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Engineering Microbial Living Therapeutics: The Synthetic Biology Toolbox

2018; Elsevier BV; Volume: 37; Issue: 1 Linguagem: Inglês

10.1016/j.tibtech.2018.09.005

0167-9430

Danielle Biscaro Pedrolli, Nathan Vinícius Ribeiro, Patrick N. Squizato, Victor Nunes de Jesus, Daniel A. Cozetto, Rafael B. Tuma, Amanda Gracindo, Mariana B. Cesar, Paulo J.C. Freire, Ana F.M. da Costa, Milca R.C.R. Lins, Graciely Gomes Corrêa, Marcel O. Cerri,

Gut microbiota and health

Living therapeutics have been engineered to diagnose diseases and produce and deliver therapeutics in situ. These therapeutics can be equipped with devices for sensing inputs, controlling gene expression, building memory, and producing and delivering an active compound. Ingenious devices responding to stress, temperature, quorum-sensing signals, and other small molecules have been built to control the production and delivery of therapeutic molecules. To deal with biosafety, some living therapeutics carry biocontainment devices based on cell auxotrophy, temperature-sensitive regulators, and toxin/antitoxin counteraction. Recent advances in synthetic biology greatly expanded the toolbox for engineering living therapeutics; however, new parts are still needed to help synthetic biologists engineer more diverse and fully functional living therapeutics. Microbes can be engineered to act like living therapeutics designed to perform specific actions in the human body. From fighting and preventing infections to eliminating tumors and treating metabolic disorders, engineered living systems are the next generation of therapeutics. In recent years, synthetic biologists have greatly expanded the genetic toolbox for microbial living therapeutics, adding sensors, regulators, memory circuits, delivery devices, and kill switches. These advances have paved the way for successful engineering of fully functional living therapeutics, with sensing, production, and biocontainment devices. However, some important tools are still missing from the box. In this review, we cover the most recent biological parts and approaches developed and describe the missing tools needed to build robust living therapeutics. Microbes can be engineered to act like living therapeutics designed to perform specific actions in the human body. From fighting and preventing infections to eliminating tumors and treating metabolic disorders, engineered living systems are the next generation of therapeutics. In recent years, synthetic biologists have greatly expanded the genetic toolbox for microbial living therapeutics, adding sensors, regulators, memory circuits, delivery devices, and kill switches. These advances have paved the way for successful engineering of fully functional living therapeutics, with sensing, production, and biocontainment devices. However, some important tools are still missing from the box. In this review, we cover the most recent biological parts and approaches developed and describe the missing tools needed to build robust living therapeutics. a biological part or device that generates an output (e.g., motility, death, antibiotic production). a cell-surface component of bacteria that promotes adhesion to other cells or to surfaces. the Pseudomonas aeruginosa QS signal N-acyl homoserine lactone. inability of an organism to synthesize a compound needed for growth. peptides that can cross tissue and cell membranes via energy-dependent or -independent mechanisms. a host cell for engineering living therapeutics. a collection of all synthetic regulatory parts (e.g., promoter, RBS, protein tags, regulatory proteins and RNAs) that determines when and how many actuators will be produced by the cell. Control devices are directly connected to sensors. genome-editing tool that relies on Cas endonuclease activity guided toward the DNA substrate by an RNA sequence. repression of gene expression by the catalytically inactive endonuclease Cas9 (dCas9), which binds to the target DNA blocking transcription by RNA polymerase. dead Cas9; a catalytically inactive form of Cas9. in a biological part or device, exportation or release of molecules to the extracellular environment. in synthetic biology, a collection of biological parts that perform a higher-order function. complex microbial behaviors can be engineered by combining regulatory devices to create logic gates for integration of different inputs. enterohemorrhagic Escherichia coli adhesin. a promoter. genetic circuit that promotes cell death when triggered. an artificial genetic circuit that alters the system behavior on transient exposure to a signal. Unlike a regular control device, a memory device will continue to exhibit the altered behavior even when the signal input is over. immunoglobulin domains of high affinity, solubility, and stability based on the variable domain of heavy chain-only antibodies. context-free behavior (i.e., the property of biological parts and devices to work independently of the biological context). in synthetic biology, a sequence of DNA that encodes a biological function (e.g., promoter, RBS, gene). live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. cell-to-cell communication through small molecules. a protein and/or nucleic acid device able to recognize a signal (usually a small molecule) and induce a cellular response (e.g., gene expression). a bistable gene-regulatory network. transistor-like device that regulates the flow of RNA polymerase along DNA.