Cell-surface display of designer cellulosomes by Lactobacillus plantarum

Ben-David, Yonit; Moraïs, Sarah; Stern, Johanna; Mizrahi, Itzhak; Bayer, Edward A.

doi:10.1016/bs.mie.2018.12.011

Cited by 6 publications

(6 citation statements)

References 36 publications

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“…Then, each signal peptide was ligated into the cut plasmid pLp_3050sGH or by the restriction‐free method (Unger et al , 2010). Since the pSIP vectors are suitable for both L. plantarum and E. coli , all cloning steps were performed using E. coli XL1 competent cells, which were grown in LB (Luria‐Bertani) medium supplemented with 200 µg ml −1 erythromycin (Ben‐David et al , 2019). The primers that were used in this study are listed in Table S2.…”

Section: Methodsmentioning

confidence: 99%

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level

Ben-David

Moraïs

Bayer

et al. 2020

Microbial Biotechnology

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The multi-enzyme cellulosome complex can mediate the valorization of lignocellulosic biomass into soluble sugars that can serve in the production of biofuels and valuable products. A potent bacterial chassis for the production of active cellulosomes displayed on the cell surface is the bacterium Lactobacillus plantarum, a lactic acid bacterium used in many applications. Here, we developed a methodological pipeline to produce improved designer cellulosomes, using a cell-consortium approach, whereby the different components self-assemble on the surface of L. plantarum. The pipeline served as a vehicle to select and optimize the secretion efficiency of potent designer cellulosome enzyme components, to screen for the most efficient enzymatic combinations and to assess attempts to grow the engineered bacterial cells on wheat straw as a sole carbon source. Using this strategy, we were able to improve the secretion efficiency of the selected enzymes and to secrete a fully functional high-molecular-weight scaffoldin component. The adaptive laboratory process served to increase significantly the enzymatic activity of the most efficient cell consortium. Internal plasmid rearrangement towards a higher enzymatic performance attested for the suitability of the approach, which suggests that this strategy represents an efficient way for microbes to adapt to changing conditions.

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Section: Methodsmentioning

confidence: 99%

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level

Ben-David

Moraïs

Bayer

et al. 2020

Microbial Biotechnology

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“…Since the cohesin-dockerin interaction is species-specific, this enables the controlled incorporation of the docking enzymes or nano-engineering of the multi-enzyme complexes. Designer cellulosomes have the potential to be used in biotechnological processes, such as the production of fermentable sugars from lignocellulosic biomass (Ben-David et al 2019;Hyeon et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode

Vanderstraeten

Lamote

Fonseca

et al. 2022

Appl Microbiol Biotechnol

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Cellulosomes are multi-enzyme complexes produced by specialised micro-organisms. The spatial proximity of synergistically acting enzymes incorporated in these naturally occurring complexes supports the efficient hydrolysis of lignocellulosic biomass. Several functional designer cellulosomes, incorporating naturally non-cellulosomal cellulases, have been constructed and can be used for cellulose saccharification. However, in lignocellulosic biomass, cellulose is tightly intertwined with several hemicelluloses and lignin. One of the most abundant hemicelluloses interacting with cellulose microfibrils is xyloglucan, and degradation of these polymers is crucial for complete saccharification. Yet, designer cellulosome studies focusing on the incorporation of hemicellulases have been limited. Here, we report the conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode. Therefore, we constructed multiple docking enzyme variants of C. japonicus endoxyloglucanase, β-1,2-galactosidase, α-1,6 xylosidase and β-1,4-glucosidase, using the combinatorial VersaTile technique dedicated to the design and optimisation of modular proteins. We individually optimised the docking enzymes to degrade the xyloglucan backbone and side chains. Finally, we show that a purified designer xyloglucanosome comprising these docking enzymes was able to release xyloglucan oligosaccharides, galactose, xylose and glucose from tamarind xyloglucan. Key points • Construction of xyloglucan-degrading designer cellulosome.• Conversion of free Cellvibrio japonicus enzymes to cellulosomal mode.• Type of linker inserted between dockerin and enzyme module affects docking enzyme activity.

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“…8,9 Artificial cellulosomes (also termed designer cellulosomes in literature) are a class of synthetic biocatalysts developed to mimic and improve the understanding of the mechanisms related to the observed high catalytic efficiency of natural cellulosomes. 10 Ongoing studies aim to modify their thermal stability and composition to target specific biomass feedstocks and to scale up synthesis for commercialization. 11 Artificial cellulosomes can be divided into two distinct groups: (i) cellulosomes made using biological mechanisms to synthesize recombinant proteins and (ii) biomimetic synthetic structures incorporating chemically synthesized building blocks.…”

Section: Introductionmentioning

confidence: 99%

“…Artificial cellulosomes (also termed designer cellulosomes in literature) are a class of synthetic biocatalysts developed to mimic and improve the understanding of the mechanisms related to the observed high catalytic efficiency of natural cellulosomes . Ongoing studies aim to modify their thermal stability and composition to target specific biomass feedstocks and to scale up synthesis for commercialization .…”

Section: Introductionmentioning

confidence: 99%

Biomimetic Cellulosomes Assembled on Molecular Brush Scaffolds: Random Complexes vs Enzyme Mixtures

Жолобко

Hammed

Zakharchenko

et al. 2021

ACS Appl. Polym. Mater.

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This work reports on the development of biomimetic polymeric cellulosomesenzyme–polymer conjugates (EPCs) generated by covalent binding of cellulase enzymes onto a molecular brush polymer scaffold synthesized by radical copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and glycidyl methacrylate (GMA). In contrast to natural cellulosomes, the EPC is generated in a random process of the conjugation of different cellulases from their mixture in the solution. The catalytic activity of the EPCs is systematically studied using carboxymethyl cellulose, microcrystalline cellulose, filter paper, and pretreated switchgrass biomass substrates. EPCs exhibit improved catalytic activity with both soluble and insoluble substrates. The synergistic effect is essential, with multifold improvement in substrate surface coverage by the enzyme being less than 50% of the saturation amount. Coarse-grained molecular dynamics simulations provide evidence for the increased concentration of EPC-delivered synergistic motifs of enzymes on the substrate as compared to the number of such motifs randomly formed by adsorption of enzymes from a mixture of free enzymes, which is in good agreement with the experiments. The catalytic activity of the EPC conjugates increases with the increased diversity of enzymes included in the EPC structure and also in the presence of free enzymes in solutions. The experiments reveal that EPCs reduce the inhibitory effect of the depolymerization product (glucose), which could facilitate higher hydrolysis rates at increased solid loadings. The experimental results demonstrate that at a random arrangement of the enzymes in the complex, the synergistic effect is substantial and comparable to that of natural cellulosomes at a low enzyme-to-substrate surface ratio due to the combination of complementary catalytic functions bound to the same EPC in close vicinity.

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Cell-surface display of designer cellulosomes by Lactobacillus plantarum

Cited by 6 publications

References 36 publications

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode

Biomimetic Cellulosomes Assembled on Molecular Brush Scaffolds: Random Complexes vs Enzyme Mixtures

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