Project: Development of the next-stage production platform for industrial enzymes

Recombinant protein production in the current production platforms often suffers from poor yields of the product. Two major reasons for this are the incompatibility of the foreign polypeptide to the host secretion system, and its sensitivity to the host proteases. The present project aims to tackle these challenges by applying the Zera peptide fusion technology in the filamentous fungus Trichoderma reesei. In this system the recombinant protein forms protein bodies inside the cell. When being inside the protein bodies, the recombinant protein is protected from host proteases. Due to the high density of the bodies, the product can be easily isolated from the cell material. The Zera peptide is derived from a maize storage protein, but it has been shown to promote protein body formation in multiple eukaryotic production systems, including Trichoderma reesei. This fungus, on the other hand, is the most efficient protein production system known to date, being able to produce more than 100 g/l of its native hydrolytic enzymes in the industrial process (Cherry and Fidantsef, 2003, Curr. Opin. Biotechnol 14, 438-443). The project aims to combine the vast protein synthesis and folding capacity of T. reesei with the benefits that the Zera technology offers. The expression of 6-8 different enzymes from different enzyme families will be studied and developed in the project. With these assets an efficient production platform will be developed for specific enzyme products._x000D_The project consortium is formed by two Ps, ERA Biotech (Barcelona, Spain) and VTT Technical Research Centre (Espoo, Finland). ERA Biotech is the holder and developer of the Zera technology, showing and excellent record of exploitation of this system in plants and animal cells. VTT is one of the leading institutes in fungal protein production research globally, and has extensive expertise and tools like efficient expression vectors and host strains for recombinant protein production in T. reesei. _x000D_

Acronym	ZERAZYME (Reference Number: 5126)
Duration	01/07/2010 - 30/06/2013
Project Topic	This project aims to set up an innovative alternative production platform combining the filamentous fungus Trichoderma reesei with Zera technology in order to create an industrially attractive production system.
Project Results (after finalisation)	IMPROVEMENT OF TRICHODERMA REESEI TECHNOLOGY_x000D_T. reesei strains carrying fluorescent markers were developed to aid in transformant selection. New cloning procedure based on Golden Gate technique was established to ease the construction of Zera-EOI expression cassettes. Transformation procedure based on particle bombardment was created to fasten the strain generation procedure. _x000D__x000D_EXPRESSION OF ZERA-ENZYME FUSIONS IN T. REESEI_x000D_Expession of intracellular ZERA-Enzyme fusions in filamentous fungus Trichoderma reesei proved to be unpredictable and highly dependent on the ZERA fusion P. Acceptable expression levels were found for two fusion Ps: glucose oxidase (GOX) and beta galactosidase (CELB). While expression of GOX was good, the stability and downstream processing of this fusion worked poorly and therefore the last quarter of the project focused on ZERA-CELB fusion. For CelB a new hyphotesis for formation of PBs out side of cells i.e. the “in vitro” self- aggregation of Zera fusion proteins in artificial PBs was tested. This was obtained by secreted version of ZERA peptide. The downstream process for secreted Zera was developed and included a high pore filtration and gravity centrifugation._x000D__x000D_PROTEIN CHARACTERIZATION_x000D_The activity of Zera-CelB PBs recovered from T. reesei mycelia was measured by standard colorimetric activity assay. The results indicate that T. reesei PBs are as active as the PBs obtained from tobacco plants (ERA). For the secreted ZeraSec-CelB produced in T. reesei the CelB activity of the in vitro aggregates was measured in several temperatures and compared to commercial CelB standards. It was found that ZeraSec-CelB aggregates had low activity in room temperature. However, in elevated temperatures at 70 and 99 C degrees, close to expected optimum temperatures, high specific activities were recorded for this thermostable enzyme._x000D__x000D_FERMENTATION, PROCESS OPTIMIZATION AND SCALE-UP_x000D_Zera-CelB was selected as the candidate with best potential for scale-up study and was selected for fermentation studies.Following parallel fermentations were performed:_x000D__x000D_-intracellular ZERA-CelB_x000D_-Intracellular parental control_x000D_-secreted CelB_x000D_-ZERAsec-CelB_x000D_-CBHI-ZERAsec-CelB_x000D_-secreted parental contol_x000D__x000D_Fermentations were performed in 1 litre stirred tanks. The fermentations were sampled daily and supernatant and intracellular fractions analysed and quantified as above. Intra cellular Zera-Cel be accumulated 150mg/l, ZERAsec-CelB was detected in culture supernatant at 100mg/l level. Secreted CelB was detected at about 1mg/l level and CBH-ZERAsec-CelB less than 1mg/l._x000D__x000D__x000D__x000D_
Network	Eurostars
Call	Eurostars Cut-Off 3

Project partner

Number	Name	Role	Country
2	ERA Biotech, S.A.	Coordinator	Spain
2	VTT Technical Research Centre of Finland	Partner	Finland