ERA-NET: Thermostable isomerase processes for biotechnology

Disulfide isomerases are important enzymes involved in directing correct oxidative folding of a vast number of proteins, particularly those that are secreted to the extracellular environment. They do so by re-arranging disulfide bridges, a special type of covalent bonds between the side chains of different cysteine residues within a polypeptide chain and by helping the protein substrate to fold and curl into its native three-dimensional conformation. This activity is known as oxidative folding and is of major importance for correct cell functioning. In the TIPs project we aim to discover and characterize novel thermostable disulfide isomerases with a potential application in the production of other industrially relevant enzymes, either improving its production or extending its shelf life. In an early phase of this project we identified some thermostable disulfide isomerase enzyme candidates in genomes of thermophilic microorganisms and in metagenomes from hot (extreme) environments. Their production in a heterologous host was optimized and their catalytic activity was partially characterized. Some of the best-performing enzyme candidates were chosen for further functional and structural studies. In collaboration with the Centre for Biocatalysis at the University of Exeter in United Kingdom, we optimized the up-scaled production, purification and crystallization conditions for four different enzyme candidates. Several crystals were obtained and subsequently subjected to X-ray diffraction analysis. Even though crystals diffracted, the resolution was poor, and thus, it was not possible to make crystal structure models even after several trials to improve the conditions for crystal formation along with seeding techniques. In parallel, synthetic biology approaches were applied for the development of a quick high-throughput activity-based screening platform for disulfide isomerases. An engineered phosphatase was used as a target substrate to screen for disulfide isomerase activity by complementation studies in an in vivo assay. Correct disulfide shuffling catalyzed by the enzyme candidate recovers the dysfunctional (engineered) phosphatase to a functional phosphatase that specifically reacts with a color-generating substrate. The platform allows for efficient functional discovery of novel disulfide isomerases and high-throughput screening of expression conditions and activity. A total of 12 genes encoding enzyme candidates originating from thermophilic bacteria thriving at different temperatures (from 50oC to above 80oC) were cloned and expressed in E. coli. The recombinant enzymes gave varying degrees of yield and solubility. Enzyme activity assays also showed varying biochemical activity. One of the enzymes (candidate C011), from a Geobacter sp. showed optimal activity between 70 and 80oC, and highest activity and stability of all the expressed candidates. Four candidates, including C011, were subjected to crystallization trials using a crystallization robot in Exeter. The quality of the X-ray diffraction was however poor (maximally 12Å resolution), which is way below the resolution needed to make structural models. The project thus did not fulfil its main goals, especially regarding structure determination. However, a range of disulfide isomerases from bacteria growing at different temperatures have been expressed, purified and preliminary characterized biochemically, and these enzymes may serve as a valuable basis for further studies or applications.

The outcomes of this project are increased preliminary knowledge of the properties of protein disulfide isomerases from a variety of thermophilic bacteria thriving at a range of different temperatures and their behaviour following heterologous expression in E. coli. Structural analyses of selected disulfide isomerases are still ongoing and will potentially yield important 3-dimensional information. In the future this basic information can improve the use of disulfide-correcting enzymes in biotechnological processes for heterologous expression and correct folding, in particular of thermophilic proteins that tend to form aggregates due to incorrectly formed disulfide bonds.

The TIPs project focuses on the provision of novel thermostable isomerases from thermophilic microorganisms and metagenomes and their biotechnological applications. Isomers are molecules with identical atomic composition but with different structural characteristics. Different isomers can show very distinctive function. The formation of isomers often reduces the productivity of biotechnological and Chemical processes because only one of the two or more isomers is utilized in biocatalytic reactions (reducing the final efficiency to below 50%). Isomerases are enzymes catalyzing the conversion between different types of isomers. Using the appropriate isomerase enzyme in the industrial process will increase production efficiency resulting in 100% conversion of a racemic substrate to the product. Thermostable isomerases are desired because they possess high resistance and durability, are able to withstand harsh industrial process conditions,including heating and organic solvents. Elevated temperatures can also enhance substrate accessibility and solubility. The proposed project includes comparative bioinformatic analyses of sequence data to identify different classes of thermostable isomerases of industrial interest which will be cloned, over-expressed, functionally and structurally characterized and optimized towards their biotechnological application. Three types of isomerases will be targeted: sugar isomerases (to produce new desirable sugars for calorie-free sweeteners and as building blocks for drugs), disulfide isomerases (to improve protein folding and stability of industrial enzymes), and chalcone isomerases (involved in the transformation of flavonoids, secondary metabolites of importance as natural colorants, anti-oxidants, anti-microbial and anti-inflammatory agents). Durable isomerases will allow new opportunities for green, competitive and sustainable biotechnological processes that can replace conventional chemical synthesis.