Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.
DNA can assume various structures as a result of interactions at atomic and molecular levels (e.g., hydrogen bonds, π–π stacking interactions, and electrostatic potentials), so understanding of the consequences of these interactions could guide development of ways to produce elaborate programmable DNA for applications in bio- and nanotechnology. We conducted advanced ab initio calculations to investigate nucleobase model structures by componentizing their donor-acceptor interactions. By unifying computational conditions, we compared the independent interactions of DNA duplexes, triplexes, and quadruplexes, which led us to evaluate a stability trend among Watson–Crick and Hoogsteen base pairing, stacking, and even ion binding. For a realistic solution-like environment, the influence of water molecules was carefully considered, and the potassium-ion preference of G-quadruplex was first analyzed at an ab initio level by considering both base-base and ion-water interactions. We devised new structure factors including hydrogen bond length, glycosidic vector angle, and twist angle, which were highly effective for comparison between computationally-predicted and experimentally-determined structures; we clarified the function of phosphate backbone during nucleobase ordering. The simulated tendency of net interaction energies agreed well with that of real world, and this agreement validates the potential of ab initio study to guide programming of complicated DNA constructs.
In guiding lipid droplets (LDs) to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids with biogenetic similarity but structural dissimilarity, we computationally and experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and β-carotene. In molecular dynamics simulations, worm-like flexible squalene is readily deformed to move through intertwined chains of triacylglycerols in the LD core, whereas rod-like rigid zeaxanthin is trapped on the LD surface due to a high free energy barrier in diffusion. By designing yeast cells with either much larger LDs or with a greater number of LDs, we observe that intracellular storage of squalene significantly increases with LD volume expansion, but that of zeaxanthin and β-carotene is enhanced through LD surface broadening; as visually evidenced, the outcomes represent internal penetration of squalene and surface localization of zeaxanthin and β-carotene. Our study shows the computational and experimental validation of selective lipid migration into a phase-separated organelle and reveals LD dynamics and functionalization.
Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells 1. We investigated whether engineered microbes could exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner 2,3. In this study, we demonstrated a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking becomes programmable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically coupled a terpene-specific protein with an export signal peptide and subsequently demonstrated exceptionally efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins could also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the first and most efficient cognate pathway for metabolite secretion by microorganisms.
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