10 research outputs found
Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase
Abstract Background In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3′-sialyllactose (3SL). Results Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5′-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. Conclusions Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production
Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency
3535 Hayden Avenue, Culver City CA 90232exterio
Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, <i>In-Situ</i> AFM Characterization of Enzymatic Cellulose Degradation
In the field of enzymatic cellulose
degradation, fundamental interactions between different enzymes and
polymorphic cellulose materials are of essential importance but still
not understood in full detail. One technology with the potential of
direct visualization of such bioprocesses is atomic force microscopy
(AFM) due to its capability of real-time <i>in situ</i> investigations
with spatial resolutions down to the molecular scale. To exploit the
full capabilities of this technology and unravel fundamental enzyme–cellulose
bioprocesses, appropriate cellulose substrates are decisive. In this
study, we introduce a semicrystalline-thin-film-cellulose (SCFTC)
substrate which fulfills the strong demands on such ideal cellulose
substrates by means of (1) tunable polymorphism via variable contents
of homogeneously sized cellulose nanocrystals embedded in an amorphous
cellulose matrix; (2) nanoflat surface topology for high-resolution
and high-speed AFM; and (3) fast, simple, and reproducible fabrication.
The study starts with a detailed description of SCTFC preparation
protocols including an in-depth material characterization. In the
second part, we demonstrate the suitability of SCTFC substrates for
enzymatic degradation studies by combined, individual, and sequential
exposure to TrCel6A/TrCel7A cellulases (<i>Trichoderma reesei</i>) to visualize synergistic effects down to the nanoscale
Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, <i>In-Situ</i> AFM Characterization of Enzymatic Cellulose Degradation
In the field of enzymatic cellulose
degradation, fundamental interactions between different enzymes and
polymorphic cellulose materials are of essential importance but still
not understood in full detail. One technology with the potential of
direct visualization of such bioprocesses is atomic force microscopy
(AFM) due to its capability of real-time <i>in situ</i> investigations
with spatial resolutions down to the molecular scale. To exploit the
full capabilities of this technology and unravel fundamental enzyme–cellulose
bioprocesses, appropriate cellulose substrates are decisive. In this
study, we introduce a semicrystalline-thin-film-cellulose (SCFTC)
substrate which fulfills the strong demands on such ideal cellulose
substrates by means of (1) tunable polymorphism via variable contents
of homogeneously sized cellulose nanocrystals embedded in an amorphous
cellulose matrix; (2) nanoflat surface topology for high-resolution
and high-speed AFM; and (3) fast, simple, and reproducible fabrication.
The study starts with a detailed description of SCTFC preparation
protocols including an in-depth material characterization. In the
second part, we demonstrate the suitability of SCTFC substrates for
enzymatic degradation studies by combined, individual, and sequential
exposure to TrCel6A/TrCel7A cellulases (<i>Trichoderma reesei</i>) to visualize synergistic effects down to the nanoscale