Adapting T. reesei cellulolytic enzymes for Simultaneous Saccharification and Fermentation – ACTIFE
Towards an economically feasible and integrated bioethanol process
Combination of lignocellulosic biomass hydrolysis and fermentation steps can lead to lower process costs for the 2nd generation bio-ethanol industry, but also brings constraints to the enzymes' activities. The objective of this project is to adapt several enzymes to these constraints.
Lowering enzyme costs and simplifying 2nd generation ethanol process configuration
The advent of a non-food biomass to ethanol industry (2nd generation ethanol) is a significant challenge for the transport sector. In this production process, pretreated plant biomass is hydrolysed by enzymes produced by a filamentous fungus, Trichoderma reesei. These enzymes cut cellulose into sugar monomers. Then, baker's yeast Saccharomyces cerevisiae ferments said glucose to ethanol. <br />This enzymatic hydrolysis step remains by far the most expensive step of the process, as a huge amount of enzyme has to be used, in part because glucose inhibits enzymes action. A possible answer to this problem is to perform the fermentation at the same time as hydrolysis so that glucose is consumed as soon as it is released by enzymes, but those two steps require different operating conditions. Today, this limits the advantages of this configuration called SSF (Simultaneous Saccharification and Fermentation). The ACTIFE project (Adaptation des Cellulases de Trichoderma reesei aux contraIntes de la Fermentation Ethanolique) aims at adapting T. reesei enzymes to fermentation constraints to lower enzyme doses to be used. The objective is to make the SSF scheme significantly attractive compared to the classical one where all reactions are separated (SHF : Separate Hydrolysis and Fermentation). <br />
The project is based on the use of directed evolution technologies. These genetic engineering technologies allow the generation of «super-enzymes« adapted to industrial conditions, with far higher performances than native enzymes. Millions of random versions of the original gene are generated through mutagenesis or recombinations and then screened for the few of them that retained the desired properties. This last step requires an adapted high-throughput screening technology, as several thousands enzyme versions are assessed. Those technologies mimic natural selection mechanisms but accelerate them and make them fit to industry's needs.
ACTIFE also make use of biochemistry techniques to identify, among the several enzymes produced by T. reesei, which ones are the limiting steps in SSF conditions. Those enzymes are enhanced using directed evolution and implanted again using genetic engineering into the original strain or the yeast S. cerevisiae. Performances of the new enzyme mixtures and strains are assessed and compared to wild-type versions. Finally the impact on ethanol production cost is evaluated by techno-economical simulation of the whole process.
The role of each main enzyme in the cocktail was studied and allowed to establish the targets for directed evolution. This particular technology was also subject to further development and important barriers were overcome in the enzyme expression systems and corresponding high-throughput screening. Standard SSF tests were set up and allowed a first evaluation of the process cost and definition of price and performance targets.
More than 10 enzyme versions adapted to a lower hydrolysis temperature are currently being introduced again in the fungus T. reesei or the yeast S. cerevisiae. Four of them have been successfully expressed and activity enhancement in these new hosts have been confirmed in several cases.
Enzymes developed in the project could be used in any process involving direct conversion of lignocellulosic biomass to biofuel or chemical where optimal microorganism fermentation conditions is an obstacle to the enzymes' activity. More generally screening methods developed could be used again for fine-tuning other enzymes properties to other process configurations.
Screening methodologies and enzymes studies will be published and presented in congresses. Enhanced enzymes will be patented. Presently we have one patent in preparation and a publication project. Other patents may be possible, depending on the new enzymes performances.
Bioethanol production from lignocellulosic biomass is one of the main topics of the ANR Bioenergy program. Enzymatic hydrolysis of lignocellulose is generally considered as one of the major bottleneck of the whole process representing an important part of the whole cost of lignocellulosic ethanol. The project deals with the 3rd thematic of the call for proposal "development of biological conversion processes". It aims at developing a cost-effective ethanol production process from lignocellulosic feedstocks by using a "Simultaneous Saccharification and Fermentation" (SSF) conversion mode. Currently, the cellulolytic fungus, Trichoderma reesei, remains the most efficient cellulase-producing micro-organism at an industrial scale and Saccharomyces cerevisiae the reference yeast for industrial ethanol fermentation. The project work program includes i) adapting major cellulolytic enzymes of Trichoderma reeseito specific SSF conditions using directed evolution protein engineering tools and ii) integrating some of these optimized enzymes in S. cerevisiae. The main goal of the project is to increase the global productivity and to decrease the enzyme loading in order to reduce the cost of the cellulose-ethanol conversion process. The scientific program is based on the development of enzymes adapted to SSF conditions which are not optimal for cellulases (low temperature, presence of ethanol which inhibits cellulolytic activities, etc.).
Project coordination
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
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