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Enzyme Discovery and Engineering CHEM-E3140 Bioprocess Technology II 30.10.2015 Ossi Turunen Why to find new enzymes for biotechnology? Existing enzymes may not be good enough in • • • • • • • • performance stability activity in solvents pH properties substrate specificity enantioselectivity resistance to inhibition etc. http://www.csa.com/discoveryguides/biofuel/review6.php READ: http://www.novozymes.com/en/about-us/brochures/Documents/Enzymes_at_work.pdf • One gram of soil contains more than 4,000 different microorganisms. • The search for the right enzyme is a highly automated process. Huge robotic systems can simultaneously test a culture of microorganisms on 12 different substrates such as starch, proteins or fats. With the help of the robots, Novozymes' researchers can scan thousands of microorganisms in just a few days. • The final test involves scanning the enzymes under the precise working conditions in which that enzyme will be used in the final product. • Data from: http://www.novozymes.com/en/about-us/our-business/what-areenzymes/Pages/finding-and-producing-enzymes.aspx Template user guide 2/19/2010 3 Yellowstone hot springs http://waynesword.palomar.edu/ploct97.htm Deep sea thermal vents http://microbes.nres.uiuc.edu/NRES512.htm T. reesei growing on a plate culture Thermophilic bacteria isolated from Bag City Vent, one of the vents that was part of the MBL/JISAO microbial diversity study. (c) Julie Huber http://www.chem.mq.edu.au/ edge/fungal-biotech.html Clockwise from top left: Amanita muscaria, a basidiomycete; Sarcoscypha coccinea, an ascomycete; bread covered in mold; a chytrid; an Aspergillus conidiophore. http://www.answers.com/topic/fungus From presentation of Maria Ciaramella Vester et al. Microbial Cell Factories 2014, 13:72. Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing • The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. • The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. • Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. Template user guide 2/19/2010 6 Huge progress in genome sequencing • Scientists needed $3 billion and 13 years to sequence the three billion base pairs encoded in a single human genome - the first time (completed in 2003). • By 2011, eight years after that first project was completed, the cost of sequencing a human genome had fallen to $5,000, in a process that took just a few weeks. • And in January, Jonathan Rothberg, a chemical engineer and the founder of the biotech company Ion Torrent, unveiled an approach that is faster and cheaper still. He says his machine will be able to sequence a human genome, some 3.2 gigabytes’ worth of data, in two hours for just $1,000. • http://www.popsci.com/science/article/2012-02/1000-genome-medicine-hasnew-problem-too-much-information Template user guide 2/19/2010 8 http://www.nature.com/news/technology-the-1-000-genome-1.14901 >3000 genomes sequenced in 2011; > 18000 in 2014 Sequencing the genome creates so much data we don’t know what to do with it. Scientists are expecting as many as 1 billion people to have their genomes sequenced by 2025. Doolittle, 2002. Nature 419, 493-494 http://www.the-scientist.com/?articles.view/articleNo/39742/title/Sequencing-the-Tree-of-Life/ Structure of eukaryotic genes Protein expression http://genmed.yolasite.com/fundamentals-of-genetics.php http://www.piercenet.com/browse.cfm?fldID=F46A98FE-C7DD-4868-AB2C-2E3C71FC84D1 DNA sequence –> amino acid sequence Codon tables Template user guide 2/19/2010 15 GenBank: 188 372 017 sequences in Oct 2015 Template user guide 2/19/2010 16 Cloning of genes From www.explorebiology.com http://www.blackwellpublishing.com/allison/docs/sample_ch8.pdf Cloning of cDNA using BamHI linkers From presentation of Linnea Fletcher BIOL 2316 http://www.ncbe.reading.ac.uk/ncbe/gmfood/chymosin.html Chymosin protease is used in cheese production to precipitate casein protein. https://en.wikipedia.org/wiki/Chymosin Gene libraries http://www.emunix.emich.edu/~rwinning/genetics/tech2.htm DNA libraries • Genomic DNA Libraries - These libraries are made from genomic DNA (all of the DNA found in the organism's nuclei). Genomic DNA molecules are very large (each chromosome in the nucleus is one such DNA molecule), so they must be fragmented into small enough pieces to insert into vectors. This is typically done through digestion with one or more appropriate restriction endonucleases, mechanical shearing, or a combination of the two processes. The DNA is then ligated into the vector, which could be a plasmid, but is more often a cosmid or a viral chromosome. • cDNA Libraries - These libraries are made from cDNA, which are DNA copies of mRNA molecules. To make cDNA, mRNA is isolated from a tissue or whole organism, and DNA is copied from the mRNA template using an enzyme called reverse transcriptase. This enzyme works like a DNA polymerase, except that is uses RNA as a template instead of DNA. The resulting cDNA molecules are then engineered so that they have restriction enzyme recognition sites at each end of every molecule, which allows them to be digested and inserted into a vector as outlined previously. http://www.emunix.emich.edu/~rwinning/genetics/tech2.htm Metagenomic libraries http://muou.sc.mahidol.ac.th/research_an.html cDNA Libraries are best for eukaryotes No introns in cDNA From presentation of Linnea Fletcher BIOL 2316 Screening for specific cDNA plasmids in a cDNA Library by using an antibody probe The antibody binds to a specific Site on a protein that is made via The inserted foreign DNA. This Is a Western Blot since it uses antibody To detect a protein. From presentation of Linnea Fletcher BIOL 2316 High throughput screening • Leemhuis et al. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity. Biotechnology Letters 25: 1643–1645, 2003. • A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly measured using liquid cultures grown in microtiter plates without the need of separation or purification steps. • http://www.youtube.com/watch?v=Ap64ruCQTOQ Screening for improved carbohydrate hydrolase activity. Starch as substrate. Increase of reducing sugars dtetcted. http://www.exptec.com/Expression%20Technologies/Host%20cells.htm Advantages of fungal expression systems • • • • • • • • • • • • High yield Stable production strains Cost effectiveness High density growth Easy scale-up Safety High expression levels Rapid growth in chemically defined media Product processing similar to mammalian cells Can handle S–S rich proteins Can assist protein folding Can glycosylate protein Sharma et al. World J Microbiol Biotechnol (2009) 25:2083–2094 Protein secretion: over 100 g/L Milestones in protein engineering Luetz et al., 2008 New Biocatalysts: biocatalysis roadmap 1999 http://www1.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf • developing biocatalysts which are better, faster and cheaper than current chemical catalysts • development of a tool box of biocatalysts, i.e., biocatalysts that can catalyze a broader range of reactions and have greater versatility than is now possible • increased temperature stability, activity, and solvent compatibility • developing molecular modeling to permit rapid de novo design of new enzymes • creating better tools for new biocatalyst development • educating the public to the societal benefits of using and creating biocatalysts New Biocatalysts: biocatalysis roadmap 1999 http://www1.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf Engineering of Good Biocatalyst Luetz et al., 2008 Protein Engineering Approaches A) Random mutagenesis – Error-prone PCR, Mutator strains – Global – Site specific (pick up some interesting sites) B) DNA shuffling – In vitro recombination between members in a protein family – Methods have been developed for non-homologous recombination Recombination effectively exploits information present in the parental sequences to form new functional sequences. C) Directed evolution – In each mutation round the best variants are selected and used for a new round of mutagenesis or DNA shuffling. – Screening method is critical for good result. D) Phage-display – A library can be made into bacteriophage genome – The expressed proteins are exposed on the surface of the bacteriophage – The bacteriophages can be screened by binding or enzymatic assays – 109 variants screened E) Rational design – Rational design utilizes information on structure-function relationships – Site-directed mutagenesis. – Sequence comparison of thermophilic and mesophilic protein family members (semi-rational design). – Consensus sequences (semi-rational design). – Computational methods F) De novo design – Artificial folded proteins. – Protein scaffolds – new activities are engineered into them. http://ocw.mit.edu/courses/biological-engineering/20-109-laboratory-fundamentals-in-biologicalengineering-spring-2010/lecture-notes/MIT20_109S10_lec_m2d2.pdf http://ocw.mit.edu/terms/ Directed evolution Screening method is critical. It determines What will be obtained by directed evolution Gene shuffling http://www.proteus.fr/en/integrated-platform/proprietary-technologies/protein-directed-evolution/ Quin, ACS Catal. 2011 Sep 2;1(9):1017-1021 Site-directed Mutagenesis Using PCR 1. Identify site for mutation 2. Denature the plasmid and anneal primers containing the desired mutation 3. Add DNA polymerase and extend the primers 4. Use Dpn1 to degrade the original plasmid. Some examples of rational design - Structure-based design Lutz. Curr Opin Biotechnol. 2010 December ; 21(6): 734–743. B factor stabilization 1TE1 Reetz et al. Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability. Angew. Chem. Int. Ed. 2006, 45, 7745 –7751 Böttcher and Bornscheuer. Current Opinion in Microbiology 2010, 13:274–282 From the presentation of Novozymes in NWBC, Helsinki 20.10.2015 Template user guide 2/19/2010 50