Browsing by Author "Valiante, Vito"
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- ItemCell-free protein synthesis and in situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion(Cambridge : RSC, 2020) Köhler, Tony; Heida, Thomas; Hoefgen, Sandra; Weigel, Niclas; Valiante, Vito; Thiele, JulianIn the present work, microgels were utilized as a cell-free reaction environment to produce a functional malonyl-CoA synthetase (deGFP-MatB) under geometry-controlled transcription and translation. Our approach combines the straight-forward optimization of overall protein yield of an E. coli-based cell-free protein synthesis (CFPS) system based on concentration screening of magnesium and potassium glutamate, DNA as well as polyethylene glycol (PEG), and its innovative usage in microgel-based production of a key enzyme of the polyketide synthesis pathway. After partial modification of the carboxyl groups of hyaluronic acid (HA) with 5′-methylfuran groups via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM)-activation, these were further functionalized with dibenzocyclooctyne (DBCO) and nitrilotriacetic acid (NTA) groups by bio-orthogonal [4+2] Diels-Alder cycloaddition to yield a bifunctional macromer. After coupling the DBCO groups with azide-functionalized DNA, containing the genetic information for deGFP-MatB, via strain-promoted azide-alkyne cycloaddition (SPAAC), the DNA-/NTA-functionalized HA macromer was utilized as base material together with maleimide-functionalized PEG (PEG-mal2) as the crosslinker to form bifunctional microgels utilizing water-in-oil (W/O) microemulsions. As-formed microgels were incubated with nickel sulfate to activate the NTA groups and provide binding sites for deGFP-MatB, which contained six histidine residues (His-tag) for that purpose. The optimized CFPS mixture was loaded into the microgels to initiate the formation of deGFP-MatB, which was detected by a clear increase in fluorescence exclusively inside the microgel volume. Functionality of both, the bound and the decoupled enzyme was proven by reaction with malonate to yield malonyl CoA, as confirmed by a colorimetric assay. © 2020 The Royal Society of Chemistry.
- ItemEngineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid(Lausanne : Frontiers Media, 2023) Riedel, Florian; Bartolomé, Maria Puertas; Enrico, Lara Luana Teruel; Fink-Straube, Claudia; Duong, Cao Nguyen; Gherlone, Fabio; Huang, Ying; Valiante, Vito; Del Campo, Aránzazu; Sankaran, ShrikrishnanMicrobial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.