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Re-factoring Pseudomonas putida for biosynthesis of vaIue-added polymers from cellulosic waste

Periodic Reporting for period 1 - FUTURE (Re-factoring Pseudomonas putida for biosynthesis of vaIue-added polymers from cellulosic waste)

Reporting period: 2016-10-01 to 2018-09-30

Lignocellulose (plant biomass made of three components - cellulose, hemicellulose, and lignin) is the most abundant organic matter on Earth and important constituent of agricultural and industrial wastes. Over 600 million tonnes of lignocellulosic and cellulosic wastes - crop residues, wood waste, paper, or food waste - are generated only in EU every year. Cellulosic sugars (such as cellulose-derived glucose or hemicellulose-derived xylose) and lignin-derived aromatic compounds can serve as a cheap substrates for clean biotechnological production of numerous value-added chemicals (VAC) that are currently being produced from oil. However, a well-defined microbial platforms that could efficiently utilize lignocellulose for biosynthesis of VAC in a single step are still missing. Pseudomonas putida KT2440, safe and robust soil bacterium with versatile metabolism, has wide potential to utilize lignocellulose-derived substrates for VAC formation but cannot de-polymerize (hemi)cellulose to monomeric sugars. This challenge could be solved by expanding the biocatalytic functions of P. putida using cellulosomes, efficient enzymatic nanomachines displayed on the surface of certain cellulolytic microorganisms. Cellulosic enzymes clustered in cellulosomes can degrade cellulose up to 50x more efficiently than free enzymes.

The major goal of the project is to construct P. putida strains with improved metabolism of glucose, displaying designer cellulosomes with cellulolytic enzymes and forming valuable biopolymers (polyhydroxyalkanoates, PHA) from cellulosic glucose (Figure 1). This project introduces P. putida as a new platform for lignocellulose biotechnology, and contributes to both understanding of fundamentals of model biological systems and corporate effort aimed at establishing FUTURE knowledge-based bio-economy in Europe.
The three FUTURE project Objectives and the progress achieved during the two-year period are as follows:

[1] Optimisation of glucose metabolism in available P. putida chassis:
During the first part of the FUTURE project, the Experienced Reseracher (ER) focused on solving the problem of non-optimal glucose utilisation by P. putida. The energetics of sugar assimilation was improved and the glycolytic flux streamlined by teaching P. putida how to utilise cellobiose (dimer of glucose, the shortest cellooligosaccharide) instead of glucose. Enzyme from cellulolytic bacterium Thermobifida fusca allowed P. putida to grow rapidly on cellobiose and use it as as a sole carbon source. The outcomes of the first project part were published in a renowned journal (Dvořák et de Lorenzo, Metab Eng. 2018 Jul;48:94-108.) and used in a patent application.

[2] Engineering surface display and excretion of designer cellulosome components in P. putida chassis:
In the second part of the project, so called scaffoldin with protein domains cohesins that can bind corresponding dockerins at cellulolytic enzymes had to be displayed on the cell surface. ER developed rapid screening system, based on dockerin-tagged beta-glucosidase and fluorescent protein, for scaffoldin display monitoring and quantification in P. putida cells. Scaffoldins containing one or two cohesins were prepared by genetic engineering and exposed on P. putida surface with very good efficiency using the display system borrowed from bacterium Escherichia coli. Dockerin-tagged cellulases, selected based on their compatibility with P. putida host, are currently being combined with cohesin displaying cells to allow degradation of cellulosic substrates to glucose. A publication summarizing the aforementioned results is in preparation.

[3] Probing PHA biosynthesis from cellulosic waste in re-factored P. putida:
Synthesis of PHA biopolymer in P. putida was previously demonstrated from fatty acids and unrelated substrates such as glucose, but never from cellooligosaccharides. Within the scope of project FUTURE, ER showed for the first time, that PHA can be produced in engineered P. putida also from biotechnologically relevant cellobiose which forms significant part of cellulosic hydrolysates. The data are included in publication in preparation.

To conclude, metabolism of P. putida was successfully engineered toward more efficient sugar utilisation. Synthetic scaffoldin with two cohesins was successfully displayed on P. putida surface and is being used for attachment of cellulases. The project has already given rise to two highly impacted publications (IF 10 and 8) and one patent application. Two more publications are in preparation. Project concept and results were presented at four renowned international scientific conferences, two student/postdoc scientific meetings, EMBO training course, one seminar for public, two university lectures, and one newspaper interview. Project was also discussed with numerous individuals visiting Receiving Laboratory, including some of the most influential personalities in the fields of synthetic biology and metabolic engineering. News on the project and its outcomes were regularly spread through the social media (Facebook, Twitter). We estimate that the project reached thousands of persons from academia, industry, and general public.
Project FUTURE has introduced P. putida as a new promising platform for plug-in of biochemical pathways for utilization and valorization of cellulosic and hemicellulosic sugars. Our data published in Metabolic Engineering journal show that engineered P. putida EM42 can co-utilise glucose or cellobiose with xylose with no sign of carbon catabolite repression - a phenomenon which prevents co-utilisation of sugar mixtures and more cost effective lignocellulose processing by traditional microbial workhorses such as E. coli or Saccharomyces cerevisiae. P. putida EM42 expressing beta-glucosidase intracellularly and capable of growth on cellobiose saves one of the three enzymes needed for cellulose depolymerization and thus represents important step toward consolidated bioprocessing of cellulosic materials with this bacterium. Remaining two enzymes - exo- and endoglucanase - can be now attached to the P. putida surface with a help of the synthetic two-cohesin scaffoldin whose display on the cells was achieved. This is the first reported example of designer scaffoldin displayed on the surface of Gram negative bacterium.

Metabolic engineering and systems biology approaches are now being employed to stream carbon from new (hemic)cellulosic substrates toward PHA, other valuable chemicals whose production was reported in P. putida, such as rhamnolipids, terpenoids, coronatines etc. can be targeted in future. Given the promising outcomes of the FUTURE project and the recent global progress in P. putida engineering for valorization of lignin, we can envision that this joint effort will lead to unique recombinant bacterial workhorse capable of simultaneous biotechnological processing of and adding value to all three lignocellulose-derived fractions. Considering the overall impact of the project, FUTURE not only opened a new career opportunity for ER, who will start his own laboratory back in the Czech Republic in 2019, but also drew more attention to the rather non-canonical host, P. putida, and its use in lignocellulose biotechnology where it can provide unorthodox solutions for the stagnating field.
Figure 1. Original schematic representation of FUTURE concept.
Scheme 1. Overview of the actions.