Current advances of biocontainment strategy in synthetic biology

Synthetic biotechnology has led to the widespread application of genetically modified organisms(GMOs)in biochemistry, bioenergy, and therapy. However, the uncontrolled spread of GMOs may lead to genetic contamination by horizontal gene transfer, resulting in unpredictable biosafety risks. To deal with these challenges, many effective methods have been developed for biocontainment. In this article, we summarize and discuss recent advances in biocontainment strategies from three aspects: DNA replication, transcriptional regulation, and protein translation. We also briefly introduce the efforts in the biocontainment convention, such as the recent publication of the Tianjin Biosecurity Guidelines for the Code of Conduct for Scientists.

Food synthetic biology-driven protein supply transition: From animal-derived production to microbial fermentation

Animal-derived protein production is one of the major traditional protein supply methods,which continues to face increasing challenges to satisfy global needs due to population growth,augmented individual protein consumption,and aggravated environmental pollution.Thus,ensuring a sustainable protein source is a considerable challenge.The emergence and development of food synthetic biology has enabled the establishment of cell factories that effectively synthesize proteins,which is an important way to solve the protein supply problem.This review aims to discuss the existing problems of traditional protein supply and to elucidate the feasibility of synthetic biology in the process of protein synthesis.Moreover,using artificial bioengineered milk and artificial bioengineered eggs as examples,the progress of food protein supply transition based on synthetic biology has been systematically summarized.Additionally,the future of food synthetic biology as a potential source of protein has been also discussed.By strengthening and innovating the application of food synthetic biology technologies,including genetic engineering and high-throughput screening methods,the current limitations of artificial foods for protein synthesis and production should be addressed.Therefore,the development and industrial production of new food resources should be explored to ensure safe,high-quality,and sustainable global protein supply.

Efficient production of chemicals from microorganism by metabolic engineering and synthetic biology

The use of traditional chemical catalysis to produce chemicals has a series of drawbacks,such as high dependence on fossil resources,high energy consumption,and environmental pollution.With the development of synthetic biology and metabolic engineering,the use of renewable biomass raw materials for chemicals synthesis by constructing efficient microbial cell factories is a green way to replace traditional chemical catalysis and traditional microbial fermentation.This review mainly summarizes several types of bulk chemicals and high value-added chemicals using metabolic engineering and synthetic biology strategies to achieve efficient microbial production.In addition,this review also summarizes several strategies for effectively regulating microbial cell metabolism.These strategies can achieve the coupling balance of material and energy by regulating intracellular material metabolism or energy metabolism,and promote the efficient production of target chemicals by microorganisms.

Cell-free synthetic biology in the new era of enzyme engineering

With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,the difficulty of controlling the reaction environment,and the toxicity of some metabolic products in traditional in vivo enzyme engineering,it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds.Recently,cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies.This cell-free method has no limitation of the cell membrane and no need to maintain cell viability,and each biosynthetic pathway is highly flexible.This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize.This article aims to discuss the latest advances in cell-free enzyme engineering,assess the trend of this developing topical filed,and analyze its prospects.

An international comprehensive benchmarking analysis of synthetic biology in China from 2015 to 2020

As a new interdisciplinary field,synthetic biology has led to valuable innovations in the fields of medicine,chemistry,agriculture,energy and environment.In this paper,we systematically review the development status of global synthetic biology in the past six years,and make an in-depth benchmarking analysis of the field in China.With the aid of Scopus and SciVal,we analyze the scholarly output of synthetic biology in the world and individual countries,including publication distribution,popular journals and eminent institutions.Furthermore,the research focus and concepts,citation impact and collaborations are also examined using numerical index methods such as the field-weighted citation impact(FWCI)and relative activity index(RAI),showing the differences between data more intuitively.This study aims to offer a comprehensive understanding of the research status of synthetic biology in China and the world,offering a benchmarked overview of the results as a reference to guide the development of this field in the future.

Synthetic Biology Construct of Ebola Virus in Bacteria Surrogate Is Stable and Safe for Rapid Detection Studies in a BSL-2 Laboratory Setting

Rapid detection of virulent pathogens during an outbreak is critical for public health advisories and control of the disease in a population. While many molecular techniques for point of care and clinical diagnosis abound, the US experience with the COVID-19 testing in the early stages of the pandemic underscores the critical importance of determining the appropriate target gene(s) with in-built controls that reliably detect pathogens with high sensitivity and specificity. Assays and research for diagnostics and therapy could be slowed during an epidemic because access to the required BSL-3 and BSL-4 laboratories are limited. So, during the 2014 West Africa Ebola outbreak, we tested the hypothesis that using synthetic cDNA of Ebolavirus in a bacteria surrogate (fit for all lab settings), would remain unmutated and safe after several generations, serving as an effective positive control in research settings, self test and point-of-care detection platforms. Primers were designed for the detection and quantification of the nucleoprotein (NP) gene of the 2014 Makona Ebola strain (KR781608.1, 733 - 1332 bp). To test the stability of artificially inserted translation arrest in the Orf of the model gene, it was edited to include three STOP codons in the RNA transcript using SNAP GENE. The segment was then spliced into a high copy number plasmid, cloned into One ShotTM TOP10 Escherichia coli (Invitrogen), and tested for stability and safety by periodic subculture, extraction and sequencing. Unlike COVID-19, rapid detection of blood-borne etiologies like Ebola requires optimized protocols for blood matrix. Using real-time PCR and newly designed primer pairs, the EBOV surrogate was detected and enumerated in human blood and regular broth and buffers. Based on aligned sequence analysis, the EBOV synthetic NP gene was stable (>99.9999% similarity coefficient) for at least 3 months. Detection sensitivity in broth and blood was at least 100 cells/ml or about 5.8 × 103 to 7.3 × 103 virion equivalents per ml. While the developments of transcription-and-replication-competent virus like particles (trVLP) have made it possible to study the infection and replication cycles of virulent pathogens in BSL-2 laboratories, the simplicity of our model and the reproducibility of detection and enumeration show the utility of synthetic bio-components as positive controls for point of care diagnostic tools. The inserted stop codons remained intact after many generations, suggesting that expressed virulent proteins can be easily silenced in synthetic biology models for research in BSL-1 and 2 and a wide range of pathogens. Synthetic bio-components can thereby aid further research by reducing costs and improving safety for workers and stakeholders.

Enabling technology and core theory of synthetic biology

Synthetic biology provides a new paradigm for life science research(“build to learn”)and opens the future journey of biotechnology(“build to use”).Here,we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology,including synthesis and assembly of a genome,DNA storage,gene editing,molecular evolution and de novo design of function proteins,cell and gene circuit engineering,cell-free synthetic biology,artificial intelligence(AI)-aided synthetic biology,as well as biofoundries.We also introduce the concept of quantitative synthetic biology,which is guiding synthetic biology towards increased accuracy and predictability or the real rational design.We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.

Synthetic biology identifies the minimal gene set required for paclitaxel biosynthesis in a plant chassis

The diterpenoid paclitaxel(Taxol)is a chemotherapy medication widely used as a first-line treatment against several types of solid cancers.The supply of paclitaxel from natural sources is limited.However,missing knowledge about the genes involved in several specific metabolic steps of paclitaxel biosynthesis has rendered it difficult to engineer the full pathway.In this study,we used a combination of transcriptomics,cell biology,metabolomics,and pathway reconstitution to identify the complete gene set required for the heterologous production of paclitaxel.We identified the missing steps from the current model of paclitaxel biosynthesis and confirmed the activity of most of the missing enzymes via heterologous expression in Nicotiana benthamiana.Notably,we identified a new C4β-C20 epoxidase that could overcome the first bottleneck of metabolic engineering.We used both previously characterized and newly identified oxomutases/epoxidases,taxane 1β-hydroxylase,taxane 9aα-hydroxylase,taxane 9α-dioxygenase,and phenylalanine-CoA ligase,to successfully biosynthesize the key intermediate baccatin Ill and to convert baccatin Ill into paclitaxel in N.benthamiana.In combination,these approaches establisha metabolic route to taxoidbiosynthesis and provide insights into the unique chemistry that plants use to generate complex bioactive metabolites.

Making small molecules in plants:A chassis for synthetic biology-based production of plant natural products

Plant natural products have been extensively exploited in food,medicine,flavor,cosmetic,renewable fuel,and other industrial sectors.Synthetic biology has recently emerged as a promising means for the cost-effective and sustainable production of natural products.Compared with engineering microbes for the production of plant natural products,the potential of plants as chassis for producing these compounds is underestimated,largely due to challenges encountered in engineering plants.Knowledge in plant engineering is instrumental for enabling the effective and efficient production of valuable phytochemicals in plants,and also paves the way for a more sustainable future agriculture.In this manuscript,we briefly recap the biosynthesis of plant natural products,focusing primarily on industrially important terpenoids,alkaloids,and phenylpropanoids.We further summarize the plant hosts and strategies that have been used to engineer the production of natural products.The challenges and opportunities of using plant synthetic biology to achieve rapid and scalable production of high-value plant natural products are also discussed.

Degradation strategies of pesticide residue:From chemicals tosynthetic biology

The past 50 years have witnessed a massive expansion in the demand and application of pesticides.However,pesticides are difficult to be completely degraded without intervention hence the pesticide residue could pose a persistent threat to non-target organisms in many aspects.To aim at the problem of the abuse of pesticide products and excessive pesticide residues in the environment,chemical and biological degradation methods are widely developed but are scaled and insufficient to solve such a pollution.In recent years,bio-degradative tools instructed by synthetic biological principles have been further studied and have paved a way for pesticide degradation.Combining the customized design strategy and standardized assembly mode,the engineering bacteria for multi-dimensional degradation has become an effective tool for pesticide residue degradation.This review introduces the mechanisms and hazards of different pesticides,summarizes the methods applied in the degradation of pesticide residues,and discusses the advantages,applications,and prospects of synthetic biology in degrading pesticide residues.

Biotechnological and food synthetic biology potential of platform strain:Bacillus licheniformis

Bacillus licheniformis is one of the most characteristic Gram-positive bacteria.Its unique genetic background and safety characteristics make it have important biologic applications in the food industry,including,the biosyn-thesis of high value-added bioproducts,probiotic functions,biological treatment of wastes derived from food production,etc.In this review,these recent advances are summarized and presented systematically for the first time.In addition,we highlight synthetic biology strategies as a potential driver of developing this strain for wider and more efficient application in the food industry.Finally,we present the current challenges faced and provide our unique perspective on relevant future research directions.In summary,this review will provide an illumi-nating and comprehensive perspective that will allow an in-depth understanding of B.licheniformis and promote its more effective development in the food industry.

Enhanced depolluting capabilities of microbial bioelectrochemical systems by synthetic biology

Microbial bioelectrochemical system(BES)is a promising sustainable technology for the electrical energy recovery and the treatment of recalcitrant and toxic pollutants.In microbial BESs,the conversion of harmful pollutants into harmless products can be catalyzed by microorganisms at the anode(Type I BES),chemical catalysts at the cathode(Type II BES)or microorganisms at the cathode(Type III BES).The application of synthetic biology in microbial BES can improve its pollutant removing capability.Synthetic biology techniques can promote EET kinetics,which is helpful for microbial anodic electro-respiration,expediting pollutant removing not only at the anode but also at the cathode.They offer tools to promote biofilm development on the electrode,enabling more microorganisms residing on the electrode for subsequent catalytic reactions,and to overexpress the pollutant removing-related genes directly in microorganisms,contributing to the pollutant decomposition.In this work,based on the summarized aspects mentioned above,we describe the major synthetic biology strategies in designing and improving the pollutant removing capabilities of microbial BES.Lastly,we discuss challenges and perspectives for future studies in the area.

From qualitative to quantitative:the state of the art and challenges for plant synthetic biology

Backgrounds:As an increasing number of synthetic switches and circuits have been created for plant systems and of synthetic products produced in plant chassis,plant synthetic biology is taking a strong foothold in agriculture and medicine.The ever-exploding data has also promoted the expansion of toolkits in this field.Genetic parts libraries and quantitative characterization approaches have been developed.However,plant synthetic biology is still in its infancy.The considerations for selecting biological parts to design and construct genetic circuits with predictable functions remain desired.Results:In this article,we review the current biotechnological progresses in field of plant synthetic biology.Assembly standardization and quantitative approaches of genetic parts and genetic circuits are discussed.We also highlight the main challenges in the iterative cycles of design-build-test-learn for introducing novel traits into plants.Conclusion:Plant synthetic biology promises to provide important solutions to many issues in agricultural production,human health care,and environmental sustainability.However,tremendous challenges exist in this field.For example,the quantitative characterization of genetic parts is limited;the orthogonality and the transfer functions of circuits are unpredictable;and also,the mathematical modeling-assisted circuits design still needs to improve predictability and reliability.These challenges are expected to be resolved in the near future as interests in this field are intensifying.

Ethical framework on risk governance of synthetic biology

Synthetic biology is an emerging multidisciplinary field that aims to design and construct new biological systems not found in nature.Whereas synthetic biology may yield tremendous benefits,it may also pose substantial risks to human health and the environment that must be addressed.In this paper,we examined the environmental risks associated with synthetic biology,including changes to or depletion of the environment,competition with native species,horizontal gene transfer,pathogenicity or toxicity,bioterrorism,and laboratory biosecurity.We highlight three approaches for assessing environmental risks in synthetic biology:solution-focused risk assessment,Bayesian networks,and network of networks for sustainable capacity building.An ethical governance framework is proposed to facilitate innovation while minimising risks.This framework emphasises the precautionary principle and balancing stakeholder interests prior to project development and commercialisation.Overall,we underscore the importance and urgency of assessing and managing the environmental risks of synthetic biology to ensure its safe and ethical development and application.

Next-generation synthetic biology approaches for the accelerated discovery of microbial natural products

Microbial natural products(NPs)and their derivates have been widely used in health care and agriculture during the past few decades.Although large-scale bacterial or fungal(meta)genomic mining has revealed the tremendous biosynthetic potentials to produce novel small molecules,there remains a lack of universal approaches to link NP biosynthetic gene clusters(BGCs)to their associated products at a large scale and speed.In the last ten years,a series of emerging technologies have been established alongside the developments in synthetic biology to engineer cryptic metabolite BGCs and edit host genomes.Diverse computational tools,such as antiSMASH and PRISM,have also been simultaneously developed to rapidly identify BGCs and predict the chemical structures of their products.This review discusses the recent developments and trends pertaining to the accelerated discovery of microbial NPs driven by a wide variety of next-generation synthetic biology approaches,with an emphasis on the in situ activation of silent BGCs at scale,the direct cloning or refactoring of BGCs of interest for heterologous expression,and the synthetic-bioinformatic natural products(syn-BNP)approach for the guided rapid access of bioactive non-ribosomal peptides.

Synthetic biology for sustainable food ingredients production:recent trends

Problems with food security result from increased population,global warming,and decrease in cultivable land.With the advancements in synthetic biology,microbial synthesis of food is considered to be an efficient alternate approach that could permit quick food biosynthesis in an eco-friendly method.Furthermore,synthetic biology can be assumed to the synthesis of healthy or specially designed food components like proteins,lipids,amino acids and vitamins and widen the consumption of feedstocks,thus offering possible resolutions to high-quality food synthesis.This review describes the impact of synthetic biology for the microbial synthesis of various food ingredients production.

Synthetic biology in the UK-An outline of plans and progress

Synthetic biology is capable of delivering new solutions to key challenges spanning the bioeconomy,both nationally and internationally.Recognising this significant potential and the associated need to facilitate its translation and commercialisation the UK government commissioned the production of a national Synthetic Biology Roadmap in 2011,and subsequently provided crucial support to assist its implementation.Critical infrastructural investments have been made,and important strides made towards the development of an effectively connected community of practitioners and interest groups.A number of Synthetic Biology Research Centres,DNA Synthesis Foundries,a Centre for Doctoral Training,and an Innovation Knowledge Centre have been established,creating a nationally distributed and integrated network of complementary facilities and expertise.The UK Synthetic Biology Leadership Council published a UK Synthetic Biology Strategic Plan in 2016,increasing focus on the processes of translation and commercialisation.Over 50 start-ups,SMEs and larger companies are actively engaged in synthetic biology in the UK,and inward investments are starting to flow.Together these initiatives provide an important foundation for stimulating innovation,actively contributing to international research and development partnerships,and helping deliver useful benefits from synthetic biology in response to local and global needs and challenges.

Synthetic biology for CO2 fixation

Recycling of carbon dioxide(CO_2) into fuels and chemicals is a potential approach to reduce CO_2 emission and fossil-fuel consumption. Autotrophic microbes can utilize energy from light, hydrogen, or sulfur to assimilate atmospheric CO_2 into organic compounds at ambient temperature and pressure. This provides a feasible way for biological production of fuels and chemicals from CO_2 under normal conditions. Recently great progress has been made in this research area, and dozens of CO_2-derived fuels and chemicals have been reported to be synthesized by autotrophic microbes. This is accompanied by investigations into natural CO_2-fixation pathways and the rapid development of new technologies in synthetic biology. This review first summarizes the six natural CO_2-fixation pathways reported to date, followed by an overview of recent progress in the design and engineering of CO_2-fixation pathways as well as energy supply patterns using the concept and tools of synthetic biology. Finally, we will discuss future prospects in biological fixation of CO_2.

Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products

The methylotrophic yeast Pichia pastoris(a.k.a.Komagataella phaffii)is one of the most commonly used hosts for industrial production of recombinant proteins.As a non-conventional yeast,P.pastoris has unique biological characteristics and its expression system has been well developed.With the advances in synthetic biology,more efforts have been devoted to developing P.pastoris into a chassis for the production of various high-value compounds,such as natural products.This review begins with the introduction of synthetic biology tools for the engineering of P.pastoris,including vectors,promoters,and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System(CRISPR/Cas)for genome editing.This review is then followed by examples of the production of value-added natural products in metabolically engineered P.pastoris strains.Finally,challenges and outlooks in developing P.pastoris as a synthetic biology chassis are prospected.

Cell-free synthetic biology:Engineering in an open world

Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.