ACS Synthetic Biology最新文献

Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7, encoding a subunit of the mitochondrial F₁F₀ ATP synthase, and overexpression of QDR2 and QDR3, two previously uncharacterized members of the 12-spanner H⁺ antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a ΔFUM1 background strain improved titers of fumaric acid production from 0.26 to 2.16 g L^–1. Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus.

An accurate deep learning predictor of enzyme optimal pH is essential to quantitatively describe how pH influences the enzyme catalytic activity. CatOpt, developed in this study, outperformed existing predictors of enzyme optimal pH (RMSE = 0.833 and R² = 0.479), and could provide good interpretability with informative residue attention weights. The classification of acidophilic and alkaliphilic enzymes and prediction of enzyme optimal pH shifts caused by point mutations showcased the capability of CatOpt as an effective computational tool for identifying enzyme pH preferences. Furthermore, a single point mutation designed with the guidance of CatOpt successfully enhanced the activity of Pyrococcus horikoshii diacetylchitobiose deacetylase at low pH (pH = 4.5/5.5) by approximately 7%, suggesting that CatOpt is a promising in silico enzyme design tool for pH-dependent enzyme activities.

There is immense potential in crafting synthetic microbial communities for application in human health, agriculture, the environment, and even biomanufacturing where an appropriately constructed consortium can be assembled with tremendous biosynthetic or degradative capabilities. In many of these cases, bacterial signaling serves as a form of intercellular information transfer that guides the collective’s behavior. Such communication is complex, as many signals, signal disruptors, microbial species, physical barriers, and spatiotemporal constraints may be involved. In this work, we demonstrate that a multisignal pathway for molecular information transfer within a consortium of several Pseudomonas spp. can be scrambled (genetically and organizationally) while the original message is still effectively conveyed. Assembled from the bottom up, we have employed two types of signaling molecules (i) a redox active secondary metabolite (rhizospheric signal, phloroglucinol), and (ii) a bacterial quorum sensing signal (3-oxo-C12 acylhomoserine lactone, AI-1). These signals can be intraconverted and acted upon by designated community members. We show how the order in which the signals are received, transduced, and subsequently transmitted can be rearranged with minimal impact on the intended outcome. In the consortial context, we found this messaging structure can be remarkably robust. Inspired by rhizospheric molecular signaling mechanisms, this work provides a conceptual framework for designing signaling and information transfer processes within assembled communities.

Rapid and decentralized vaccine production is essential to ensure global preparedness against emerging and re-emerging infectious diseases. Cell-free gene expression systems, which can be freeze-dried for long-term storage and reactivated for point-of-use synthesis, offer a promising solution to address this need. However, scalable cell-free production of conjugate vaccines─highly effective tools against bacterial infections─has been hindered by low yields and inefficient glycosylation. To address these challenges, we developed a modular, cell-free platform for the synthesis and purification of conjugate vaccines. By decoupling cell-free protein expression from in vitro glycosylation in a two-step approach, we achieved >85% glycosylation efficiency and up to ∼450 mg/L of glycoprotein. We applied this platform to manufacture protein–polysaccharide conjugates composed of vaccine carrier proteins covalently modified with polysaccharide antigens from enterotoxigenic Escherichia coli O78 and Streptococcus pneumoniae serotype 4. Our workflow produced conjugate vaccine candidates in under 5 days with >87% product purity and low endotoxin levels suitable for preclinical evaluation. Immunization of mice with the pneumococcal conjugate vaccine induced a strong IgG response against S. pneumoniae serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.

Sesquiterpenes are a diverse class of natural products characterized by complex structures and broad biological activities with their core scaffolds formed by sesquiterpene synthase. In this study, a novel sesquiterpene synthase, NmSTS, from the fungus Neophaeococcomyces mojaviensis was functionally characterized. NmSTS catalyzes the cyclization of (E,E)-farnesyl pyrophosphate ((E,E)-FPP) to produce the bridged ring sesquiterpene α-santalene, a compound widely used in the fragrance and flavor industries. Additionally, NmSTS also exhibits substrate promiscuity, converting (E)-geranyl pyrophosphate ((E)-GPP) and (E,E,E)-geranylgeranyl pyrophosphate ((E,E,E)-GGPP) into linear terpenes. Through protein modeling and site-directed mutagenesis, ten key amino acid residues essential for catalytic activity were identified. Among these, the R187A and Y302A variants enable the transformation of the sesquiterpene product from a bridged ring to hydroxylated tri- and acyclic skeletons, respectively. These findings enhance our understanding of sesquiterpene biosynthetic mechanisms and provide valuable genetic tools for the engineered microbial production of valuable sesquiterpenes.

The emergence of multidrug resistance in Gram-negative bacteria poses a significant challenge to global health, necessitating the development of effective antibiotics and therapeutic approaches. Endolysins derived from phages exhibit specificity toward bacteria, making them desirable candidates for the treatment of infectious bacteria. Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) with unique side chains, endowing proteins with new functions and properties. In this study, we applied genetic code expansion to introduce ncAAs into endolysin LysPA26, which targeted a broad spectrum of Gram-negative bacteria. Incorporating p-azido-l-phenylalanine (pAzF) at position R16 in LysPA26 increased its broad-spectrum bacteriolytic activity without an apparent change in its secondary structure. Furthermore, the engineered LysPA26-R16pAzF variant exhibited higher bacteriolytic activity than the wild type after exposure to varying temperatures (4 to 70 °C) or freezing (−25 °C). Additionally, the incorporation of another azido-containing ncAA, azidonor-leucine (AnzL), at position R16 of LysPA26 also improved bacteriolytic activity. Using a similar strategy, the insertion of pAzF into LysDLP1, a lysin from Acinetobacter phage vB AbaM DLP1, yielded a variant with enhanced bacteriolytic activity. These results collectively indicate that ncAAs can be effectively incorporated to engineer endolysins with enhanced antimicrobial performance, offering significant implications for lysin engineering and the development of antibiotics.

CRISPR-dCas tools have widespread applications for rapidly manipulating and dissecting gene function across the microbial tree of life. However, despite their theoretical suitability for use in a broad range of species, CRISPR-dCas tools that are often initially optimized for use in model cell lines and model organisms still frequently require extensive modifications to enable their application in specific microbial organisms. Here, we review different iterations of CRISPR-dCas in microbes and the application of these techniques. We further discuss common obstacles faced and troubleshooting approaches while developing and applying CRISPR-dCas systems to a microbial organism. Finally, we suggest enhancements that can be made that may help improve the applicability of a CRISPR-dCas tool developed for nonmodel microbial organisms.

Iron is a critical micronutrient for both plants and humans, yet its declining availability across agricultural systems threatens global food security and health. Biofortification of food crops has emerged as a promising strategy to combat iron deficiency and anemia, leveraging both crop breeding and microbiome-based approaches to enhance iron mobilization and uptake. Advances in plant and bacterial synthetic biology could enable the precise programming of iron homeostasis and acquisition mechanisms, offering tailored solutions across diverse species and environments. Here, we outline key biomolecules, genes, and biosynthetic and transport pathways that represent underexplored synthetic biology targets for improving crop iron acquisition. We highlight opportunities to tune expression strength, tissue specificity, and cross-host pathway transfer to enhance chelation- and reduction-mediated solubilization of soil iron and augment plant uptake. Finally, we emphasize the broader importance of developing plant–microbe–metal actuators as modular components in genetic circuit design and discuss how their deployment across diverse plant and microbial chassis could accelerate agricultural biofortification and improve global nutrition.

Cell separation and purification techniques are crucial in modern biomedical research and clinical applications. Endogenous RNA, which reflects a cell’s genetic and physiological characteristics, provides a new way to determine cell identity at the transcriptional level. Here, we utilize RNA editing technology based on adenosine deaminase acting on RNA (ADAR) to design a dual-switch genetic circuit capable of detecting unique RNA biomarkers for cell separation and purification. The circuit incorporates a kill switch driven by barnase, which selectively eliminates nontarget cells, and a recognition switch, precisely regulated by ADAR editing, to control the expression of the MS2 bacteriophage coat protein (MCP) and barstar that inhibit barnase expression and activity. By temporally regulating these switches, our approach achieves purification efficiencies of 93–97% for HepG2, A549, and HER2-overexpressing SK-BR-3 cells in mixed populations, surpassing traditional methods. Furthermore, utilizing standard cell culture protocols, our approach simplifies cell identification and purification without interfering with the normal gene expression of target cells, ensuring robustness and safety. We believe that this ADAR-assisted genetic circuit holds great potential for applications in cell therapy and biopharmaceutical manufacturing.

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency, setting the stage for automated recommendations based on actual track records. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.