Conjugated Protein Domains as Engineered Scaffold Proteins

Lemmens, Lenne J M; Ottmann, C.; Brunsveld, Luc

doi:10.1021/acs.bioconjchem.0c00183

Cited by 12 publications

(11 citation statements)

References 68 publications

(123 reference statements)

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“…This kind of phenomenon has inspired researchers to artificially combine biocatalysts together. By adding multiple enzymes in the same reaction vessel, a multi-step enzymatic reaction could be performed in vitro [ 20 , 21 , 22 , 23 ]. Compared with the method of using the metabolic pathways in the microbial host, the reaction conditions are easy to control.…”

Section: Discussionmentioning

confidence: 99%

“…In order to improve the multi-enzyme reaction rates and reaction efficiency, scaffold proteins are designed to combine multienzymes to produce functional metabolites through the substrate channels [ 23 , 26 , 27 , 28 , 29 ]. Scaffold proteins are not randomly aggregated but are usually used to organize multiple binding partners to promote their interaction and function [ 26 , 27 , 28 , 29 ].…”

Section: Discussionmentioning

confidence: 99%

“…Using similar multidomain scaffolds, researchers synthesized glucaric acid, resveratrol, butyrate, and L-serine. This technology showed great potential in the biosynthesis industry [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

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Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

Yang

et al. 2021

Biomolecules

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Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

Yang

et al. 2021

Biomolecules

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“…Aside from avoiding stress, such mechanisms are also ideal for avoiding unwanted intrinsic down-regulation of biosynthetic networks by key intermediates, which often occurs in biosynthetic pathways and impairs product yield. Another emerging strategy to achieve a higher and more balanced flux is to spatially organise enzymes within synthetic scaffolds [164,165] to create high local concentrations of metabolites and enzymes, with well-defined stoichiometry to support immediate conversion without diffusion of intermediates. All these strategies (Figure 3) together with an increasing understanding of tolerance traits will be instrumental for effective production applications and expanding the catalytic landscape of Pseudomonas towards novel chemistries [12,166].…”

Section: Engineering Of Optimal Flux In Cell Factories With Novel Pathwaysmentioning

confidence: 99%

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Bitzenhofer

Kruse

Thies

et al. 2021

Essays in Biochemistry

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Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

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“…The functional protein subunits are attached to the scaffold, whereby their proximity and alignment results in emergent properties of the whole assembly. The alignment of functional domains on protein nanostructures includes enzymes for multistep biocatalysis pathways (1–3), cell signaling domains for tissue engineering (4), and metalloproteins on filaments to produce electronically conductive nanowires (5), However, the fabrication of multicomponent protein complexes is laborious, as the different components must be recombinantly expressed and purified separately before assembly of the functional nanostructure can occur (6).…”

Section: Introductionmentioning

confidence: 99%

Single-step purification of functionalized protein nanostructures using multimodal chromatography

Winter

Lebhar

McCluskey

et al. 2022

Preprint

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Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Here, we developed an efficient approach to purify functionalized protein filament assemblies directly from bacterial lysates in a single step through a type of multimodal chromatography that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. In this approach, the ultrastable filamentous protein gamma-prefoldin was employed as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was optimized across a wide range of salt concentrations and pH. Subsequently, functionalized protein assemblies were purified from bacterial lysates using MMC in a single step and shown to befree of unincorporated subunits. The assembly and purification of protein nanostructures with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.

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Conjugated Protein Domains as Engineered Scaffold Proteins

Cited by 12 publications

References 68 publications

Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Single-step purification of functionalized protein nanostructures using multimodal chromatography

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