Programming Dynamic Division of Labor Using Horizontal Gene Transfer.

The metabolic engineering of microbes has broad applications, including biomanufacturing, bioprocessing, and environmental remediation. The introduction of a complex, multistep pathway often imposes a substantial metabolic burden on the host cell, restraining the accumulation of productive biomass and limiting pathway efficiency. One strategy to alleviate metabolic burden is the division of labor (DOL) in which different subpopulations carry out different parts of the pathway and work together to convert a substrate into a final product. However, the maintenance of different engineered subpopulations is challenging due to competition and convoluted interstrain population dynamics. Through modeling, we show that dynamic division of labor (DDOL), which we define as the DOL between indiscrete populations capable of dynamic and reversible interchange, can overcome these limitations and enable the robust maintenance of burdensome, multistep pathways. We propose that DDOL can be mediated by horizontal gene transfer (HGT) and use plasmid genomics to uncover evidence that DDOL is a strategy utilized by natural microbial communities. Our work suggests that bioengineers can harness HGT to stabilize synthetic metabolic pathways in microbial communities, enabling the development of robust engineered systems for deployment in a variety of contexts.

Duplicated antibiotic resistance genes reveal ongoing selection and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) and gene duplication are often considered as separate mechanisms driving the evolution of new functions. However, the mobile genetic elements (MGEs) implicated in HGT can copy themselves, so positive selection on MGEs could drive gene duplications. Here, we use a combination of modeling and experimental evolution to examine this hypothesis and use long-read genome sequences of tens of thousands of bacterial isolates to examine its generality in nature. Modeling and experiments show that antibiotic selection can drive the evolution of duplicated antibiotic resistance genes (ARGs) through MGE transposition. A key implication is that duplicated ARGs should be enriched in environments associated with antibiotic use. To test this, we examined the distribution of duplicated ARGs in 18,938 complete bacterial genomes with ecological metadata. Duplicated ARGs are highly enriched in bacteria isolated from humans and livestock. Duplicated ARGs are further enriched in an independent set of 321 antibiotic-resistant clinical isolates. Our findings indicate that duplicated genes often encode functions undergoing positive selection and horizontal gene transfer in microbial communities.

Programming dynamic division of labor using horizontal gene transfer.

The metabolic engineering of microbes has broad applications, including in biomanufacturing, bioprocessing, and environmental remediation. The introduction of a complex, multi-step pathway often imposes a substantial metabolic burden on the host cell, restraining the accumulation of productive biomass and limiting pathway efficiency. One strategy to alleviate metabolic burden is division of labor (DOL), in which different subpopulations carry out different parts of the pathway and work together to convert a substrate into a final product. However, the maintenance of different engineered subpopulations is challenging due to competition and convoluted inter-strain population dynamics. Through modeling, we show that dynamic division of labor (DDOL) mediated by horizontal gene transfer (HGT) can overcome these limitations and enable the robust maintenance of burdensome, multi-step pathways. We also use plasmid genomics to uncover evidence that DDOL is a strategy utilized by natural microbial communities. Our work suggests that bioengineers can harness HGT to stabilize synthetic metabolic pathways in microbial communities, enabling the development of robust engineered systems for deployment in a variety of contexts.

Programmable synthetic biomolecular condensates for cellular control.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.

Design patterns for engineering genetic stability.

The past 20 years have witnessed enormous progress in synthetic biology in the development of engineered cells for diverse applications, including biomanufacturing, materials fabrication, and potential therapeutics and diagnostics. However, it still remains a major challenge to maintain long-term performance of synthetic gene circuits, due to the emergence of mutants that lose circuit function. Here, we highlight major vulnerabilities of synthetic gene circuits resulting in circuit failure and mutant escape. We also discuss engineering strategies to enhance long-term circuit stability and performance. These approaches can be divided into two strategies: the suppression of the emergence of mutants and the suppression of their relative fitness if mutants do emerge. We anticipate that mechanistic understanding of the modes of circuit failure will facilitate future efforts to design evolutionarily robust synthetic biology-inspired applications.

Acetylated tau destabilizes the cytoskeleton in the axon initial segment and is mislocalized to the somatodendritic compartment.

BACKGROUND: Neurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer's disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood. RESULTS: Here we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and ßIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures. CONCLUSIONS: Together, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS.

Microglial AGE-albumin is critical in promoting alcohol-induced neurodegeneration in rats and humans.

Alcohol is a neurotoxic agent, since long-term heavy ingestion of alcohol can cause various neural diseases including fetal alcohol syndrome, cerebellar degeneracy and alcoholic dementia. However, the molecular mechanisms of alcohol-induced neurotoxicity are still poorly understood despite numerous studies. Thus, we hypothesized that activated microglial cells with elevated AGE-albumin levels play an important role in promoting alcohol-induced neurodegeneration. Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex following alcohol treatment in a rat model. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the mitogen activated protein kinase pathway. Treatment with soluble RAGE or AGE inhibitors significantly diminished neuronal damage in the animal model. Furthermore, the levels of activated microglial cells, AGE-albumin and neuronal loss were significantly elevated in human brains from alcoholic indivisuals compared to normal controls. Taken together, our data suggest that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration.