Developing the cellulolytic bacteria Clostridium phytofermentans and Clostridium cellulolyticum as catalysts to convert plant biomass into higher alcohols – Phytocell

Phytocell is organized into 4 connected research projects:

*Project 1 Expression of synthetic mini-cellulosomes composed of selected cellulases derived from C. cellulolyticum in C. phytofermentans to improve its capacity to degrade and grow on recalcitrant cellulose.

*Project 2 Implementation of a synthetic butanol synthesis pathway in C. phytofermentans and C. cellulolyticum. Phases include pathway construction, implementation in WT strains, implementation in evolved strains, and CBP optimization by removal of alternative fermentation products.

*Project 3 Improvement of butanol resistance using a GM3 automat. It includes directed evolution of C. phytofermentans and C. cellulolyticum and genome sequencing/phenotyping of evolved strains.

*Project 4 is the conception of foundational genetic tools to enhance these clostridia as CBP organisms. Phases include plasmid construction, methods for genomic insertion and deletion in both strains, genomic insertion of mini-cellulosomes in C. phytofermentans, and deletion of alternative fermentation pathways from the final CBP candidate.

*We have implemented a complete butanol synthesis pathway in C. cellulolyticum and C. phytofermentans. The pathway is functional in C. cellulolytcium, but produced only weak amounts of butanol, and requires further improvements to increase this yield, including for evolved butanol-tolerant strains.

*Both bacteria were evolved to grow in 2.5% (v/v) butanol in the growth medium, which exceeds the production potential of any known microorganism.

*We developed a series of genetic tools to facilitate the modification of the genomes of both bacteria.

In this project, we have made several critical advances for development of microbial biocatalysts for the transformation of biomass into butanol.
We have successfully introduced a complete butanol pathway in cellulolytic clostridia. The improvement of the butanol yield is now possible thanks to the genetic tools developed during the project, tools which can also be used for the production of other compounds of interest by these clostridia.
The directed evolution protocol we have developed can be used to adapt both bacteria to other toxic compounds.

Cerisy T., Iglesias A., Rostain W., Boutard M., Pelle C., Perret A., Salanoubat M., Fierobe H.-P., Tolonen A. C. (2019) ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans. J. Bacteriol. 201:e00241-19

Kampik C., Liu N., Mroueh M., Franche N., Borne R., Denis Y., Gagnot S., Tardif C., Pagès S., Perret S., Vita N., de Philip P., Fierobe H.-P. (2021) Handling several sugars at a time: the case study of xyloglucan utilization by Ruminiclostridium cellulolyticum. in revision at mBio. (in revision)

Rostain W, Boutard M, Tabuteau S, Sanitha M,Tolonen AC. Tuning of gene expression in Clostridium phytofermentans using synthetic promoters and CRISPRi. 2021. (Submitted).

Cerisy T, Rostain W, Chhun A, Boutard M, Salanoubat M, Tolonen AC. A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia. mSphere. 2019 Dec 11;4(6):e00710-19. doi: 10.1128/mSphere.00710-19.

Cerisy T, Souterre T, Torres-Romero I, Boutard M, Dubois I, Patrouix J, Labadie K, Berrabah W, Salanoubat M, Doring V, Tolonen AC. Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics. Appl Environ Microbiol . 2017 May 17;83(11):e00289-17. doi: 10.1128/AEM.00289-17.

We propose a four-year project to develop Clostridium phytofermentans and Clostridium cellulolyticum as microbial platforms for the conversion of plant biomass into high-order alcohols. These two bacterial taxa naturally degrade and ferment cellulose and related plant polysaccharides, making them ideal candidates for direct transformation of plant biomass into fuels and biocommodities by Consolidated Bioprocessing (CBP). In CBP, enzyme production, hydrolysis, and fermentation occur in a single reactor, leading to savings in capital and operating costs. Here we focus on transformation of biomass to n-butanol (hereafter called butanol), which is itself an attractive fuel and solvent and a new process can catalytically convert butanol to jet and diesel fuel at high yield. We will modify each organism to increase butanol tolerance and to express a synthetic butanol production pathway. We will evaluate the potential of each organism to produce butanol from biomass, and the higher performing strain will be further modified to fulfill the requirements for Consolidated BioProcessing (CBP) of plant biomass to low-cost butanol.

This proposal leverages the complementary expertise of academic groups at the Genoscope-CEA in Évry and the Laboratoire de Chimie Bactérienne in Marseille to study and engineer plant-fermenting clostridia. As such, it significantly diverges from most cellulosic biofuel projects in the USA, Europe, and Asia that attempt to enable biomass degradation in model organisms that ferment sugars to ethanol (Escherichia coli, Saccharomyces cerevisiae) or butanol (Clostridium acetobutylicum). None of these projects have led to a truly cellulolytic organism, likely because efficient degradation of lignocellulosic biomass requires adaptations including adhesion to insoluble plant substrates, coordinated expression of many plant-degrading enzymes, and resistance to plant-derived inhibitors. Thus, we approach this problem from a different perspective by modifying naturally lignocellulose-degrading bacteria to produce higher-order alcohols.

Clostridium phytofermentans and Clostridium cellulolyticum are both mesophilic, obligately anaerobic bacteria that ferment cellulose and other plant cell wall polysaccharides such as hemicellulose and pectin. The complete genomes of both strains are available, which exhibit similar GC content and codon bias. However, these bacteria also display significant differences. C. phytofermentans ferments cellulose primarily to ethanol at yields of ~70% the theoretical maximum with titers reaching 7 g L-1 ethanol. In contrast, C. cellulolyticum ferments cellulose primarily to lactate and acetate with ethanol titers reaching only 0.7 g L-1. However,  C. cellulolyticum degrades crystalline cellulose more efficiently than C. phytofermentans, likely because C. phytofermentans freely secretes its cellulases while C. cellulolyticum gathers extracellular cellulases into an extracellular complex, the cellulosome. Formation of a cellulosome has been shown to dramatically boost cellulase activities and improve degradation of cellulose and raw lignocellulosic biomass. By combining research on two plant-fermenting bacteria, one that  is specialized for cellulose degradation and the other for alcohol formation, this project will integrate the strengths of both organisms to develop novel strains with enhanced conversion of cellulosic biomass to butanol.