SynBio, Volume 3, Issue 1 (March 2025) – 5 articles

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step. Full article

The increasing prevalence of multi-drug-resistant (MDR) bacterial pathogens presents a critical global health threat, highlighting the urgent need for innovative approaches to understanding bacterial pathogenesis and developing effective therapies. This review underscores the potential of synthetic biology in elucidating host–pathogen interactions and facilitating the creation of advanced diagnostic tools and targeted therapies to combat MDR infections. We first explore CRISPR-based strategies that modulate essential gene expression, providing insights into the molecular mechanisms underlying host–pathogen interactions. Next, we discuss engineered microbial synthetic circuits for rapid pathogen detection by identifying molecular signatures involved in interspecies communication and facilitating swift pathogen elimination. Additionally, we explore phage therapy (PT), which leverages bacteriophages to selectively target and eliminate specific bacterial pathogens, presenting a targeted and promising approach to combat MDR infections. Finally, we review the application of organ-on-a-chip (OOAC) technology, which overcomes the limitations of animal models in predicting human immune responses by using microfluidic devices that simulate organ-level physiology and pathophysiology, thereby enabling more accurate disease modeling, drug testing, and the development of personalized medicine. Collectively, these synthetic biology tools provide transformative insights into the molecular mechanisms of host–pathogen interactions, advancing the development of precise diagnostic and therapeutic strategies against MDR infections. Full article

Synechocystis sp. PCC 6803 is a highly promising organism for the production of diverse recombinant compounds, including biofuels. However, conventional genetic engineering in Synechocystis presents challenges due to its highly polyploid genome, which not only results in low product yields but also compromises the reliability of recombinant strains for biomanufacturing applications. The CRISPR/Cas9 system, renowned for its precision, efficiency, and versatility across a wide range of chassis, offers significant potential to address the limitations posed by polyploid genomes. In this study, we developed and optimized an effective sgRNA for the targeted knock-in of nucleotide sequences of varying lengths into the neutral locus slr0168 of polyploid Synechocystis using CRISPR/Cas9. The gene encoding di-geranylgeranylglycerophospholipid reductase from Sulfolobus acidocaldarius and the methyl ketone operon from Solanum habrochaites were chosen as the exemplar nucleotide sequences for incorporation into the chromosome of Synechocystis. Our results demonstrate that the designed sgRNA effectively facilitated both knock-in events and that CRISPR/Cas9 enabled complete mutant segregation in a single round of selection and induction. Full article

Atherosclerosis remains a major driver for cardiovascular disease (CVD), despite advancements in traditional risk factor management therapies. Recent evidence emphasizes the crucial role of the gut microbiome in the progression of atherosclerosis and plaque rupture, highlighting a promising therapeutic avenue. This review focuses on the intertwined relationship between the gut microbiome, its metabolites, and atherosclerosis and CVD, also highlighting the potential therapeutic role of probiotics and prebiotics. Given the diverse and unique gut microbiota signatures among individuals, a one-size-fits-all therapeutic approach is unlikely to be effective. Personalized treatment strategies are therefore necessary. Here, we discussed how Artificial Intelligence (AI) can be leveraged to analyze individual gut microbiome profiles from microbiome sequencing, predict treatment response, and optimize therapeutic strategies based on individual patients, which would significantly improve outcomes of the treatment for atherosclerosis patients. Full article

Pancreatic cancer is the result of mutations in crucial genetic markers like KRAS and TP53 that make treatment challenging. This article discusses how CRISPR Cas9 technology can be combined with these markers to create treatments. CRISPR allows for the alteration or repair of these mutations, with the aim of restoring gene function or blocking cancer-causing pathways. For instance, CRISPR has the potential to fix mutations in TP53 or CDKN2A genes and restore SMAD4 signaling or target the KRAS oncogene in the body’s cells. However promising, it may be that CRISPR encounters obstacles like unintentional effects and challenges in effectively delivering it to pancreatic tumor cells. Furthermore, ethical concerns, especially related to the editing of the germline, need consideration. As techniques based on CRISPR advance, there is a chance for them to transform the treatment landscape for cancer by offering personalized therapies. More studies are needed to enhance how treatments are administered accurately and safely through methods and targeted testing for effectiveness. Full article