creation of a cellulosome-bearing strain of E. coli for production of cellulosic butanol – cellutanol

The bacterium Escherichia coli is neither cellulolytic, nor solvantogenic. The introduction of these two phenotypes therefore requires large scale engineering. Indeed, a large number of heterologous genes had to be expressed in this bacterium by means of plasmids or by integration in its genome. Besides, a number of chromosomic genes also had to be inactivated, to prevent the consumption of glucose for production of by-products other than butanol (succinate…). The growth rate of the resulting strain being coupled to the rate of glucose consumption and butanol production, an in vivo directed evolution strategy was applied and led to the selection of evolved clones producing higher amounts of butanol. The complete sequencing of the genomes of these mutants identified the genetic targets leading to the improved phenotype. Besides, to increase the ability of the bacterium to secrete heterologous proteins (cellulases), genetic engineering was employed to improve its secretion machinery and modify, among others, some components of its outer membrane.

Though the final goal of the Cellutanol project, the creation of a bacterium able to convert cellulose into butanol, was not reached, CNRS achieved a strain that rapidly catabolizes cellobiose and grows on amorphous cellulose. INSA successfully introduced an optimized pathway for butanol production and selected more efficient evolved clones. Furthermore, an E. coli strain whose envelope can be functionalized at will was developed by CNRS, leading to a contract with the Diamidex firm, and INSA will soon start a collaboration with the Rice University (Houston, USA).
The data obtained by both groups allowed to identify unexpected bottlenecks such as the interconnection between some metabolic pathways or the difficulty to induce an important secretion of cellulases by Escherichia coli. The results also allowed to specify the role of various metabolic enzymes, as well as that of LamB in the import of cellobiose, or the massive surface-exposure of a cellulosomal scaffoldin allowing the functionalization of its envelope with biotechnological applications in various sectors.

The development of a new E. coli strain suitable for functional screening and in vivo directed evolution of Ferredoxin NAD(P)+ reductases constitutes an efficient tool for understanding sequence-structure-function relationships in these enzymes, but can also be used for a larger screen of a library of genes encoding NAD(P)+ réductases, or ferrédoxins (partners of these enzymes). These approaches will be developed in partnership with the Rice University (Houston, USA).

The data generated during the Cellutanol project led to 6 publications (3 in Biotechnology for Biofuels, 1 in Scientific Report, 1 in FEBS letters, 1 in FEBS Journal), including a mutual CNRS-INSA publication (in Biotechnology for Biofuels). Four new publications are also planned to be submitted by the end of 2019.
Partner INSA has obtained one European patent in 2015 (EP15306225), and Partner CNRS has filed a European patent application in November 2018.

The final goal of the Cellutanol proposal is to build within fours years an Escherichia coli strain which will directly convert crystalline cellulose in butanol at a high yield and can be used for production of third generation biofuel with a better octane rating than ethanol. The proposal relies on the complementary expertise of two academic groups in the fields of cellulolysis (Partner 1, CNRS, Marseille) and synthetic biology coupled to metabolic engineering (Partner 2, INSA, Toulouse). The originality of the proposal lays in the selected host which is neither cellulolytic, nor produces butanol. Most of the competing projects aim at using a natural butanol producer such as Clostridium acetobutylicum to ferment plant cell wall hydrolysates generated by Trichoderma reesei commercial enzyme cocktails. Although some progresses were made over the past decade (reduced production of byproducts, improvement of commercial cocktails’ efficiency), this process remains far from being economically viable. Recently, Partner 2 engineered and patented an E. coli strain expressing a new metabolic pathway allowing to produce in batch cultures 9.5 g/L of butanol and 0.9 g/L of ethanol from glucose at a yield of 0.3 g of butanol/g of glucose, corresponding to 73% of the maximal theoretical yield. To drastically decrease the overall cost, the present proposal aims at improving the butanol yield, titer and productivity of the already E. coli obtained by Partner 2 and introducing an efficient cellulolytic system for direct conversion of cellulose to butanol in a Consolidated BioProcessing. The simultaneous introduction of both phenotypes (cellulolysis and butanol overproduction) in E. coli was never reported and will require large scale engineering. Nevertheless, this micro-organism is the most well known and characterized bacterium, grows rapidly, and can be easily modified thanks to the numerous available genetic tools. Furthermore, to our knowledge the construction of a cellulolytic E. coli for the production of butanol has not generated any patent application to date.
The project involves i) introducing an optimized cellulolytic system based on the artificial cellulosomes set up (in vitro) by Partner 1 in the strain already engineered by Partner 2, and make these very active complexes secreted (or cell surface exposed) by the selected host ii) increasing the butanol yield by inactivation of newly discovered competing pathways (in addition to those already deleted) and iii) improving the butanol tolerance in order to produce Butanol in continuous culture at a titer higher or equal to 10 g/l. In the newly engineered homo-butanologenic E. coli strain, growth and glucose consumption are linked to butanol production, and the strain could then be subsequently evolved in vivo to improve its efficiency regarding butanol titer, tolerance and productivity. When the improved phenotype is obtained, one or several clones will be isolated, characterized and their genomic DNA will be sequenced. This strategy will lead to the identification of the genetic targets of the evolution and the contribution of each single mutation identified to the improved phenotype will be evaluated and patented. Moreover, to attain a sufficient extracellular production of the cellulosomes to engender a cellulolytic phenotype, five different strategies for secretion/exposure that may possibly be combined are planned. Finally, the last stage of the project will involve the combination of both phenotypes by integration of all necessary heterologous genes for cellulolysis in the chromosome of the butanol overproducing strain.
The final deliverable will be a prototype strain of E. coli that secretes an efficient cellulolytic system, exhibits a high butanol titer, tolerance and productivity and generates nearly no byproduct.