Research

Engineering environmental bacteria for utilization and valorization of lignocellulosic substrates

The non-eatable portion of plant biomass, lignocellulose (LC), is the most abundant organic matter on the Earth and represents a literally indefinite source of carbon and energy embedded in sugars and aromatic chemicals that form its three major fractions - cellulose, hemicellulose, and lignin. The efficient processing of LC waste for the bioproduction of diverse valuable chemicals (biofuels, biopolymers, or pharmaceuticals) is one of the major technological challenges of our time. Traditional microbial hosts that have been tested for this task are sensitive to the toxic effects of inhibitory products resulting from LC pre-treatment and cannot assimilate LC sugars simultaneously due to the genetically encoded mechanisms causing carbon catabolite repression. Alternative robust microorganisms are therefore sought.

Pseudomonas putida KT2440, the best-characterized safe pseudomonad, belongs to the most promising bacterial workhorses for synthetic biology and biotechnology endeavors. This soil bacterium typically thrives in polluted sites and is thus endowed with a number of traits desirable for harsh biotransformations. The metabolic versatility and nutritional specialization of P. putida KT2440 were recently proven useful for biotechnological processing of lignin-derived aromatics or cellulosic glucose towards the production of valuable chemicals such as polyhydroxyalkanoates (PHA, bacterial bioplastics), cis,cis-muconic acid, or rhamnolipids. Another attractive microbial host for lignocellulose biotechnology is the thermophilic environmental bacterium Caldimonas thermodepolymerans which possesses enzymatic complement for both synthesis and depolymerization of PHA. It can perform conversions of lignocellulosic sugars into PHA at a temperature of around 50°C and is thus a promising candidate for New Generation Biotechnologies that should be more efficient and resistant to contaminations. 

In MBL, we aim to study P. putida´s and C. depolymerans´ physiology and metabolism and engineer these bacteria to remove their remaining limitations. We want to prepare cell factories for efficient co-utilization and co-valorization of major lignocellulose-derived substrates (carbohydrates and aromatics) using state-of-the-art tools and approaches of synthetic biology, metabolic engineering, and protein engineering. This endeavor provides valuable insight into how the metabolism of the host cell manages carbon fluxes from native and non-native substrates. 

Responsible persons: Anastasiia Ieremenko, Martin Benešík, Barbora Popelářová, Barbora Burýšková, Kristýna Lipovská, Denis Pšenka, Barbora Gavendová, Hana Majerová, Miroslav Rosputinský, Ondřej Kavka

Designer catalytic scaffolds on bacterial surfaces

A major obstacle to the wider commercialization of lignocellulose-derived bioproducts is the high cost of degradation of raw polymeric material into simpler sugars which is typically achieved using expensive soluble cellulolytic enzymes. Cellulases and hemicellulases are derived from certain fungi and bacteria that are often difficult to engineer and handle at an industrial scale. Accordingly, methods to recombinantly express cellulolytic enzymes are under constant investigation. The construction of well-defined recombinant microbes that can degrade biomass and convert it efficiently into bioproducts without externally added enzymes is a holy grail of biotechnology and an essential step toward so-called consolidated bioprocessing.  

Natural cellulose degraders often incorporate cellulolytic enzymes into cell-surface attached cellulosomes through highly specific interactions of cohesin-dockerin domain pairs. Such clustered cellulases anchored to the substrate through carbohydrate-binding modules can degrade cellulose several fold more efficiently than free enzymes. Natural cellulosome producers are often difficult to genetically manipulate or cultivate and their cellulosomes are too large and complicated. Hence, synthetic designer cellulosomes have been assembled either in vitro using purified enzymes or by self-assembly in recombinant bioethanol-producing yeast. Concerning Gram-negative biotechnological workhorses, display and excretion of only single free enzymes have been achieved for proof-of-concept biofuel production from plant biomass in engineered Escherichia coli, thus far.

Pseudomonas putida KT2440 is currently gaining considerable interest as a promising microbial platform for the biotechnological valorization of lignocellulosic feedstocks. However, P. putida on its own cannot make much use of such complex substrates mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to meet this challenge by adopting a recombinant CATalytic scaFFOLDS (CATFFOLDS) strategy mimicking natural cellulosomes for this attractive host. The whole process encompasses three essential steps: (i) display of designer protein scaffolds with cohesins on P. putida surface, (ii) parallel release of cellulolytic enzymes with dockerins into the medium, and (iii) self-assembly of the CATFFOLDS through bonding of scaffoldin-born cohesins and enzyme-born dockerins. The resulting P. putida biocatalysts will be capable of efficient degradation and valorization of lignocellulosic feedstocks and the established platform can be adopted and modified also for processing of other types of polymeric or toxic waste (e.g., synthetic plastics, anthropogenic toxic chemicals).

Responsible persons: Barbora Jankovičová, Miguel Silva, Matúš Pešta, Pavel Dvořák

Synthetic bacterial consortia for efficient biocatalysis

In the last decades, new tools and approaches of metabolic engineering, systems and synthetic biology allowed unprecedented progress in designing microbial hosts for the production of bulk and fine chemicals or biodegradation of anthropogenic pollutants. Many fundamental and technical problems have been solved but new questions have arisen during the attempts to tame the cells for directed bioprocesses. One of the most prominent recent disputes in microbial biotechnology is whether a plethora of desired functions can be reliably performed by a single engineered cell, let’s call it „an individualist“, or whether it is better to distribute the burden and tasks among the members of the microbial community in which specialist strains work together in co-operation. 

Microbial individualists or “superbugs” have been prepared in laboratories since the emergence of genetic engineering tools in the 1970’s. Synthetic microbial consortia have drawn the attention of the researchers more recently. Natural microbial communities that take part in complicated biocatalytic tasks such as lignocellulose degradation, wastewater purification, or intestinal food digestion are being intensively studied and works demonstrating the potential of engineered consortia for synthesis of valuable products or decontamination of polluted sites already exist. Examples of direct functional comparison and competition of the two above-mentioned paradigms are nonetheless scarce. We want to fill in this knowledge gap by comparing a single recombinant Pseudomonas putida individualist with a P. putida-based synthetic consortia used for desirable biotechnological tasks. 

Responsible persons: Barbora Burýšková, Miguel Silva, Matúš Pešta