Genetically engineering cyanobacteria to convert CO2, water, and light into the long-chain hydrocarbon farnesene.

Genetically engineered cyanobacteria offer a shortcut to convert CO2 and H2O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C15H24), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO2, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1 ± 1.8 µg·L(-1)·O.D.(-1)·d(-1). Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading, and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT.

Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture.

The nuclear envelope (NE) creates a barrier between the cytosol and nucleus during interphase that is key for cellular compartmentalization and protecting genomic DNA. NE rupture can expose genomic DNA to the cytosol and allow admixture of the nuclear and cytosolic constituents, a proposed mechanism of cancer and NE-associated diseases. Barrier-to-autointegration factor (BAF) is a DNA-binding protein that localizes to NE ruptures where it recruits LEM-domain proteins, A-type lamins, and participates in rupture repair. To further reveal the mechanisms by which BAF responds to and aids in repairing NE ruptures, we investigated known properties of BAF including LEM domain binding, lamin binding, compartmentalization, phosphoregulation of DNA binding, and BAF dimerization. We demonstrate that it is the cytosolic population of BAF that functionally repairs NE ruptures, and phosphoregulation of BAF's DNA-binding that enables its ability to facilitate that repair. Interestingly, BAF's LEM or lamin binding activity appears dispensable for its role in functional repair. Furthermore, we demonstrate that BAF functions to reduce the extent of leakage though NE ruptures, suggesting that BAF effectively forms a diffusion barrier prior to NE repair. Collectively, these results enhances our knowledge of the mechanisms by which BAF responds to NE ruptures and facilitates their repair.

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT.

A BioID-Derived Proximity Interactome for SARS-CoV-2 Proteins.

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus-host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.

Barrier-to-autointegration factor: a first responder for repair of nuclear ruptures.

The nuclear envelope (NE) is a critical barrier between the cytosol and nucleus that is key for compartmentalization within the cell and serves an essential role in organizing and protecting genomic DNA. Rupturing of the NE through loss of constitutive NE proteins and/or mechanical force applied to the nucleus results in the unregulated mixing of cytosolic and nuclear compartments, leading to DNA damage and genomic instability. Nuclear rupture has recently gained interest as a mechanism that may participate in various NE-associated diseases as well as cancer. Remarkably, these rupturing events are often transient, with cells being capable of rapidly repairing nuclear ruptures. Recently, we identified Barrier-to-Autointegration Factor (BAF), a DNA-binding protein involved in post-mitotic NE reformation and cytosolic viral regulation, as an essential protein for nuclear rupture repair. During interphase, the highly mobile cytosolic BAF is primed to monitor for a compromised NE by rapidly binding to newly exposed nuclear DNA and subsequently recruiting the factors necessary for NE repair. This review highlights the recent findings of BAF's roles in rupture repair, and offers perspectives on how regulatory factors that control BAF activity may potentially alter the cellular response to nuclear ruptures and how BAF may participate in human disease.

A BioID-derived proximity interactome for SARS-CoV-2 proteins.

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.

Repair of nuclear ruptures requires barrier-to-autointegration factor.

Cell nuclei rupture following exposure to mechanical force and/or upon weakening of nuclear integrity, but nuclear ruptures are repairable. Barrier-to-autointegration factor (BAF), a small DNA-binding protein, rapidly localizes to nuclear ruptures; however, its role at these rupture sites is unknown. Here, we show that it is predominantly a nonphosphorylated cytoplasmic population of BAF that binds nuclear DNA to rapidly and transiently localize to the sites of nuclear rupture, resulting in BAF accumulation in the nucleus. BAF subsequently recruits transmembrane LEM-domain proteins, causing their accumulation at rupture sites. Loss of BAF impairs recruitment of LEM-domain proteins and nuclear envelope membranes to nuclear rupture sites and prevents nuclear envelope barrier function restoration. Simultaneous depletion of multiple LEM-domain proteins similarly inhibits rupture repair. LEMD2 is required for recruitment of the ESCRT-III membrane repair machinery to ruptures; however, neither LEMD2 nor ESCRT-III is required to repair ruptures. These results reveal a new role for BAF in the response to and repair of nuclear ruptures.