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CellulosomePlus Report Summary

Project ID: 604530
Funded under: FP7-NMP
Country: Spain

Periodic Report Summary 2 - CELLULOSOMEPLUS (Boosting Lignocellulose Biomass Deconstruction with Designer Cellulosomes for Industrial Applications)

Project Context and Objectives:
A reliable supply of sustainable energy is critical for the healthy, wealthy and peaceful future of our planet. Energy is the world’s largest market, with a political and strategic impact that is unmatched by any other sector. Most countries, including European nations, are currently highly dependent on the finite and non-renewable resources of fossil fuels for their energy needs. This allows countries rich in such resources to become major players in world politics, frequently at the expense of countries that lack them.
Biofuels constitute a major alternative to face this problem. For their production, among all the catalysts, nanocatalysts are very attractive ones as they greatly increase the surface-to-volume ratio compared to bulk materials. Recent advances in nanocatalysis have prompted a persistent shift in the economic and political balance of the fossil fuels market. Furthermore, there are alternative energy sources (some of them renewable and sustainable) that remain to be exploited. One of them is fiber, the non-edible plant cell wall cellulosic biomass, which is the source for so-called second generation biofuels.
The major source of carbon and energy in the biosphere is fiber, the plant cell wall formed by polysaccharides primarily composed of cellulose and hemicellulose. These two compounds are the first and second most abundant organic molecules on Earth, respectively, and offer a renewable and seemingly inexhaustible feedstock not only for the production of biofuels but also for a variety of fine chemicals. Most cellulose and hemicellulose is found in plant fiber, specifically in the primary cell wall of plants. The secondary cell wall (produced after the cell has stopped growing) also contains polysaccharides and is strengthened by polymeric (non-polysaccharide) lignin. The major bottleneck for plant biomass processing is fibre saccharification: the conversion of cell wall lignocellulosic biomass into fermentable sugars (in route to production of value-added chemicals like second generation biofuels). Some microbes enhance this step by using natural self-assembling proteinaceous nanocatalists known as cellulosomes.
CellulosomePlus targets rational design of optimized cellulosomes (Designer Cellulosomes, DCs) to overcome this problem. This would allow efficient production of biofuels from low-value raw materials like inedible parts of plants and industrial residues (which are all renewable, sustainable and inexpensive). The CellulosomePlus consortium will achieve this final aim through the following main objectives:
1. Characterization of natural cellulosomes and the selected substrate. CellulosomePlus will produce the basic components of natural cellulosomes as well as other lignocellulosic enzymes and characterize the hydrolysis (by the cellulosome, its components and by the DCs to be developed in this project) of the substrate of interest: organic fraction of municipal solid waste, OFMSW.
2. Modelling the cellulosome for in silico knowledge integration. Multi-scale modelling (from atomic to supramolecular levels) will provide crucial support for the synthesis, self-assembly and characterization tasks, supplying detailed structural and energetic information that will aid in the design and interpretation of the experiments.
3. The integration of the acquired knowledge from these two strategies will lead to the Rational design and mass production of DCs that will provide a platform to test the goal of constructing final DCs (carrying both cellulosomal and noncellulosomal components) optimized for the degradation of the selected industrial substrate, and validated at the laboratory-scale.

Project Results:
During this second project period, most of the planned activities were successfully carried out as follows:
We have provided deeper insights into the mechanical properties of cohesins, complementing previous results.
Novel enzymes, cohesin and dockerin components were discovered by bioinformatics approaches. Databases were constructed, and a large variety of selected components were used for the development of novel DCs. The performance of the resultant DCs has been consistently improved. Analytical tools for evaluation of the DCs and their component parts were devised.
Thermostable designer cellulosomes (DCs) exhibited enhanced cellulose degradation. Compared to conventional DCs, the use of a thermostabilized scaffoldin proved critical under conditions of high temperatures.
In order to test a larger number of cohesin-dockerin interactions with force assays protocols were developed to obtain reliable and high-yield single-molecule force measurements. Furthermore, new automated measurement and data analysis software, provides us with an unparalleled statistical depth of our data sets. For assembly and hydrolysis quantification, a new assay using fluorescent hydrogels was developed.
A complete trivalent DC structure, containing ~5 million atoms, was generated using all atom model. The multimodular Cel9R enzyme, found in the trivalent DC, was studied in detail to understand how it approaches different cellulose defects. CBM and expansin binding specificity to different types of cellulosic material at the atomistic level were studied. Results provide a comprehensive overview of candidate residues for site-directed mutagenesis.
We have undertaken crystallographic 3D structure determination and small angle X-ray scattering of ‘nanosomes’ of various compositions. Data will be used for performing dynamic molecular modeling. The challenge of obtaining an experimental SAXS curve for a truncated CipA scaffoldin consisting of 9 cohesins in complex with 9 Cel8A enzymes has been achieved, and data is being analysed.
Models of 2 multimodular cellulosomal enzymes have been developed and characterized. We have used all-atom simulations to derive effective parameters for a coarse-grained description of the crystalline cellulose Iα. We have combined experimental and theoretical methods to assess the effect of a set of single-site mutations on the highly mechanostable cohesin c7A. We have re-examined mechanostability of the cohesin-dockerin complexes computationally.
A detailed low throughput assay protocol was developed for the characterization of cellulosomal and non-cellulosomal fractions of cellulosome-producing bacteria. This can be used to distinguish between endo- or exo-acting cellulases, in a simple and precise manner. Moreover, an additional protocol is provided for the detection of xylanases derived from cellulosome-producing bacteria.
A low throughput assay was developed to analyze the enzymatic activity on industrial substrate provided by. The results indicate that the cellulosome hydrolyzes the lignocellulosic biomass in a more efficient manner than a single purified enzyme or a mixture of enzymes.
A robust assay for the measurement of binding solid, recalcitrant cellulosic biomass with soluble proteins was developed based on titration of enzyme on a specific amount of cellulose, which provides qualitative binding information.
To establish a medium-to-high throughput assay, the previously developed assay for analysis of cellulolytic activities using insoluble cellulose and lignocellulosic biomass was adapted to be compatible with liquid handling robotics. The production of eight cellulosomal proteins was scaled-up in a laboratory scale bioreactor and some were already purified and validated.
Abengoa has been working on optimization of pre-treatment, pre-industrial scaling-up and pretreated biowaste conditioning. Analysis of the yield of each process has been carried out.
Most of scientific articles describing the results obtained were published in high-ranking journals. Our methodology and results have merited an invitation to publish a 2016 review on our approach, with all the CellulosomePlus partners as co-authors, in Advanced Materials a very prestigious, high-impact factor, journal in the field (28:5619-47. Nanoscale Engineering of Designer Cellulosomes. IF:18.96).
Potential Impact:
At the end of this reporting period the Abengoa (ABNT, partner 8) has terminated his participation in the CellulosomePlus consortium due to financial problems, which ended in a internal re-structuring of the company. Still, they have been able to provide the substrate samples to the partners and the delay experienced has not affected significantly the global activity of the consortium. Its remaining activities have been fully transferred to the partner Biopolis (BIO, partner 9), which is one of the world-leading groups in the field of cellulose active enzymes, fully equipped and experienced to perform these activities. However, they will need extra time to accommodate the new activities in their agenda. As a result, the consortium requests an extension of 12 months (filed within a 3rd Amendment of the Grant Agreement) in order to be able to fully cover our objectives and assure the expected impact of this ambitious project. We have already the first prototypes of these novel enzymatic complexes, and when successfully validated at a larger scale, will be commercially attractive biocatalysts. It is also expected that in this last period of the project, industrial property rights are accomplished through patent application among others when all technological information has been gathered.
If this extension is granted we expect to deliver the proposed pre-industrial results, which should impact industry and society in turn. We expect that the use of self-assembled DCs as bio-inspired nanocatalysts produced by our consortium will reduce significantly both the cost of the saccharification step and the environmental impact of the whole process. These DCs should be marketable by European biotechnology industries working in the transport-related sector in addition to other chemical industries to process urban waste and residues from the agro-food, paper, and forestry based industries. This should reduce Europe’s reliance on oil, strengthen SMEs from the EU, stimulate job creation and reduce the environmental impact of the second generation biofuel sector.
From our recent activities in the field, BIOPOLIS, an active member of the European BioBased Industry Consortium (full member since 2015), estimates a return on investment of 100k/year to benefit all contributing partners, if any of these enzymatic complexes is successfully launched to the market, for research/laboratory scale applications. In case any of the products reach full market scale for bulk applications, such as enzymatic hydrolysis of lignocellulosic materials—sugar platform, or improved fermentation through consolidated bioprocessing, a 0.1% share of the global biofuel enzymes market is estimated as 300k/year (>USD 1.500 million, >USD 300 million for cellulases in 2016, growing at >7% CAGR).
The major expected impact of CellulosomePlus is that the rational design of DCs will enable fast industrialization of materials of very high activity and selectivity, and minimum energy use in their preparation and during the work cycle. DCs have the potential of improving the performance of existing industrial processes including energy production and to lead to exploitation of renewable, efficient and inexpensive sources for biotechnological processes.
The expected success of CellulosomePlus will significantly advance European cutting edge research in bio-molecular science and technology. The impact of CellulosomePlus is indeed envisaged as to be long-term not only by providing new tools for scientists, but also by making possible the rational design of DCs based on their true bio-molecular mechanisms.
Furthermore, the introduction of new nanotechnology into established industry will promote transformation of the industry to high value-added production. The education level of the work force is expected to increase significantly and together with more challenging tasks in high-technology industry will increase employee job satisfaction.
Finally, regarding the environmental impact, CellulosomePlus will certainly have positive environmental impact both in terms of new and precise methods for analyses and novel biological catalysts. The analysis of biological nanosystems by experiment and modelling should allow unprecedented insight into the true biomolecular mechanism. It therefore provides new means to control and analyse biological (e.g. enzymatic) processes. In addition, the CellulosomePlus systems that spatiotemporally control cascade reactions will greatly increase the selectivity due to the nanoscale dimensions as well as the efficiency of reactions due to the reduction in the entropy of the system. This will also result in new and more efficient industrial enzymes. The CellulosomePlus technology platform will make it possible for the industry to use less raw materials, produce less waste, and apply a more environmentally friendly production technology.
Summarizing, the CellulosomePlus Project will take the promising technologies in development from the partners involved in this consortium formed by a research-industry complementation in order to provide an increase in the Technological Readiness Level toward a pre-commercial development. It will enable the European industries to better bridge the 'innovation gap' and the "valley of death" between technology development and commercialisation. Both very pertinently apply to the field of biorefineries which require significant investments, typically beyond the financial reach of individual private companies. Public intervention is required to foster industry leadership and to promote long-term industry commitment in research and innovation related to bio-based industries.

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