Synthetic Biology Engineering Research Articles

Advances on the production of organic acids by yeast

Organic acids are organic compounds that can be synthesized using biological systems. They often contain one or more low molecular weight acidic groups, such as carboxyl group and sulphonic group. Organic acids are widely used in food, agriculture, medicine, bio-based materials industry and other fields. Yeast has unique advantages of biosafety, strong stress resistance, wide substrate spectrum, convenient genetic transformation, and mature large-scale culture technology. Therefore, it is appealing to produce organic acids by yeast. However, challenges such as low concentration, many by-products and low fermentation efficiency still exist. With the development of yeast metabolic engineering and synthetic biology technology, rapid progress has been made in this field recently. Here we summarize the progress of biosynthesis of 11 organic acids by yeast. These organic acids include bulk carboxylic acids and high-value organic acids that can be produced naturally or heterologously. Finally, future prospects in this field were proposed.

Preface for special issue on chemical bioproduction

Engineering efficient enzymes or microbial cell factories should help to establish green bio-manufacturing process for chemical overproduction. The rapid advances and development in synthetic biology, systems biology and enzymatic engineering accerleate the establishing feasbile bioprocess for chemical biosynthesis, including expanding the chemical kingdom and improving the productivity. To consolidate the latest advances in chemical biosynthesis and promote green bio-manufaturing, we organized a special issue on chemical bioproduction that including review or original research papers about enzymatic biosynthesis, cell factory, one-carbon based biorefinery and feasible strategies. These papers comprehensively discussed the latest advaces, the challenges as well as the possible solutions in chemical biomanufacturing.

Multi-compartmental MOF microreactors derived from Pickering double emulsions for chemo-enzymatic cascade catalysis

Bioinspired multi-compartment architectures are desired in synthetic biology and metabolic engineering, as credited by their cell-like structures and intrinsic ability of assembling catalytic species for spatiotemporal control over cascade reactions like in living systems. Herein, we describe a general Pickering double emulsion-directed interfacial synthesis method for the fabrication of multicompartmental MOF microreactors. This approach employs multiple liquid–liquid interfaces as a controllable platform for the self-completing growth of dense MOF layers, enabling the microreactor with tailor-made inner architectures and selective permeability. Importantly, simultaneous encapsulation of incompatible functionalities, including hydrophilic enzyme and hydrophobic molecular catalyst, can be realized in a single MOF microreactor for operating chemo-enzymatic cascade reactions. As exemplified by the Grubb’ catalyst/CALB lipase driven olefin metathesis/ transesterification cascade reaction and glucose oxidase (GOx)/Fe-porphyrin catalyzed oxidation reaction, the multicompartmental microreactor exhibits 2.24–5.81 folds enhancement in cascade reaction efficiency in comparison to the homogeneous counterparts or physical mixture of individual analogues, due to the restrained mutual inactivation and substrate channelling effects. Our study prompts further design of multicompartment systems and the development of artificial cells capable of complex cellular transformations.

Taking Synthetic Biology to the Seas - From Blue Chassis Organisms to Marine Aquaforming.

Oceans cover 71% of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example for the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed.

Biological Databases and Resources: Their Engineering and Applications in Synthetic Biology

Biological Databases and Resources: Their Engineering and Applications in Synthetic Biology

Metabolic Engineering and Synthetic Biology Approaches for the Heterologous Production of Aromatic Polyketides.

Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.

Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics.

Genetically encoded biosensors are powerful tools for product-driven high-throughput screening in synthetic biology and metabolic engineering. However, most biosensors can only properly function in a limited concentration cutoff, and the incompatible performance characteristics of biosensors will lead to false positives or failure in screening. The transcription factor (TF)-based biosensors are usually organized in modular architecture and function in a regulator-depended manner, whose performance properties can be fine-tuned by modifying the expression level of the TF. In this study, we modulated the performance characteristics, including sensitivity and operating range, of an MphR-based erythromycin biosensor by fine-adjusting regulator expression levels via ribosome-binding site (RBS) engineering and obtained a panel of biosensors with varied sensitivities by iterative fluorescence-assisted cell sorting (FACS) in Escherichia coli to accommodate different screening purposes. To exemplify their application potential, two engineered biosensors with 10-fold different sensitivities were employed in the precise high-throughput screening by microfluidic-based fluorescence-activated droplet sorting (FADS) of Saccharopolyspora erythraea mutant libraries with different starting erythromycin productions, and mutants representing as high as 6.8 folds and over 100% of production improvements were obtained starting from the wild-type strain and the high-producing industrial strain, respectively. This work demonstrated a simple strategy to engineer biosensor performance properties, which was significant to stepwise strain engineering and production improvement.

Using Synthetic Biology Technology as an Avenue to Improve Diagnosis and Treatment in Women’s Health

Using Synthetic Biology Technology as an Avenue to Improve Diagnosis and Treatment in Women’s Health

Application of Synthetic Biology in SARS-CoV-2 Vaccine Development

The novel coronavirus SARS-CoV-2 has been spreading at an extraordinary rate since it first appeared in late 2019, causing a large number of infections and deaths. The COVID-19 pan-demic can be successfully controlled most effectively with vaccines, and efforts are being made globally to create new vaccines. Synthetic biology is a design-oriented field that focus-es on creating novel biological functions by discovering, analyzing, and repurposing biologi-cal components. Many SARS-CoV-2 vaccines have been developed using synthetic biology, and the recent approval of a few more vaccines is evidence of the field's continuous growth. This review discusses the use of synthetic biology technology in the creation of several COVID-19 vaccines, concentrating on how to produce vaccines that are safer and more effec-tive through genome optimization and delivery mechanism optimization. Finally, this article highlights some existing problems of synthetic biology technology and provides an outlook on the future development of application this technology in COVID-19 vaccines.

Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing

BackgroundThe main commercially available methods for detecting small molecules of mycotoxins in traditional Chinese medicine (TCM) and functional foods are enzyme-linked immunosorbent assay and mass spectrometry. Regarding the development of diagnostic antibody reagents, effective methods for the rapid preparation of specific monoclonal antibodies are inadequate.MethodsIn this study, a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library with a glove-like cavity configuration was established using phage display technology in synthetic biology. We applied this unique SynaGG library on the small molecule aflatoxin B1 (AFB1), which has strong hepatotoxicity, to isolate specific nanobodies with high affinity for AFB1.ResultThese nanobodies exhibit no cross-reactivity with the hapten methotrexate, which is recognized by the original antibody template. By binding to AFB1, two nanobodies can neutralize AFB1-induced hepatocyte growth inhibition. Using molecular docking, we found that the unique non-hypervariable complementarity-determining region 4 (CDR4) loop region of the nanobody was involved in the interaction with AFB1. Specifically, the CDR4’s positively charged amino acid arginine directed the binding interaction between the nanobody and AFB1. We then rationally optimized the interaction between AFB1 and the nanobody by mutating serine at position 2 into valine. The binding affinity of the nanobody to AFB1 was effectively improved, and this result supported the use of molecular structure simulation for antibody optimization.ConclusionIn summary, this study revealed that the novel SynaGG library, which was constructed through computer-aided design, can be used to isolate nanobodies that specifically bind to small molecules. The results of this study could facilitate the development of nanobody materials to detect small molecules for the rapid screening of TCM materials and foods in the future.

Synthetic bacteria for the detection and bioremediation of heavy metals.

Toxic heavy metal accumulation is one of anthropogenic environmental pollutions, which poses risks to human health and ecological systems. Conventional heavy metal remediation approaches rely on expensive chemical and physical processes leading to the formation and release of other toxic waste products. Instead, microbial bioremediation has gained interest as a promising and cost-effective alternative to conventional methods, but the genetic complexity of microorganisms and the lack of appropriate genetic engineering technologies have impeded the development of bioremediating microorganisms. Recently, the emerging synthetic biology opened a new avenue for microbial bioremediation research and development by addressing the challenges and providing novel tools for constructing bacteria with enhanced capabilities: rapid detection and degradation of heavy metals while enhanced tolerance to toxic heavy metals. Moreover, synthetic biology also offers new technologies to meet biosafety regulations since genetically modified microorganisms may disrupt natural ecosystems. In this review, we introduce the use of microorganisms developed based on synthetic biology technologies for the detection and detoxification of heavy metals. Additionally, this review explores the technical strategies developed to overcome the biosafety requirements associated with the use of genetically modified microorganisms.

L-lactate production in engineered Saccharomyces cerevisiae using a multistage multiobjective automated design framework.

The design of alternative biodegradable polymers has the potential of severely reducing the environmental impact, cost and production time currently associated with the petrochemical industry. In fact, growing demand for renewable feedstock has recently brought to the fore synthetic biology and metabolic engineering. These two interdependent research areas focus on the study of microbial conversion of organic acids, with the aim of replacing their petrochemical-derived equivalents with more sustainable and efficient processes. The particular case of Lactic acid (LA) production has been the subject of extensive research because of its role as an essential component for developing an eco-friendly biodegradable plastic-widely used in industrial biotechnological applications. Because of its resistance to acidic environments, among the many LA-producing microbes, Saccharomyces cerevisiae has been the main focus of research into related biocatalysts. In this study, we present an extensive in silico investigation of S. cerevisiae cell metabolism (modeled with Flux Balance Analysis) with the overall aim of maximizing its LA production yield. We focus on the yeast 8.3 steady-state metabolic model and analyze it under the impact of different engineering strategies including: gene knock-in, gene knock-out, gene regulation and medium optimization; as well as a comparison between results in aerobic and anaerobic conditions. We designed ad-hoc constrained multiobjective evolutionary algorithms to automate the engineering process and developed a specific postprocessing methodology to analyze the genetic manipulation results obtained. The in silico results reported in this paper empirically show that our method is able to automatically select a small number of promising genetic and metabolic manipulations, deriving competitive strains that promise to impact microorganisms design in the production of sustainable chemicals.

Structure-driven protein engineering for production of valuable natural products.

Proteins are the most frequently used biocatalysts, and their structures determine their functions. Modifying the functions of proteins on the basis of their structures lies at the heart of protein engineering, opening a new horizon for metabolic engineering by efficiently generating stable enzymes. Many attempts at classical metabolic engineering have focused on improving specific metabolic fluxes and producing more valuable natural products by increasing gene expression levels and enzyme concentrations. However, most naturally occurring enzymes show limitations, and such limitations have hindered practical applications. Here we review recent advances in protein engineering in synthetic biology, chemoenzymatic synthesis, and plant metabolic engineering and describe opportunities for designing and constructing novel enzymes or proteins with desirable properties to obtain more active natural products.

Biocontainment Techniques and Applications for Yeast Biotechnology

Biocontainment techniques for genetically modified yeasts (GMYs) are pivotal due to the importance of these organisms for biotechnological processes and also due to the design of new yeast strains by using synthetic biology tools and technologies. Due to the large genetic modifications that many yeast strains display, it is highly desirable to avoid the leakage of GMY cells into natural environments and, consequently, the spread of synthetic genes and circuits by horizontal or vertical gene transfer mechanisms within the microorganisms. Moreover, it is also desirable to avoid patented yeast gene technologies spreading outside the production facility. In this review, the different biocontainment technologies currently available for GMYs were evaluated. Interestingly, uniplex-type biocontainment approaches (UTBAs), which rely on nutrient auxotrophies induced by gene mutation or deletion or the expression of the simple kill switches apparatus, are still the major biocontainment approaches in use with GMY. While bacteria such as Escherichia coli account for advanced biocontainment technologies based on synthetic biology and multiplex-type biocontainment approaches (MTBAs), GMYs are distant from this scenario due to many reasons. Thus, a comparison of different UTBAs and MTBAs applied for GMY and genetically engineered microorganisms (GEMs) was made, indicating the major advances of biocontainment techniques for GMYs.

Engineering Escherichia coli to Utilize Erythritol as Sole Carbon Source.

Erythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Humans themselves are not able to catabolize erythritol and their gut microbes lack related catabolic pathways either to metabolize erythritol. Here, Escherichia coli (E. coli) is engineered to utilize erythritol as sole carbon source aiming for defined applications. First, the erythritol metabolic gene cluster is isolated and the erythritol-binding transcriptional repressor and its DNA-binding site are experimentally characterized. Transcriptome analysis suggests that carbohydrate metabolism-related genes in the engineered E. coli are overall upregulated. In particular, the enzymes of transaldolase (talA and talB) and transketolase (tktA and tktB) are notably overexpressed (e.g., the expression of tktB is improved by nearly sixfold). By overexpression of the four genes, cell growth can be increased as high as threetimes compared to the cell cultivation without overexpression. Finally, engineered E. coli strains can be used as a living detector to distinguish erythritol-containing soda soft drinks and can grow in the simulated intestinal fluid supplemented with erythritol. This work is expected to inspire the engineering of more hosts to respond and utilize erythritol for broad applications in metabolic engineering, synthetic biology, and biomedical engineering.

Bioengineering for the Microbial Degradation of Petroleum Hydrocarbon Contaminants

Petroleum hydrocarbons are relatively recalcitrant compounds, and as contaminants, they are one of the most serious environmental problems. n-Alkanes are important constituents of petroleum hydrocarbons. Advances in synthetic biology and metabolic engineering strategies have made n-alkane biodegradation more designable and maneuverable for solving environmental pollution problems. In the microbial degradation of n-alkanes, more and more degradation pathways, related genes, microbes, and alkane hydroxylases have been discovered, which provide a theoretical basis for the further construction of degrading strains and microbial communities. In this review, the current advances in the microbial degradation of n-alkanes under aerobic condition are summarized in four aspects, including the biodegradation pathways and related genes, alkane hydroxylases, engineered microbial chassis, and microbial community. Especially, the microbial communities of "Alkane-degrader and Alkane-degrader" and "Alkane-degrader and Helper" provide new ideas for the degradation of petroleum hydrocarbons. Surfactant producers and nitrogen providers as a "Helper" are discussed in depth. This review will be helpful to further achieve bioremediation of oil-polluted environments rapidly.

Engineered bacteria combined with doxorubicin nanoparticles suppress angiogenesis and metastasis in murine melanoma models.

Methods capable of distributing antitumour therapeutics uniformly throughout an entire tumour and that can suppress metastasis at the same time, would be of great significance in improving cancer treatment. Bacteria-mediated synergistic therapies have been explored for better specificity, temporal and spatial controllability, as well for providing regulation of the immune microenvironment, in order to provide improved cancer treatment. To achieve this goal, here we developed an engineered bacteria delivery system (GDOX@HSEc) using synthetic biology and interfacial chemistry technologies. The engineered bacteria were concurrently modified to express heparin sulfatase 1 (HSulf-1) inside (HSEc), to attach doxorubicin-loaded glycogen nanoparticles (GDOX NPs) on their surface. Here we demonstrate that HSEc can actively target and colonise tumour sites resulting in HSulf-1 overexpression, thereby suppressing angiogenesis and metastasis. Simultaneously, the GDOX NPs were able to penetrate into tumour cells, leading to intracellular DNA damage. Our results confirmed that a combination of biotherapy and chemotherapy using GDOX@HSEc resulted in significant melanoma suppression in murine models, with reduced side effects. This study provides a powerful platform for the simultaneous delivery of biomacromolecules and chemotherapeutic drugs to tumours, representing an innovative strategy potentially more effective in treating solid tumours. STATEMENT OF SIGNIFICANCE: An original engineered bacteria-based system (GDOX@HSEc) was developed using synthetic biology and interfacial chemistry technologies to concurrently produce naturally occurring heparin sulfatase 1 (HSulf-1) inside and anchor doxorubicin-loaded glycogen nanoparticles on the surface. GDOX@HSEc allowed for combined local delivery of chemotherapeutic agents along with the enzymes and immunostimulatory bacterial adjuvants, which resulted in a synergistic action in the inhibition of tumour growth and metastasis. The study provides a potential therapeutic approach that allows therapeutic agents to be distributed in a spatiotemporally controllable manner in tumours for combinatorial enhanced therapy.

Expanding the genetic code: a non-natural amino acid story

From enzymes to hormones, proteins are the most versatile macromolecules that serve a vital function throughout all biological systems. In nature, organisms are restricted to a 20 amino acid repertoire, which in turn limits the chemistry. Nature evolves and adapts but currently does so under a “chemistry-limited” circumstance. This in turn has limited the aspirations of protein designers and engineers that aim to expand the functional and structural properties of proteins into new and exciting realms. One way nature has circumvented this problem is to recruit non-proteinaceous cofactors. Another way is to change one of the most fundamental concepts underlying biology: the genetic code. Pyrrolysine and selenocysteine are the two main genetically encodable proteinogenic “21st” amino acids; stop codons are recruited to encode the incorporation during ribosomal polypeptide synthesis. This ability of nature to go beyond the standard 20 amino acid repertoire inspired researchers to engineer the fundamental elements of ribosomal polypeptide synthesis and allow full genetic encoding of non-natural amino acids. This in turn helped advance synthetic biology and protein engineering in ways that were not possible by using the standard 20 amino acids. To this day, a vast repertoire of non-natural amino acids is available and is continuously expanding with increasing scientific needs.

Petroleum Hydrocarbon Catabolic Pathways as Targets for Metabolic Engineering Strategies for Enhanced Bioremediation of Crude-Oil-Contaminated Environments

Anthropogenic activities and industrial effluents are the major sources of petroleum hydrocarbon contamination in different environments. Microbe-based remediation techniques are known to be effective, inexpensive, and environmentally safe. In this review, the metabolic-target-specific pathway engineering processes used for improving the bioremediation of hydrocarbon-contaminated environments have been described. The microbiomes are characterised using environmental genomics approaches that can provide a means to determine the unique structural, functional, and metabolic pathways used by the microbial community for the degradation of contaminants. The bacterial metabolism of aromatic hydrocarbons has been explained via peripheral pathways by the catabolic actions of enzymes, such as dehydrogenases, hydrolases, oxygenases, and isomerases. We proposed that by using microbiome engineering techniques, specific pathways in an environment can be detected and manipulated as targets. Using the combination of metabolic engineering with synthetic biology, systemic biology, and evolutionary engineering approaches, highly efficient microbial strains may be utilised to facilitate the target-dependent bioprocessing and degradation of petroleum hydrocarbons. Moreover, the use of CRISPR-cas and genetic engineering methods for editing metabolic genes and modifying degradation pathways leads to the selection of recombinants that have improved degradation abilities. The idea of growing metabolically engineered microbial communities, which play a crucial role in breaking down a range of pollutants, has also been explained. However, the limitations of the in-situ implementation of genetically modified organisms pose a challenge that needs to be addressed in future research.

A novel synthetic sRNA promoting protein overexpression in cell-free systems.

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non-Hfq-dependent sRNA scaffold that uses a modifiable 20 nucleotide antisense binding region to target mRNAs selectively and influence protein expression. The system was developed for regulation of a fluorescent reporter in vivo using Escherichia coli, but the system was found to be more responsive and produced statistically significant results when applied to protein synthesis using in vitro cell-free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high-energy stem loop structure and the 3' end of mRNA yielded protein expression knock-downs that approached 70%. Notably, targeting a low-energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in protein expression (p < 0.05). These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in a CFS. It is possible the designs developed here can be applied to improve/regulate expression of other proteins in a CFS.