human health, with significant demand across the food, pharmaceutical, and animal feed industries. Currently, the market primarily relies on chemical synthesis and natural extraction methods, which face challenges such as low synthesis efficiency and complex extraction processes. Advances in synthetic biology have enabled vitamin A biosynthesis using microbial cell factories, offering a promising and sustainable solution to meet the increasing market demands. This review introduces the key enzymes involved in the biosynthesis of vitamin A from β-carotene, evaluates achievements in vitamin A production using various microbial hosts, and summarizes strategies for optimizing vitamin A biosynthesis. Additionally, we outline the remaining challenges and propose future directions for the biotechnological production of vitamin A.

Yeast Surface-Displayed Quenchbody as a Novel Whole-Cell Biosensor for One-Step Detection of Influenza A (H1N1) Virus.

Timely surveillance of airborne pathogens is essential to preventing the spread of infectious diseases and safeguard human health. Methods for sensitive, efficient, and cost-effective detection of airborne viruses are needed. With advances in synthetic biology, whole-cell biosensors have emerged as promising platforms for environmental monitoring and medical diagnostics. However, the current design paradigm of whole-cell biosensors is mostly based on intracellular detection of analytes that can transport across the cell membrane, which presents a critical challenge for viral pathogens and large biomolecules. To address this challenge, we developed a new type of whole-cell biosensor by expressing and displaying VHH-based quenchbody (Q-body) on the surface of the yeast Saccharomyces cerevisiae for simple one-step detection of influenza A (H1N1) virus. Seventeen VHH antibody fragments targeting the hemagglutinin protein H1N1-HA were displayed on the yeast cells and screened for the H1N1-HA binding affinity. The functionally displayed VHHs were selected to create surface-displayed Q-body biosensors. The surface-displayed Q-body exhibiting the highest quenching and dequenching efficiency was identified. The biosensor quantitatively detected H1N1-HA in a range from 0.5 to 16 μg/mL, with a half-maximal concentration of 2.60 μg/mL. The biosensor exhibited high specificity for H1N1-HA over other hemagglutinin proteins from various influenza A virus subtypes. Moreover, the biosensor succeeded in detecting the H1N1 virus at concentrations from 2.4 × 104 to 1.5 × 107 PFU/mL. The results from this study demonstrated a new whole-cell biosensor design that circumvents the need for transport of analytes into biosensor cells, enabling efficient detection of the target virus particles.

Current Progress on Scale‐Up and Commercialization of Microbially‐Produced Silk

AbstractOver the past two decades, significant advancements have been made in the scalable production and commercialization of microbially‐produced recombinant protein polymers. This perspective presents the evolution from early research efforts to the development of market‐ready products, with a focus on recombinant silk‐like proteins. Initial attempts to synthesize spider silk proteins in microbial hosts faced challenges with solubility, stability, and yield. Recent advancements in synthetic biology, protein engineering, and bioprocess development have enabled the substantial progress on these challenges. Early commercial efforts highlight the complexities and high costs involved in silk production and more recent strategies have shifted toward processes with better scalability, techno‐economics, and product properties. Significant commercial progress has been made, with products launched in textiles and personal care. Although market penetration is limited so far, substantial groundwork is laid for future success. Key challenges remain, such as continued high production costs and the need for cost‐effective purification and fiber spinning techniques. However, the convergence of scientific, technological, and market developments –including a growing number of product launches – suggests that recombinant silk and protein polymers can soon become widespread sustainable materials across various industries.

Advancements in Synthetic Biology for Enhancing Cyanobacterial Capabilities in Sustainable Plastic Production: A Green Horizon Perspective

This comprehensive review investigates the potential of cyanobacteria, particularly nitrogen-fixing strains, in addressing global challenges pertaining to plastic pollution and carbon emissions. By analyzing the distinctive characteristics of cyanobacteria, including their minimal growth requirements, high photosynthetic efficiency, and rapid growth rates, this study elucidates their crucial role in transforming carbon sequestration, biofuel generation, and biodegradable plastic production. The investigation emphasizes cyanobacteria’s efficiency in photosynthesis, positioning them as optimal candidates for cost-effective bioplastic production with minimized land usage. Furthermore, the study explores their unconventional yet promising utilization in biodiesel production, mitigating environmental concerns such as sulfur emissions and the presence of aromatic hydrocarbons. The resulting biodiesel exhibits significant combustion potential, establishing cyanobacteria as a viable option for sustainable biofuel production. Through a comprehensive assessment of both achievements and challenges encountered during the commercialization process, this review offers valuable insights into the diverse contributions of cyanobacteria. Its objective is to provide guidance to researchers, policymakers, and industries interested in harnessing bio-inspired approaches for structural and sustainable applications, thereby advancing global efforts towards environmentally conscious plastic and biofuel production.

Breaking the Degeneracy of Sense Codons – How Far Can We Go?

AbstractGenetic code expansion aims to incorporate non‐canonical amino acids (ncAAs) into biological systems, enhancing protein functionality or enabling the in vitro selection of peptides from diverse mRNA displayed libraries. Typically, genetic code expansion has involved reassignment of stop codons to ncAAs through orthogonal translation systems. This review instead focuses on efforts to expand the genetic code by breaking the redundancy of sense codons in vitro and in vivo. In vivo, orthogonal aminoacyl‐tRNA synthetase (AARS)/tRNA/AA systems are able to compete with endogenous machinery, enabling partial to full codon reassignment. Recent approaches, like genome recoding, offer potential solutions to reduce competition. In vitro studies utilize cell extract‐based or reconstituted translation systems, allowing precise control of codon usage via gene design and tRNA addition, making breaking of sense degeneracy easier. In these systems several unsplit codon boxes have been successfully reassigned multiple to ncAAs. These efforts showcase both the successes and challenges in achieving orthogonality and selective codon decoding and point towards a future where the 64 codons can encode more than 30 monomers, enabling new advances in synthetic biology and drug discovery.

Commands of Synthetic Biology to Modernize and Re-design the Biological Systems

The scope of synthetic biology continues to expand and has encompassed a huge number of biological features. Its scope starts from scratch, enabling the de novo synthesis of biological systems. It has re-designed the biological systems and empowered the production of synthetic genes, RNA, DNA and proteins by undertaking the control of pathways involved in genetic regulation. It has increased the production of nano-scale RNA architectures and synthetic biological circuits which either have therapeutic or other productive uses. Furthermore, advancements in synthetic biology have enabled the generation of diversity through methods such as epPCR and site-directed mutagenesis, allowing for the creation of complex genetic variations. Additionally, synthetic biology intersects with computer engineering to design functional biological devices and circuits, utilizing computational analysis to guide the design process. Moreover, ethical and regulatory considerations are paramount in synthetic biology, with careful examination required to address dual-use concerns, environmental impacts, and issues of social justice and equitable access to benefits. As synthetic biology continues to advance, it presents opportunities to address pressing challenges in fields ranging from medicine and agriculture to environmental conservation and beyond. Thus, the fusion of synthetic biology with other scientific disciplines holds promise for transformative innovation and societal benefit. The present discussion enlightened the core of generating complex biological systems and has given a brief overview on the fusion of synthetic biology with other fields of science.Keywords: Biological Systems; Genetic Regulation; Synthetic biology circuits

Engineering cyanobacteria as a new platform for producing taxol precursors directly from carbon dioxide

Taxol serves as an efficient natural anticancer agent with extensive applications in the treatment of diverse malignancies. Although advances in synthetic biology have enabled the de novo synthesis of taxol precursors in various microbial chassis, the total biosynthesis of taxol remains challengable owing to the restricted oxidation efficiency in heterotrophic microbes. Here, we engineered Synechocystis sp. PCC 6803 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and taxol biosynthetic enzymes for production of taxadiene-5α-ol (T5α-ol), the key oxygenated intermediate of taxol. The best strain DIGT-P560 produced up to 17.43 mg/L of oxygenated taxanes and 4.32 mg/L of T5α-ol. Moreover, transcriptomic analysis of DIGT-P560 revealed that establishing a oxygenated taxane flux may enhance photosynthetic electron transfer efficiency and central metabolism in the engineered strain to ameliorate the metabolic disturbances triggered by the incorporation of exogenous genes. This is the first demonstration of photosynthetic production of taxadiene-5α-ol from CO2 in cyanobacteria, highlighting the broad prospects of engineered cyanobacteria as bio-solar cell factories for valuable terpenoids production and expanding the ideas for further rational engineering and optimization.

Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology.

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.

Graduate teaching reform on synthetic biology featuring the discipline of fermentation engineering

Industrial biotechnology is regarded as the most promising technology for sustainable industrial development. The advancement of synthetic biology creates new opportunities and infinite possibilities for the progress of industrial biotechnology. Fermentation engineering is the grab and foothold of the industrialization of all the biotechnologies. Our teaching team optimized the teaching content and innovated the teaching mode to establish a teaching system of synthetic biology matching fermentation engineering. We highlighted the teaching characteristics (telling fermentation story cultivated the craftsmanship spirit; bioeconomic education strengthened the engineering thinking; bioethics and safety education fostered a sense of responsibility), then we summarized and prospected the teaching reform of this course. We believe that the teaching reform of synthetic biology will improve the learning performance of postgraduates, provide a reference for the teaching of synthetic biology in related fields, and promote the development of industrial biotechnology (strengthening the innovation capability in biological manufacturing and cultivating new momentum for bioeconomy).

Advances in biosynthesis and metabolic engineering strategies of cordycepin.

Cordyceps militaris, also called as bei-chong-cao, is an insect-pathogenic fungus from the Ascomycota phylum and the Clavicipitaceae family. It is a valuable filamentous fungus with medicinal and edible properties that has been utilized in traditional Chinese medicine (TCM) and as a nutritious food. Cordycepin is the bioactive compound firstly isolated from C. militaris and has a variety of nutraceutical and health-promoting properties, making it widely employed in nutraceutical and pharmaceutical fields. Due to the low composition and paucity of wild resources, its availability from natural sources is limited. With the elucidation of the cordycepin biosynthetic pathway and the advent of synthetic biology, a green cordycepin biosynthesis in Saccharomyces cerevisiae and Metarhizium robertsii has been developed, indicating a potential sustainable production method of cordycepin. Given that, this review primarily focused on the metabolic engineering and heterologous biosynthesis strategies of cordycepin.

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways.

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.