生物设计研究(英文)最新文献

The site-specific incorporation of the noncanonical amino acid (ncAA) into proteins via genetic code expansion (GCE) has enabled the development of new and powerful ways to learn, regulate, and evolve biological functions in vivo. However, cellular biosynthesis of ncAA-containing proteins with high efficiency and fidelity is a formidable challenge. In this review, we summarize up-to-date progress towards improving the efficiency and orthogonality of GCE and enhancing intracellular compatibility of introduced translation machinery in the living cells by creation and optimization of orthogonal translation components, constructing genomically recoded organism (GRO), utilization of unnatural base pairs (UBP) and quadruplet codons (four-base codons), and spatial separation of orthogonal translation.

The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.

Advancing biotechnologies are revolutionizing not only health and medicine, but also many different sectors such as agriculture, energy, chemistry, and textiles. As synthetic biology is leveraged as a programmable platform for the creation and biodesign of high-value biological medicines, foods, and commodities, the world is facing new territory in terms of ensuring the safety and security of both novel and engineered biological organisms, as well as the biological and digital platforms in which they are designed. Biosecurity practices and policies have traditionally revolved around preventing the misuse of biological pathogens, primarily through controlling access to pathogens. The advent of biodesign capabilities, such as gene editors, gene synthesis capabilities, and genetic engineering, requires a reevaluation of traditional biosecurity policies to mitigate risks associated with such engineering of biological entities. Here, features of "Biosecurity by Design" approaches are described, including the application of risk/benefit analysis and risk mitigation, post-COVID opportunities, and ethical global norms in the progression of biodesign and growing bioeconomies.

Viral diseases have contributed significantly to worldwide morbidity and mortality throughout history. Despite the existence of therapeutic treatments for many viral infections, antiviral resistance and the threat posed by novel viruses highlight the need for an increased number of effective therapeutics. In addition to small molecule drugs and biologics, antimicrobial peptides (AMPs) represent an emerging class of potential antiviral therapeutics. While AMPs have traditionally been regarded in the context of their antibacterial activities, many AMPs are now known to be antiviral. These antiviral peptides (AVPs) have been shown to target and perturb viral membrane envelopes and inhibit various stages of the viral life cycle, from preattachment inhibition through viral release from infected host cells. Rational design of AMPs has also proven effective in identifying highly active and specific peptides and can aid in the discovery of lead peptides with high therapeutic selectivity. In this review, we highlight AVPs with strong antiviral activity largely curated from a publicly available AMP database. We then compile the sequences present in our AVP database to generate structural predictions of generic AVP motifs. Finally, we cover the rational design approaches available for AVPs taking into account approaches currently used for the rational design of AMPs.

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal and the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the "serine shunt" which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution study using an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

A major advancement has recently occurred in the ability to predict protein secondary structure from sequence using artificial neural networks. This new accessibility to high-quality predicted structures provides a big opportunity for the protein design community. It is particularly welcome for membrane protein design, where the scarcity of solved structures has been a major limitation of the field for decades. Here, we review the work done to date on the membrane protein design and set out established and emerging tools that can be used to most effectively exploit this new access to structures.

Recent years have witnessed a rise in methods for accurate prediction of structure and design of novel functional proteins. Design of functional protein fragments and peptides occupy a small, albeit unique, space within the general field of protein design. While the smaller size of these peptides allows for more exhaustive computational methods, flexibility in their structure and sparsity of data compared to proteins, as well as presence of noncanonical building blocks, add additional challenges to their design. This review summarizes the current advances in the design of protein fragments and peptides for binding to targets and discusses the challenges in the field, with an eye toward future directions.

The international Genetically Engineered Machine (iGEM) Foundation has continued to promote synthetic biology education throughout its 2021 competition. The 2021 Virtual iGEM Jamboree was the culmination of the competition's growth, with 350 projects from 7314 innovators globally. Collegiate, high school, and community lab teams applied their ideas to the Registry of Standard Biological Parts, designing biological systems that provide solutions to an international scope of issues. The environmental, diagnostics, and therapeutics tracks continue to be the most prevalent focal points for projects, as students devise approaches to detrimental impacts of climate change and the COVID-19 pandemic. The competition exemplifies high standards of human practices, biosafety, and biosecurity through responsible biological engineering. As the iGEM Foundation continues pioneering STEM education into the future, equal developments of the competition's economic accessibility, global diversity, and long-term impact are necessary to allow a larger range of thinkers to access the power of synthetic biology.

The overarching goal of computational protein design is to gain complete control over protein structure and function. The majority of sophisticated binders and enzymes, however, are large and exhibit diverse and complex folds that defy atomistic design calculations. Encouragingly, recent strategies that combine evolutionary constraints from natural homologs with atomistic calculations have significantly improved design accuracy. In these approaches, evolutionary constraints mitigate the risk from misfolding and aggregation, focusing atomistic design calculations on a small but highly enriched sequence subspace. Such methods have dramatically optimized diverse proteins, including vaccine immunogens, enzymes for sustainable chemistry, and proteins with therapeutic potential. The new generation of deep learning-based ab initio structure predictors can be combined with these methods to extend the scope of protein design, in principle, to any natural protein of known sequence. We envision that protein engineering will come to rely on completely computational methods to efficiently discover and optimize biomolecular activities.

Photosynthetic terpene production represents one of the most carbon and energy-efficient routes for converting CO₂ into hydrocarbon. In photosynthetic organisms, metabolic engineering has led to limited success in enhancing terpene productivity, partially due to the low carbon partitioning. In this study, we employed systems biology analysis to reveal the strong competition for carbon substrates between primary metabolism (e.g., sucrose, glycogen, and protein synthesis) and terpene biosynthesis in Synechococcus elongatus PCC 7942. We then engineered key "source" and "sink" enzymes. The "source" limitation was overcome by knocking out either sucrose or glycogen biosynthesis to significantly enhance limonene production via altered carbon partitioning. Moreover, a fusion enzyme complex with geranyl diphosphate synthase (GPPS) and limonene synthase (LS) was designed to further improve pathway kinetics and substrate channeling. The synergy between "source" and "sink" achieved a limonene titer of 21.0 mg/L. Overall, the study demonstrates that balancing carbon flux between primary and secondary metabolism can be an effective approach to enhance terpene bioproduction in cyanobacteria. The design of "source" and "sink" synergy has significant potential in improving natural product yield in photosynthetic species.