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id="ProfileCheckPaperUpdate-react-component-bfd690dd-d33e-4e4a-b040-3fafa349de61"></div> <div class="DesignSystem"><div class="onsite-ping" id="onsite-ping"></div></div><div class="profile-user-info DesignSystem"><div class="social-profile-container"><div class="left-panel-container"><div class="user-info-component-wrapper"><div class="user-summary-cta-container"><div class="user-summary-container"><div class="social-profile-avatar-container"><img class="profile-avatar u-positionAbsolute" border="0" alt="" src="//a.academia-assets.com/images/s200_no_pic.png" /></div><div class="title-container"><h1 class="ds2-5-heading-sans-serif-sm">Lorenzo Camisi</h1><div class="affiliations-container fake-truncate js-profile-affiliations"><div><a class="u-tcGrayDarker" href="https://unibo.academia.edu/">Universit脿 di Bologna</a>, <a class="u-tcGrayDarker" href="https://unibo.academia.edu/Departments/Pharmacy_and_Biotechnology/Documents">Pharmacy and Biotechnology</a>, <span 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However, for many industrial reactions there are no naturally occurring enzymes, and so many different engineering approaches have been used to address this problem. Engineering methods have been used to alter the stability, substrate specificity and stereospecificity of aldolases to produce excellent enzymes for biocatalytic processes. Recently greater understanding of the aldolase mechanism has allowed many successes with both rational engineering approaches and computational design of aldolases. Rational engineering approaches have produced desired enzymes quickly and efficiently while combination of computational design with laboratory methods has created enzymes with activity approaching that of natural enzymes.","grobid_abstract_attachment_id":62459854},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307715/Engineering_aldolases_as_biocatalysts","translated_internal_url":"","created_at":"2020-03-24T09:24:03.386-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459854,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459854/thumbnails/1.jpg","file_name":"2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6.pdf","download_url":"https://www.academia.edu/attachments/62459854/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Engineering_aldolases_as_biocatalysts.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459854/2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DEngineering_aldolases_as_biocatalysts.pdf\u0026Expires=1732778827\u0026Signature=CiZeRULhOaBO~NtR-V9dNPpr1ATdd7m-nXcjt3uLeudwrik109-TDqxM-tZ0Zj8ZcyKsWwjT7eoJUSIHj-j2obAb28Wb7K8qnfltBGkEda5rA5d3~CnvbR2djDzHpXpiMK4PTKIrA1PgBW8pLG3CdsvJj8y~a1hXrl-i0V9aKf0hn8-FCeVL3mvlX1HylruEb34G8qvXJtCjI2nY3gxUM2KwLpdlZh5Cj7Z0fe6rd4ImbJw9JnAPdpTUnQyngzjeb7n8Tzs8KKmhu7kuS5dJFgTPNQUVv0wQCB9YUmIhXJS7OQlXn9IbP7YVe4WulBLg0uN8JLtRa~X3ZXLbzItV5A__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Engineering_aldolases_as_biocatalysts","translated_slug":"","page_count":9,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459854,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459854/thumbnails/1.jpg","file_name":"2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6.pdf","download_url":"https://www.academia.edu/attachments/62459854/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Engineering_aldolases_as_biocatalysts.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459854/2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DEngineering_aldolases_as_biocatalysts.pdf\u0026Expires=1732778827\u0026Signature=CiZeRULhOaBO~NtR-V9dNPpr1ATdd7m-nXcjt3uLeudwrik109-TDqxM-tZ0Zj8ZcyKsWwjT7eoJUSIHj-j2obAb28Wb7K8qnfltBGkEda5rA5d3~CnvbR2djDzHpXpiMK4PTKIrA1PgBW8pLG3CdsvJj8y~a1hXrl-i0V9aKf0hn8-FCeVL3mvlX1HylruEb34G8qvXJtCjI2nY3gxUM2KwLpdlZh5Cj7Z0fe6rd4ImbJw9JnAPdpTUnQyngzjeb7n8Tzs8KKmhu7kuS5dJFgTPNQUVv0wQCB9YUmIhXJS7OQlXn9IbP7YVe4WulBLg0uN8JLtRa~X3ZXLbzItV5A__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retroaldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model. E nzymes are excellent catalysts, and the ability to design new active enzymes could have applications in drug production (1), green chemistry (2), and bioremediation of xenobiotic pollutants (3). To date, most enzyme design efforts have used selection methodologies to retrieve very rare active catalysts from large libraries of candidate protein variants (4-7). Recent advances in computational protein design have made it possible to design new protein folds (8) and binding interactions (9) and have opened the door to the possibility of computationally designing enzymatic catalysts for any chemical reaction. Despite recent progress (10, 11), creating enzymes for chemical transformations not efficiently catalyzed by naturally occurring enzymes remains a major challenge. Here, we describe (i) general computational methods for constructing active sites for multistep reactions consisting of superimposed reaction intermediates and transition states (TS) surrounded by protein functional groups in orientations optimal for catalysis ( and (ii) the use of this methodology to design novel catalysts for a retro-aldol reaction in which a carbon-carbon bond is broken in a nonnatural (i.e., not found in biological systems) substrate: 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone ( (12).","grobid_abstract_attachment_id":62459844},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307714/De_Novo_Computational_Design_of_Retro_Aldol_Enzymes_Downloaded_from","translated_internal_url":"","created_at":"2020-03-24T09:24:03.233-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459844,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459844/thumbnails/1.jpg","file_name":"2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2.pdf","download_url":"https://www.academia.edu/attachments/62459844/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"De_Novo_Computational_Design_of_Retro_Al.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459844/2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2-libre.pdf?1585128336=\u0026response-content-disposition=attachment%3B+filename%3DDe_Novo_Computational_Design_of_Retro_Al.pdf\u0026Expires=1732778828\u0026Signature=cUZPy3gTLZgDy8y6GoNfH8gTt-cknQPw48ZeNvVWhSkookrw4xmcOjoFKKCkWWDbFZvgnWUy7bxW3v2oVZaQ9SB6axJyHmEY9ueY4ZS-HEUI3usvKo3fhl210~C0aSohyteucpFORSJdNA6zPLQxJ3zVSYOP3-oG~t3Y6oxI8Krg4V79Q8qKYZ5ANJeZGrM9Ehs-t2AAARMTLwm4EeaTbPVbN2NPLFQ7zIKf2E9wiYuGBh1sOQPimhN6HLF9QbgUPDO2OR0bwdvBhCmX1LwMkrfcEngHq9oxGz8LbnyCem55MwsWieIR9zllmBXjU3TmwB9sJSd9ZK2mNm2FqnWFYQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"De_Novo_Computational_Design_of_Retro_Aldol_Enzymes_Downloaded_from","translated_slug":"","page_count":6,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459844,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459844/thumbnails/1.jpg","file_name":"2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2.pdf","download_url":"https://www.academia.edu/attachments/62459844/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"De_Novo_Computational_Design_of_Retro_Al.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459844/2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2-libre.pdf?1585128336=\u0026response-content-disposition=attachment%3B+filename%3DDe_Novo_Computational_Design_of_Retro_Al.pdf\u0026Expires=1732778828\u0026Signature=cUZPy3gTLZgDy8y6GoNfH8gTt-cknQPw48ZeNvVWhSkookrw4xmcOjoFKKCkWWDbFZvgnWUy7bxW3v2oVZaQ9SB6axJyHmEY9ueY4ZS-HEUI3usvKo3fhl210~C0aSohyteucpFORSJdNA6zPLQxJ3zVSYOP3-oG~t3Y6oxI8Krg4V79Q8qKYZ5ANJeZGrM9Ehs-t2AAARMTLwm4EeaTbPVbN2NPLFQ7zIKf2E9wiYuGBh1sOQPimhN6HLF9QbgUPDO2OR0bwdvBhCmX1LwMkrfcEngHq9oxGz8LbnyCem55MwsWieIR9zllmBXjU3TmwB9sJSd9ZK2mNm2FqnWFYQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.","grobid_abstract_attachment_id":62459852},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307713/Robust_design_and_optimization_of_retroaldol_enzymes","translated_internal_url":"","created_at":"2020-03-24T09:24:03.131-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459852,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459852/thumbnails/1.jpg","file_name":"2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet.pdf","download_url":"https://www.academia.edu/attachments/62459852/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Robust_design_and_optimization_of_retroa.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459852/2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DRobust_design_and_optimization_of_retroa.pdf\u0026Expires=1732778828\u0026Signature=GNSCNBOlbgPKFY9Mybw4PB9XWfMzDKPWqO4OJKEFrwFynHNc0qJwec5GafcaPSD8F8BeD5YZU6pXrkdVZTeIz113bm8ZY4W7QsbLMBhY4syOvdbrVbeYNWDyKbBwBjNz7G0JmUarftt47Whkm95g7RJbPJFrezIPxHfyU3SOr4TrJKfVXegRekkjKZ0ZtGbbRl6cF7ntN-gqWdCYXwRk0DymZLm26erGknWhvZsCdhXHOH~XaCGj43VuZQ4VrvVdnZNikfd4nmLIYhnrVMK9RC4x9szKh-4kKH6G1PpM7kAsIvv12ujcZHL4Nyk8IOs7M8d31gqyJbp0xOcU-D6Usw__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Robust_design_and_optimization_of_retroaldol_enzymes","translated_slug":"","page_count":10,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459852,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459852/thumbnails/1.jpg","file_name":"2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet.pdf","download_url":"https://www.academia.edu/attachments/62459852/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Robust_design_and_optimization_of_retroa.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459852/2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DRobust_design_and_optimization_of_retroa.pdf\u0026Expires=1732778828\u0026Signature=GNSCNBOlbgPKFY9Mybw4PB9XWfMzDKPWqO4OJKEFrwFynHNc0qJwec5GafcaPSD8F8BeD5YZU6pXrkdVZTeIz113bm8ZY4W7QsbLMBhY4syOvdbrVbeYNWDyKbBwBjNz7G0JmUarftt47Whkm95g7RJbPJFrezIPxHfyU3SOr4TrJKfVXegRekkjKZ0ZtGbbRl6cF7ntN-gqWdCYXwRk0DymZLm26erGknWhvZsCdhXHOH~XaCGj43VuZQ4VrvVdnZNikfd4nmLIYhnrVMK9RC4x9szKh-4kKH6G1PpM7kAsIvv12ujcZHL4Nyk8IOs7M8d31gqyJbp0xOcU-D6Usw__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Computational methods can be principally grouped into three main categories: bioinformatics; molecular modelling; and de novo design. Particularly de novo protein design is experiencing rapid development, resulting in more robust and reliable predictions. A recent trend in the field is to combine several computational approaches in an interactive manner and to complement them with structural analysis and directed evolution. 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The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s) with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi), to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. 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Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. 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window.Academia.workViewCountsFetcher.queue(workId, function (count) { var description = window.$h.commaizeInt(count) + " " + window.$h.pluralize(count, 'View'); $(".js-view-count[data-work-id=42307705]").text(description); $(".js-view-count[data-work-id=42307705]").attr('title', description).tooltip(); }); });</script></span></span><span><span class="percentile-widget hidden"><span class="u-mr2x work-percentile"></span></span><script>$(function () { var workId = 42307705; window.Academia.workPercentilesFetcher.queue(workId, function (percentileText) { var container = $(".js-work-strip[data-work-id='42307705']"); container.find('.work-percentile').text(percentileText.charAt(0).toUpperCase() + percentileText.slice(1)); container.find('.percentile-widget').show(); container.find('.percentile-widget').removeClass('hidden'); }); });</script></span><span><script>$(function() { new Works.PaperRankView({ workId: 42307705, container: "", }); });</script></span></div><div id="work-strip-premium-row-container"></div></div></div><script> require.config({ waitSeconds: 90 })(["https://a.academia-assets.com/assets/wow_profile-f77ea15d77ce96025a6048a514272ad8becbad23c641fc2b3bd6e24ca6ff1932.js","https://a.academia-assets.com/assets/work_edit-ad038b8c047c1a8d4fa01b402d530ff93c45fee2137a149a4a5398bc8ad67560.js"], function() { // from javascript_helper.rb var dispatcherData = {} if (true){ window.WowProfile.dispatcher = window.WowProfile.dispatcher || _.clone(Backbone.Events); 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Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating \"smart\" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.","grobid_abstract_attachment_id":62459830},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307705/Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution","translated_internal_url":"","created_at":"2020-03-24T09:24:02.167-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459830,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459830/thumbnails/1.jpg","file_name":"2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x.pdf","download_url":"https://www.academia.edu/attachments/62459830/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Polishing_the_craft_of_genetic_diversity.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459830/2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x-libre.pdf?1585128367=\u0026response-content-disposition=attachment%3B+filename%3DPolishing_the_craft_of_genetic_diversity.pdf\u0026Expires=1732778828\u0026Signature=aX2NUmpL2bsU9mUi4wWqrOeWeORefcAegMH5cjCd8xJwZLlj7ZjzUOly4KzrIdE95iU8cphhQZ6VBAGbVI0C3rfvCMykWPb3piVy1cYkb-QNRUgoVfaUYVRUU4VEqQ8EHZxuXdW9oaZ8cmCTTUUDxd-E7PDodubmroW9TptcK2O3Ye~Fg09GnsHUa565xcUAc0pv213STlCmQi~ziozSQYggiuIWkaw1WTMYeXi81YcGwwJ6Ol1I96mAdY8TzpSV-fSTjpdJ~8W2thSuFmDeD-P0rFOCYhDVofR1DDcrzoY5YlJ0ql0n1wFI3CC3bgiLSrqXOik4qL-KzV5eWuPxCA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution","translated_slug":"","page_count":15,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459830,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459830/thumbnails/1.jpg","file_name":"2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x.pdf","download_url":"https://www.academia.edu/attachments/62459830/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Polishing_the_craft_of_genetic_diversity.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459830/2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x-libre.pdf?1585128367=\u0026response-content-disposition=attachment%3B+filename%3DPolishing_the_craft_of_genetic_diversity.pdf\u0026Expires=1732778828\u0026Signature=aX2NUmpL2bsU9mUi4wWqrOeWeORefcAegMH5cjCd8xJwZLlj7ZjzUOly4KzrIdE95iU8cphhQZ6VBAGbVI0C3rfvCMykWPb3piVy1cYkb-QNRUgoVfaUYVRUU4VEqQ8EHZxuXdW9oaZ8cmCTTUUDxd-E7PDodubmroW9TptcK2O3Ye~Fg09GnsHUa565xcUAc0pv213STlCmQi~ziozSQYggiuIWkaw1WTMYeXi81YcGwwJ6Ol1I96mAdY8TzpSV-fSTjpdJ~8W2thSuFmDeD-P0rFOCYhDVofR1DDcrzoY5YlJ0ql0n1wFI3CC3bgiLSrqXOik4qL-KzV5eWuPxCA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); $(this).data('initialized', true); } }); $a.trackClickSource(".js-work-strip-work-link", "profile_work_strip") }); </script> <div class="js-work-strip profile--work_container" data-work-id="42307704"><div class="profile--work_thumbnail hidden-xs"><a class="js-work-strip-work-link" data-click-track="profile-work-strip-thumbnail" href="https://www.academia.edu/42307704/Strategy_and_success_for_the_directed_evolution_of_enzymes"><img alt="Research paper thumbnail of Strategy and success for the directed evolution of enzymes" class="work-thumbnail" src="https://attachments.academia-assets.com/62459826/thumbnails/1.jpg" /></a></div><div class="wp-workCard wp-workCard_itemContainer"><div class="wp-workCard_item wp-workCard--title"><a class="js-work-strip-work-link text-gray-darker" data-click-track="profile-work-strip-title" href="https://www.academia.edu/42307704/Strategy_and_success_for_the_directed_evolution_of_enzymes">Strategy and success for the directed evolution of enzymes</a></div><div class="wp-workCard_item wp-workCard--actions"><span class="work-strip-bookmark-button-container"></span><a id="dde309827642dd570e1aea38f25dfb3a" class="wp-workCard--action" rel="nofollow" data-click-track="profile-work-strip-download" data-download="{"attachment_id":62459826,"asset_id":42307704,"asset_type":"Work","button_location":"profile"}" href="https://www.academia.edu/attachments/62459826/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&s=profile"><span><i class="fa fa-arrow-down"></i></span><span>Download</span></a><span class="wp-workCard--action visible-if-viewed-by-owner inline-block" style="display: none;"><span class="js-profile-work-strip-edit-button-wrapper profile-work-strip-edit-button-wrapper" data-work-id="42307704"><a class="js-profile-work-strip-edit-button" tabindex="0"><span><i class="fa fa-pencil"></i></span><span>Edit</span></a></span></span><span id="work-strip-rankings-button-container"></span></div><div class="wp-workCard_item wp-workCard--stats"><span><span><span class="js-view-count view-count u-mr2x" data-work-id="42307704"><i class="fa fa-spinner fa-spin"></i></span><script>$(function () { var workId = 42307704; 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Molecular biology advances present many new strategies for directed evolution. Commonly used techniques have led to many successful examples of enzyme improvement, yet there is still a need to improve both the efficiency and capability of directed evolution. Recent strategies aimed at making directed evolution faster and more efficient take better advantage of available structural and sequence information. The underlying principles that lead to early dead-ends for directed evolution experiments are also discussed along with recent strategies designed to by-pass them. Several emerging methods for creating novel enzymes are also discussed including examples of catalytic activity for which there is no precedent in nature. 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At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population. Insufficient knowledge about protein traits, mutational robustness of protein folds and technological limitations in diversity generating methods are main challenges for managing the complexity of protein sequence space. This review covers computational and experimental advances for high quality mutant library generation that have been achieved in the past two years.","grobid_abstract_attachment_id":62459819},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307699/Advances_in_generating_functional_diversity_for_directed_protein_evolution","translated_internal_url":"","created_at":"2020-03-24T09:24:01.331-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459819,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459819/thumbnails/1.jpg","file_name":"2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf.pdf","download_url":"https://www.academia.edu/attachments/62459819/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Advances_in_generating_functional_divers.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459819/2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf-libre.pdf?1585128370=\u0026response-content-disposition=attachment%3B+filename%3DAdvances_in_generating_functional_divers.pdf\u0026Expires=1732778828\u0026Signature=aVeWdVmlFr0Y1Yx2E2dkfnwAsfP~rQOt0Bi-lr0QHsNL9D9692-Gscq~GEqQZYph5nVAlFuuPZAuzZwQv0LnsM9w2LvueaF29dkPvAQc2Jz-WsHsig1Vz3Re~x3hkJe-XxkCMOAUM4BGXEaA9B~hr9iWexhv7E~N0~5DU9BsPXQQJ9IKJSK-IBXNUvaFmSzy5ZcCDUB~Mcq9ssMKxUVhYcN-s-gO2mFp~~t-yM-FtA5HNIL2rUdu~FZtLJ-BbotBCAYsIGDQ2-Rq8VCQTLiObxPd4KyP5lIt1y2z3R66E2yNP64vdC~z3~x5Y9OAImxEmfvqHvCr74Z6myQBnGKQbA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Advances_in_generating_functional_diversity_for_directed_protein_evolution","translated_slug":"","page_count":7,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459819,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459819/thumbnails/1.jpg","file_name":"2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf.pdf","download_url":"https://www.academia.edu/attachments/62459819/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Advances_in_generating_functional_divers.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459819/2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf-libre.pdf?1585128370=\u0026response-content-disposition=attachment%3B+filename%3DAdvances_in_generating_functional_divers.pdf\u0026Expires=1732778828\u0026Signature=aVeWdVmlFr0Y1Yx2E2dkfnwAsfP~rQOt0Bi-lr0QHsNL9D9692-Gscq~GEqQZYph5nVAlFuuPZAuzZwQv0LnsM9w2LvueaF29dkPvAQc2Jz-WsHsig1Vz3Re~x3hkJe-XxkCMOAUM4BGXEaA9B~hr9iWexhv7E~N0~5DU9BsPXQQJ9IKJSK-IBXNUvaFmSzy5ZcCDUB~Mcq9ssMKxUVhYcN-s-gO2mFp~~t-yM-FtA5HNIL2rUdu~FZtLJ-BbotBCAYsIGDQ2-Rq8VCQTLiObxPd4KyP5lIt1y2z3R66E2yNP64vdC~z3~x5Y9OAImxEmfvqHvCr74Z6myQBnGKQbA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.","grobid_abstract_attachment_id":62459817},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307698/Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries","translated_internal_url":"","created_at":"2020-03-24T09:24:01.182-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459817,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459817/thumbnails/1.jpg","file_name":"2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp.pdf","download_url":"https://www.academia.edu/attachments/62459817/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Steering_directed_protein_evolution_stra.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459817/2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp-libre.pdf?1585128374=\u0026response-content-disposition=attachment%3B+filename%3DSteering_directed_protein_evolution_stra.pdf\u0026Expires=1732778828\u0026Signature=HABOdCOGVWbx564c~D~BBQ422~hayjuPLg9yqVLf7K2o2nntHBLXpTOGJMQ~GQkdvupLqQIxJk4bK8O~uUDh~bna5HX8FALQGciPvKKY1oa6KCzpJudYFqcA40TWEvGgq094nYdyDhsk6CommMLFNDbiC-upO3rC1KjTz1cQDDbBuXKR365TPcqRN5Ars-VHsDLXCA8~wFwzin0xRBhbB~rzIUx3EqhjjYJuRbDD2DlLwzHwW5hKYLmoHWdRDw3FApWkkdVGu2kKhclMBm74bGU9vmSAczHLfdTOj7lRMNCyw8IdAezmhT47jZFn2H3hNelseNPz-YEvwefALrb8QQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries","translated_slug":"","page_count":15,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459817,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459817/thumbnails/1.jpg","file_name":"2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp.pdf","download_url":"https://www.academia.edu/attachments/62459817/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Steering_directed_protein_evolution_stra.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459817/2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp-libre.pdf?1585128374=\u0026response-content-disposition=attachment%3B+filename%3DSteering_directed_protein_evolution_stra.pdf\u0026Expires=1732778828\u0026Signature=HABOdCOGVWbx564c~D~BBQ422~hayjuPLg9yqVLf7K2o2nntHBLXpTOGJMQ~GQkdvupLqQIxJk4bK8O~uUDh~bna5HX8FALQGciPvKKY1oa6KCzpJudYFqcA40TWEvGgq094nYdyDhsk6CommMLFNDbiC-upO3rC1KjTz1cQDDbBuXKR365TPcqRN5Ars-VHsDLXCA8~wFwzin0xRBhbB~rzIUx3EqhjjYJuRbDD2DlLwzHwW5hKYLmoHWdRDw3FApWkkdVGu2kKhclMBm74bGU9vmSAczHLfdTOj7lRMNCyw8IdAezmhT47jZFn2H3hNelseNPz-YEvwefALrb8QQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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One illustration of the efficiency of a data-driven approach is the work of Parikh and Matsumura [1], who showed that a single round of structure-guided site-saturation mutagenesis on 尾-galactosidase using the crystal structure with a bound substrate analog identified a variant that was 180-fold more active and 700 000-fold more specific for fucoside substrates than the wild-type enzyme. Previous mutants identified in seven rounds of directed evolution and screening were only 10-fold more active and 2-fold more specific than wild type, and required considerably more time and effort to screen [1, 2]. summarizes recent work in data-driven protein design. For each type of design outlined below, one specific example is dis-cussed in detail. The other examples are provided in the table to give a general summary (rather than the \"bestcase scenario\") of the work in this area and to direct interested readers towards other references worth examining.","grobid_abstract_attachment_id":62459815},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307697/Better_library_design_data_driven_protein_engineering","translated_internal_url":"","created_at":"2020-03-24T09:24:01.119-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459815,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459815/thumbnails/1.jpg","file_name":"2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e.pdf","download_url":"https://www.academia.edu/attachments/62459815/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Better_library_design_data_driven_protei.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459815/2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e-libre.pdf?1585128372=\u0026response-content-disposition=attachment%3B+filename%3DBetter_library_design_data_driven_protei.pdf\u0026Expires=1732778828\u0026Signature=fIb5OPPmx5tlYxZt~XONQ8AM2wrmYfdbPUGhVj3JomN7vexCpbnu9FBZGfCFmDo3mhWfyHSbRd0TbivUiXtWTIhEISnhDaUp2aRVkPruj7LaKZubfRTz5h94PPi3Z4yF1KYR1P-Zh7NtWWZ-VWsOO6fxZet3pW55gHkQBunyAzV9ghzkQxc9BtBMb2pWIrgsFoZar-dB9AjgkNNrZlefFyywLn9P8EswGiGpTtxcTMDJ6Rh23TSeFDjPsoInj33FDX4vZk4d6bq2kG59KA9U3EVM5c6RClQOXi5pK3bsBjfcbmpDrXuhL-k5zsY8esHg2SGcr5CUV3nSny80FKQT2Q__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Better_library_design_data_driven_protein_engineering","translated_slug":"","page_count":12,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459815,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459815/thumbnails/1.jpg","file_name":"2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e.pdf","download_url":"https://www.academia.edu/attachments/62459815/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Better_library_design_data_driven_protei.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459815/2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e-libre.pdf?1585128372=\u0026response-content-disposition=attachment%3B+filename%3DBetter_library_design_data_driven_protei.pdf\u0026Expires=1732778828\u0026Signature=fIb5OPPmx5tlYxZt~XONQ8AM2wrmYfdbPUGhVj3JomN7vexCpbnu9FBZGfCFmDo3mhWfyHSbRd0TbivUiXtWTIhEISnhDaUp2aRVkPruj7LaKZubfRTz5h94PPi3Z4yF1KYR1P-Zh7NtWWZ-VWsOO6fxZet3pW55gHkQBunyAzV9ghzkQxc9BtBMb2pWIrgsFoZar-dB9AjgkNNrZlefFyywLn9P8EswGiGpTtxcTMDJ6Rh23TSeFDjPsoInj33FDX4vZk4d6bq2kG59KA9U3EVM5c6RClQOXi5pK3bsBjfcbmpDrXuhL-k5zsY8esHg2SGcr5CUV3nSny80FKQT2Q__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, stereo-, and regioselectivity), and/or stability (temperature, organic solvents, pH) is just limited by the throughput of the genetic selection, display, or screening system that is available for a given protein. Sometimes it is possible to analyze millions of protein variants from combinatorial libraries per day. In other cases, however, only a few hundred variants can be screened in a single day, and thus the creation of smaller yet smarter libraries is needed. Different strategies have been developed to create these libraries. One approach is to perform mutational scanning or to construct \"mutability landscapes\" in order to understand sequence-function relationships that can guide the actual directed evolution process. Herein we provide a protocol for economically constructing scanning mutagenesis libraries using a cytochrome P450 enzyme in a high-throughput manner. The goal is to engineer activity, regioselectivity, and stereoselectivity in the oxidative hydroxylation of a steroid, a challenging reaction in synthetic organic chemistry. Libraries based on mutability landscapes can be used to engineer any fitness trait of interest. The protocol is also useful for constructing gene libraries for deep mutational scanning experiments.","grobid_abstract_attachment_id":62459825},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307696/Chapter_6_Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning","translated_internal_url":"","created_at":"2020-03-24T09:24:00.901-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459825,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459825/thumbnails/1.jpg","file_name":"2017__Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning20200324-64578-j04kxq.pdf","download_url":"https://www.academia.edu/attachments/62459825/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Chapter_6_Directed_Evolution_of_Proteins.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459825/2017__Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning20200324-64578-j04kxq-libre.pdf?1585128375=\u0026response-content-disposition=attachment%3B+filename%3DChapter_6_Directed_Evolution_of_Proteins.pdf\u0026Expires=1732778828\u0026Signature=O3dKAseS~eJuiwAlO-Bg5JXl54JGz8WtiR9QhhHFKQZDlayDhoxa9rLDLH1lymBIvH5EzS5jey4n6PezstfEXXI7fvmxDxq9XTpI68rpt-7EQLcuplILG-MoHJBsEweSx-7AVmpLrT2T~ely1UQD9Z~9XM2838yea53y2JY5sbcPpoKSusPTkiNlULbI0DpJL-oXWaHjs3oMwco5Y7WLXp4vl2nTdeARIBeWokB0g8bhw3aXhHJimS8YsvbR2~OYgipLxJC1vIZ7qMlXX~04kbtVSysiKHSpJ9bPr2xBHa-haKqbo9Ttw70UP49JO5vffnfOr~DbKqFYax3GRiSewA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Chapter_6_Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning","translated_slug":"","page_count":42,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459825,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459825/thumbnails/1.jpg","file_name":"2017__Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning20200324-64578-j04kxq.pdf","download_url":"https://www.academia.edu/attachments/62459825/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Chapter_6_Directed_Evolution_of_Proteins.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459825/2017__Directed_Evolution_of_Proteins_Based_on_Mutational_Scanning20200324-64578-j04kxq-libre.pdf?1585128375=\u0026response-content-disposition=attachment%3B+filename%3DChapter_6_Directed_Evolution_of_Proteins.pdf\u0026Expires=1732778828\u0026Signature=O3dKAseS~eJuiwAlO-Bg5JXl54JGz8WtiR9QhhHFKQZDlayDhoxa9rLDLH1lymBIvH5EzS5jey4n6PezstfEXXI7fvmxDxq9XTpI68rpt-7EQLcuplILG-MoHJBsEweSx-7AVmpLrT2T~ely1UQD9Z~9XM2838yea53y2JY5sbcPpoKSusPTkiNlULbI0DpJL-oXWaHjs3oMwco5Y7WLXp4vl2nTdeARIBeWokB0g8bhw3aXhHJimS8YsvbR2~OYgipLxJC1vIZ7qMlXX~04kbtVSysiKHSpJ9bPr2xBHa-haKqbo9Ttw70UP49JO5vffnfOr~DbKqFYax3GRiSewA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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However, for many industrial reactions there are no naturally occurring enzymes, and so many different engineering approaches have been used to address this problem. Engineering methods have been used to alter the stability, substrate specificity and stereospecificity of aldolases to produce excellent enzymes for biocatalytic processes. Recently greater understanding of the aldolase mechanism has allowed many successes with both rational engineering approaches and computational design of aldolases. Rational engineering approaches have produced desired enzymes quickly and efficiently while combination of computational design with laboratory methods has created enzymes with activity approaching that of natural enzymes.","grobid_abstract_attachment_id":62459854},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307715/Engineering_aldolases_as_biocatalysts","translated_internal_url":"","created_at":"2020-03-24T09:24:03.386-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459854,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459854/thumbnails/1.jpg","file_name":"2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6.pdf","download_url":"https://www.academia.edu/attachments/62459854/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Engineering_aldolases_as_biocatalysts.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459854/2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DEngineering_aldolases_as_biocatalysts.pdf\u0026Expires=1732778827\u0026Signature=CiZeRULhOaBO~NtR-V9dNPpr1ATdd7m-nXcjt3uLeudwrik109-TDqxM-tZ0Zj8ZcyKsWwjT7eoJUSIHj-j2obAb28Wb7K8qnfltBGkEda5rA5d3~CnvbR2djDzHpXpiMK4PTKIrA1PgBW8pLG3CdsvJj8y~a1hXrl-i0V9aKf0hn8-FCeVL3mvlX1HylruEb34G8qvXJtCjI2nY3gxUM2KwLpdlZh5Cj7Z0fe6rd4ImbJw9JnAPdpTUnQyngzjeb7n8Tzs8KKmhu7kuS5dJFgTPNQUVv0wQCB9YUmIhXJS7OQlXn9IbP7YVe4WulBLg0uN8JLtRa~X3ZXLbzItV5A__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Engineering_aldolases_as_biocatalysts","translated_slug":"","page_count":9,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459854,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459854/thumbnails/1.jpg","file_name":"2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6.pdf","download_url":"https://www.academia.edu/attachments/62459854/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Engineering_aldolases_as_biocatalysts.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459854/2014_Engineering_aldolases_as_biocatalysts20200324-131026-yz2bs6-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DEngineering_aldolases_as_biocatalysts.pdf\u0026Expires=1732778827\u0026Signature=CiZeRULhOaBO~NtR-V9dNPpr1ATdd7m-nXcjt3uLeudwrik109-TDqxM-tZ0Zj8ZcyKsWwjT7eoJUSIHj-j2obAb28Wb7K8qnfltBGkEda5rA5d3~CnvbR2djDzHpXpiMK4PTKIrA1PgBW8pLG3CdsvJj8y~a1hXrl-i0V9aKf0hn8-FCeVL3mvlX1HylruEb34G8qvXJtCjI2nY3gxUM2KwLpdlZh5Cj7Z0fe6rd4ImbJw9JnAPdpTUnQyngzjeb7n8Tzs8KKmhu7kuS5dJFgTPNQUVv0wQCB9YUmIhXJS7OQlXn9IbP7YVe4WulBLg0uN8JLtRa~X3ZXLbzItV5A__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retroaldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model. E nzymes are excellent catalysts, and the ability to design new active enzymes could have applications in drug production (1), green chemistry (2), and bioremediation of xenobiotic pollutants (3). To date, most enzyme design efforts have used selection methodologies to retrieve very rare active catalysts from large libraries of candidate protein variants (4-7). Recent advances in computational protein design have made it possible to design new protein folds (8) and binding interactions (9) and have opened the door to the possibility of computationally designing enzymatic catalysts for any chemical reaction. Despite recent progress (10, 11), creating enzymes for chemical transformations not efficiently catalyzed by naturally occurring enzymes remains a major challenge. Here, we describe (i) general computational methods for constructing active sites for multistep reactions consisting of superimposed reaction intermediates and transition states (TS) surrounded by protein functional groups in orientations optimal for catalysis ( and (ii) the use of this methodology to design novel catalysts for a retro-aldol reaction in which a carbon-carbon bond is broken in a nonnatural (i.e., not found in biological systems) substrate: 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone ( (12).","grobid_abstract_attachment_id":62459844},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307714/De_Novo_Computational_Design_of_Retro_Aldol_Enzymes_Downloaded_from","translated_internal_url":"","created_at":"2020-03-24T09:24:03.233-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459844,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459844/thumbnails/1.jpg","file_name":"2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2.pdf","download_url":"https://www.academia.edu/attachments/62459844/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"De_Novo_Computational_Design_of_Retro_Al.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459844/2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2-libre.pdf?1585128336=\u0026response-content-disposition=attachment%3B+filename%3DDe_Novo_Computational_Design_of_Retro_Al.pdf\u0026Expires=1732778828\u0026Signature=cUZPy3gTLZgDy8y6GoNfH8gTt-cknQPw48ZeNvVWhSkookrw4xmcOjoFKKCkWWDbFZvgnWUy7bxW3v2oVZaQ9SB6axJyHmEY9ueY4ZS-HEUI3usvKo3fhl210~C0aSohyteucpFORSJdNA6zPLQxJ3zVSYOP3-oG~t3Y6oxI8Krg4V79Q8qKYZ5ANJeZGrM9Ehs-t2AAARMTLwm4EeaTbPVbN2NPLFQ7zIKf2E9wiYuGBh1sOQPimhN6HLF9QbgUPDO2OR0bwdvBhCmX1LwMkrfcEngHq9oxGz8LbnyCem55MwsWieIR9zllmBXjU3TmwB9sJSd9ZK2mNm2FqnWFYQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"De_Novo_Computational_Design_of_Retro_Aldol_Enzymes_Downloaded_from","translated_slug":"","page_count":6,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459844,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459844/thumbnails/1.jpg","file_name":"2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2.pdf","download_url":"https://www.academia.edu/attachments/62459844/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"De_Novo_Computational_Design_of_Retro_Al.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459844/2008_De_Novo_Computational_Design_of_Retro-Aldol_Enzymes20200324-10947-htypw2-libre.pdf?1585128336=\u0026response-content-disposition=attachment%3B+filename%3DDe_Novo_Computational_Design_of_Retro_Al.pdf\u0026Expires=1732778828\u0026Signature=cUZPy3gTLZgDy8y6GoNfH8gTt-cknQPw48ZeNvVWhSkookrw4xmcOjoFKKCkWWDbFZvgnWUy7bxW3v2oVZaQ9SB6axJyHmEY9ueY4ZS-HEUI3usvKo3fhl210~C0aSohyteucpFORSJdNA6zPLQxJ3zVSYOP3-oG~t3Y6oxI8Krg4V79Q8qKYZ5ANJeZGrM9Ehs-t2AAARMTLwm4EeaTbPVbN2NPLFQ7zIKf2E9wiYuGBh1sOQPimhN6HLF9QbgUPDO2OR0bwdvBhCmX1LwMkrfcEngHq9oxGz8LbnyCem55MwsWieIR9zllmBXjU3TmwB9sJSd9ZK2mNm2FqnWFYQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.","grobid_abstract_attachment_id":62459852},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307713/Robust_design_and_optimization_of_retroaldol_enzymes","translated_internal_url":"","created_at":"2020-03-24T09:24:03.131-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459852,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459852/thumbnails/1.jpg","file_name":"2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet.pdf","download_url":"https://www.academia.edu/attachments/62459852/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Robust_design_and_optimization_of_retroa.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459852/2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DRobust_design_and_optimization_of_retroa.pdf\u0026Expires=1732778828\u0026Signature=GNSCNBOlbgPKFY9Mybw4PB9XWfMzDKPWqO4OJKEFrwFynHNc0qJwec5GafcaPSD8F8BeD5YZU6pXrkdVZTeIz113bm8ZY4W7QsbLMBhY4syOvdbrVbeYNWDyKbBwBjNz7G0JmUarftt47Whkm95g7RJbPJFrezIPxHfyU3SOr4TrJKfVXegRekkjKZ0ZtGbbRl6cF7ntN-gqWdCYXwRk0DymZLm26erGknWhvZsCdhXHOH~XaCGj43VuZQ4VrvVdnZNikfd4nmLIYhnrVMK9RC4x9szKh-4kKH6G1PpM7kAsIvv12ujcZHL4Nyk8IOs7M8d31gqyJbp0xOcU-D6Usw__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Robust_design_and_optimization_of_retroaldol_enzymes","translated_slug":"","page_count":10,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459852,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459852/thumbnails/1.jpg","file_name":"2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet.pdf","download_url":"https://www.academia.edu/attachments/62459852/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Robust_design_and_optimization_of_retroa.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459852/2012_Robust_design_and_optimization_of_retroaldol_enzymes20200324-68722-1m5ctet-libre.pdf?1585128736=\u0026response-content-disposition=attachment%3B+filename%3DRobust_design_and_optimization_of_retroa.pdf\u0026Expires=1732778828\u0026Signature=GNSCNBOlbgPKFY9Mybw4PB9XWfMzDKPWqO4OJKEFrwFynHNc0qJwec5GafcaPSD8F8BeD5YZU6pXrkdVZTeIz113bm8ZY4W7QsbLMBhY4syOvdbrVbeYNWDyKbBwBjNz7G0JmUarftt47Whkm95g7RJbPJFrezIPxHfyU3SOr4TrJKfVXegRekkjKZ0ZtGbbRl6cF7ntN-gqWdCYXwRk0DymZLm26erGknWhvZsCdhXHOH~XaCGj43VuZQ4VrvVdnZNikfd4nmLIYhnrVMK9RC4x9szKh-4kKH6G1PpM7kAsIvv12ujcZHL4Nyk8IOs7M8d31gqyJbp0xOcU-D6Usw__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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Computational methods can be principally grouped into three main categories: bioinformatics; molecular modelling; and de novo design. Particularly de novo protein design is experiencing rapid development, resulting in more robust and reliable predictions. A recent trend in the field is to combine several computational approaches in an interactive manner and to complement them with structural analysis and directed evolution. 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The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s) with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi), to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. 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Different methods of enzyme engineering have been used in the past in an attempt to produce enzymes with improved functions and properties. Recent advancement in the field of random mutagenesis, screening, selection and computational design increased the versatility and the rapid development of enzymes under strong selection pressure with directed evolution experiments. Techniques of directed evolution improve enzymes fitness without understanding them in great detail and clearly demonstrate its future role in adapting enzymes for use in industry. Despite significant advances to date regarding biocatalyst improvement, there still remains a need to improve mutagenesis strategies and development of easy screening and selection tools without significant human intervention. 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window.Academia.workViewCountsFetcher.queue(workId, function (count) { var description = window.$h.commaizeInt(count) + " " + window.$h.pluralize(count, 'View'); $(".js-view-count[data-work-id=42307705]").text(description); $(".js-view-count[data-work-id=42307705]").attr('title', description).tooltip(); }); });</script></span></span><span><span class="percentile-widget hidden"><span class="u-mr2x work-percentile"></span></span><script>$(function () { var workId = 42307705; window.Academia.workPercentilesFetcher.queue(workId, function (percentileText) { var container = $(".js-work-strip[data-work-id='42307705']"); container.find('.work-percentile').text(percentileText.charAt(0).toUpperCase() + percentileText.slice(1)); container.find('.percentile-widget').show(); container.find('.percentile-widget').removeClass('hidden'); }); });</script></span><span><script>$(function() { new Works.PaperRankView({ workId: 42307705, container: "", }); });</script></span></div><div id="work-strip-premium-row-container"></div></div></div><script> require.config({ waitSeconds: 90 })(["https://a.academia-assets.com/assets/wow_profile-f77ea15d77ce96025a6048a514272ad8becbad23c641fc2b3bd6e24ca6ff1932.js","https://a.academia-assets.com/assets/work_edit-ad038b8c047c1a8d4fa01b402d530ff93c45fee2137a149a4a5398bc8ad67560.js"], function() { // from javascript_helper.rb var dispatcherData = {} if (true){ window.WowProfile.dispatcher = window.WowProfile.dispatcher || _.clone(Backbone.Events); 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Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating \"smart\" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.","grobid_abstract_attachment_id":62459830},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307705/Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution","translated_internal_url":"","created_at":"2020-03-24T09:24:02.167-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459830,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459830/thumbnails/1.jpg","file_name":"2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x.pdf","download_url":"https://www.academia.edu/attachments/62459830/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Polishing_the_craft_of_genetic_diversity.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459830/2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x-libre.pdf?1585128367=\u0026response-content-disposition=attachment%3B+filename%3DPolishing_the_craft_of_genetic_diversity.pdf\u0026Expires=1732778828\u0026Signature=aX2NUmpL2bsU9mUi4wWqrOeWeORefcAegMH5cjCd8xJwZLlj7ZjzUOly4KzrIdE95iU8cphhQZ6VBAGbVI0C3rfvCMykWPb3piVy1cYkb-QNRUgoVfaUYVRUU4VEqQ8EHZxuXdW9oaZ8cmCTTUUDxd-E7PDodubmroW9TptcK2O3Ye~Fg09GnsHUa565xcUAc0pv213STlCmQi~ziozSQYggiuIWkaw1WTMYeXi81YcGwwJ6Ol1I96mAdY8TzpSV-fSTjpdJ~8W2thSuFmDeD-P0rFOCYhDVofR1DDcrzoY5YlJ0ql0n1wFI3CC3bgiLSrqXOik4qL-KzV5eWuPxCA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution","translated_slug":"","page_count":15,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459830,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459830/thumbnails/1.jpg","file_name":"2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x.pdf","download_url":"https://www.academia.edu/attachments/62459830/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Polishing_the_craft_of_genetic_diversity.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459830/2013_Polishing_the_craft_of_genetic_diversity_creation_in_directed_evolution20200324-129876-1mi250x-libre.pdf?1585128367=\u0026response-content-disposition=attachment%3B+filename%3DPolishing_the_craft_of_genetic_diversity.pdf\u0026Expires=1732778828\u0026Signature=aX2NUmpL2bsU9mUi4wWqrOeWeORefcAegMH5cjCd8xJwZLlj7ZjzUOly4KzrIdE95iU8cphhQZ6VBAGbVI0C3rfvCMykWPb3piVy1cYkb-QNRUgoVfaUYVRUU4VEqQ8EHZxuXdW9oaZ8cmCTTUUDxd-E7PDodubmroW9TptcK2O3Ye~Fg09GnsHUa565xcUAc0pv213STlCmQi~ziozSQYggiuIWkaw1WTMYeXi81YcGwwJ6Ol1I96mAdY8TzpSV-fSTjpdJ~8W2thSuFmDeD-P0rFOCYhDVofR1DDcrzoY5YlJ0ql0n1wFI3CC3bgiLSrqXOik4qL-KzV5eWuPxCA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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window.Academia.workViewCountsFetcher.queue(workId, function (count) { var description = window.$h.commaizeInt(count) + " " + window.$h.pluralize(count, 'View'); $(".js-view-count[data-work-id=42307704]").text(description); $(".js-view-count[data-work-id=42307704]").attr('title', description).tooltip(); }); });</script></span></span><span><span class="percentile-widget hidden"><span class="u-mr2x work-percentile"></span></span><script>$(function () { var workId = 42307704; window.Academia.workPercentilesFetcher.queue(workId, function (percentileText) { var container = $(".js-work-strip[data-work-id='42307704']"); container.find('.work-percentile').text(percentileText.charAt(0).toUpperCase() + percentileText.slice(1)); container.find('.percentile-widget').show(); container.find('.percentile-widget').removeClass('hidden'); }); });</script></span><span><script>$(function() { new Works.PaperRankView({ workId: 42307704, container: "", }); });</script></span></div><div id="work-strip-premium-row-container"></div></div></div><script> require.config({ waitSeconds: 90 })(["https://a.academia-assets.com/assets/wow_profile-f77ea15d77ce96025a6048a514272ad8becbad23c641fc2b3bd6e24ca6ff1932.js","https://a.academia-assets.com/assets/work_edit-ad038b8c047c1a8d4fa01b402d530ff93c45fee2137a149a4a5398bc8ad67560.js"], function() { // from javascript_helper.rb var dispatcherData = {} if (true){ window.WowProfile.dispatcher = window.WowProfile.dispatcher || _.clone(Backbone.Events); dispatcherData = { dispatcher: window.WowProfile.dispatcher, downloadLinkId: "dde309827642dd570e1aea38f25dfb3a" } } $('.js-work-strip[data-work-id=42307704]').each(function() { if (!$(this).data('initialized')) { new WowProfile.WorkStripView({ el: this, workJSON: {"id":42307704,"title":"Strategy and success for the directed evolution of enzymes","translated_title":"","metadata":{"grobid_abstract":"Directed evolution is widely used to improve enzymes, particularly for industrial biocatalytic processes. Molecular biology advances present many new strategies for directed evolution. Commonly used techniques have led to many successful examples of enzyme improvement, yet there is still a need to improve both the efficiency and capability of directed evolution. Recent strategies aimed at making directed evolution faster and more efficient take better advantage of available structural and sequence information. The underlying principles that lead to early dead-ends for directed evolution experiments are also discussed along with recent strategies designed to by-pass them. Several emerging methods for creating novel enzymes are also discussed including examples of catalytic activity for which there is no precedent in nature. 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This was verified by sequencing 95 members from 11 independent libraries within the gene encoding alkene reductase OYE 2.6 from Pichia stipitis. Correct PCR primer design as well as a variety of factors that increase transformation efficiency were critical contributors to the method's overall success. We also developed a quantitative analysis of library quality (Q-values) that defines library degeneracy. Q-values can be calculated from standard fluorescence sequencing data (capillary electropherograms) and the degeneracy predicted from an early stage of library construction (pooled plasmids from the initial transformation) closely matched that observed after ca. 1000 library members were sequenced. Based on this experience, we suggest that this analysis can be a useful guide when applying our optimized protocol to new systems, allowing one to focus only on good-quality libraries and reject substandard libraries at an early stage. 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Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. 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At the current state of random mutagenesis technologies mutation frequencies have often been adjusted to values that cause a limited number of amino acid changes (often one to four amino acid changes per protein). For harvesting the power of directed evolution algorithms it is therefore important that generated mutant libraries are rich in diversity and enriched in active population. Insufficient knowledge about protein traits, mutational robustness of protein folds and technological limitations in diversity generating methods are main challenges for managing the complexity of protein sequence space. This review covers computational and experimental advances for high quality mutant library generation that have been achieved in the past two years.","grobid_abstract_attachment_id":62459819},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307699/Advances_in_generating_functional_diversity_for_directed_protein_evolution","translated_internal_url":"","created_at":"2020-03-24T09:24:01.331-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459819,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459819/thumbnails/1.jpg","file_name":"2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf.pdf","download_url":"https://www.academia.edu/attachments/62459819/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Advances_in_generating_functional_divers.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459819/2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf-libre.pdf?1585128370=\u0026response-content-disposition=attachment%3B+filename%3DAdvances_in_generating_functional_divers.pdf\u0026Expires=1732778828\u0026Signature=aVeWdVmlFr0Y1Yx2E2dkfnwAsfP~rQOt0Bi-lr0QHsNL9D9692-Gscq~GEqQZYph5nVAlFuuPZAuzZwQv0LnsM9w2LvueaF29dkPvAQc2Jz-WsHsig1Vz3Re~x3hkJe-XxkCMOAUM4BGXEaA9B~hr9iWexhv7E~N0~5DU9BsPXQQJ9IKJSK-IBXNUvaFmSzy5ZcCDUB~Mcq9ssMKxUVhYcN-s-gO2mFp~~t-yM-FtA5HNIL2rUdu~FZtLJ-BbotBCAYsIGDQ2-Rq8VCQTLiObxPd4KyP5lIt1y2z3R66E2yNP64vdC~z3~x5Y9OAImxEmfvqHvCr74Z6myQBnGKQbA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Advances_in_generating_functional_diversity_for_directed_protein_evolution","translated_slug":"","page_count":7,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459819,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459819/thumbnails/1.jpg","file_name":"2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf.pdf","download_url":"https://www.academia.edu/attachments/62459819/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Advances_in_generating_functional_divers.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459819/2009_Advances_in_generating_functional_diversity_for_directed_protein_evolution20200324-4002-a3dtf-libre.pdf?1585128370=\u0026response-content-disposition=attachment%3B+filename%3DAdvances_in_generating_functional_divers.pdf\u0026Expires=1732778828\u0026Signature=aVeWdVmlFr0Y1Yx2E2dkfnwAsfP~rQOt0Bi-lr0QHsNL9D9692-Gscq~GEqQZYph5nVAlFuuPZAuzZwQv0LnsM9w2LvueaF29dkPvAQc2Jz-WsHsig1Vz3Re~x3hkJe-XxkCMOAUM4BGXEaA9B~hr9iWexhv7E~N0~5DU9BsPXQQJ9IKJSK-IBXNUvaFmSzy5ZcCDUB~Mcq9ssMKxUVhYcN-s-gO2mFp~~t-yM-FtA5HNIL2rUdu~FZtLJ-BbotBCAYsIGDQ2-Rq8VCQTLiObxPd4KyP5lIt1y2z3R66E2yNP64vdC~z3~x5Y9OAImxEmfvqHvCr74Z6myQBnGKQbA__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.","grobid_abstract_attachment_id":62459817},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307698/Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries","translated_internal_url":"","created_at":"2020-03-24T09:24:01.182-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459817,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459817/thumbnails/1.jpg","file_name":"2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp.pdf","download_url":"https://www.academia.edu/attachments/62459817/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Steering_directed_protein_evolution_stra.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459817/2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp-libre.pdf?1585128374=\u0026response-content-disposition=attachment%3B+filename%3DSteering_directed_protein_evolution_stra.pdf\u0026Expires=1732778828\u0026Signature=HABOdCOGVWbx564c~D~BBQ422~hayjuPLg9yqVLf7K2o2nntHBLXpTOGJMQ~GQkdvupLqQIxJk4bK8O~uUDh~bna5HX8FALQGciPvKKY1oa6KCzpJudYFqcA40TWEvGgq094nYdyDhsk6CommMLFNDbiC-upO3rC1KjTz1cQDDbBuXKR365TPcqRN5Ars-VHsDLXCA8~wFwzin0xRBhbB~rzIUx3EqhjjYJuRbDD2DlLwzHwW5hKYLmoHWdRDw3FApWkkdVGu2kKhclMBm74bGU9vmSAczHLfdTOj7lRMNCyw8IdAezmhT47jZFn2H3hNelseNPz-YEvwefALrb8QQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Steering_directed_protein_evolution_strategies_to_manage_combinatorial_complexity_of_mutant_libraries","translated_slug":"","page_count":15,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459817,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459817/thumbnails/1.jpg","file_name":"2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp.pdf","download_url":"https://www.academia.edu/attachments/62459817/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Steering_directed_protein_evolution_stra.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459817/2007_Steering_directed_protein_evolution__strategies_to_manage_combinatorial_complexity_of_mutant_libraries20200324-8006-1loonqp-libre.pdf?1585128374=\u0026response-content-disposition=attachment%3B+filename%3DSteering_directed_protein_evolution_stra.pdf\u0026Expires=1732778828\u0026Signature=HABOdCOGVWbx564c~D~BBQ422~hayjuPLg9yqVLf7K2o2nntHBLXpTOGJMQ~GQkdvupLqQIxJk4bK8O~uUDh~bna5HX8FALQGciPvKKY1oa6KCzpJudYFqcA40TWEvGgq094nYdyDhsk6CommMLFNDbiC-upO3rC1KjTz1cQDDbBuXKR365TPcqRN5Ars-VHsDLXCA8~wFwzin0xRBhbB~rzIUx3EqhjjYJuRbDD2DlLwzHwW5hKYLmoHWdRDw3FApWkkdVGu2kKhclMBm74bGU9vmSAczHLfdTOj7lRMNCyw8IdAezmhT47jZFn2H3hNelseNPz-YEvwefALrb8QQ__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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One illustration of the efficiency of a data-driven approach is the work of Parikh and Matsumura [1], who showed that a single round of structure-guided site-saturation mutagenesis on 尾-galactosidase using the crystal structure with a bound substrate analog identified a variant that was 180-fold more active and 700 000-fold more specific for fucoside substrates than the wild-type enzyme. Previous mutants identified in seven rounds of directed evolution and screening were only 10-fold more active and 2-fold more specific than wild type, and required considerably more time and effort to screen [1, 2]. summarizes recent work in data-driven protein design. For each type of design outlined below, one specific example is dis-cussed in detail. The other examples are provided in the table to give a general summary (rather than the \"bestcase scenario\") of the work in this area and to direct interested readers towards other references worth examining.","grobid_abstract_attachment_id":62459815},"translated_abstract":null,"internal_url":"https://www.academia.edu/42307697/Better_library_design_data_driven_protein_engineering","translated_internal_url":"","created_at":"2020-03-24T09:24:01.119-07:00","preview_url":null,"current_user_can_edit":null,"current_user_is_owner":null,"owner_id":124399282,"coauthors_can_edit":true,"document_type":"paper","co_author_tags":[],"downloadable_attachments":[{"id":62459815,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459815/thumbnails/1.jpg","file_name":"2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e.pdf","download_url":"https://www.academia.edu/attachments/62459815/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Better_library_design_data_driven_protei.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459815/2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e-libre.pdf?1585128372=\u0026response-content-disposition=attachment%3B+filename%3DBetter_library_design_data_driven_protei.pdf\u0026Expires=1732778828\u0026Signature=fIb5OPPmx5tlYxZt~XONQ8AM2wrmYfdbPUGhVj3JomN7vexCpbnu9FBZGfCFmDo3mhWfyHSbRd0TbivUiXtWTIhEISnhDaUp2aRVkPruj7LaKZubfRTz5h94PPi3Z4yF1KYR1P-Zh7NtWWZ-VWsOO6fxZet3pW55gHkQBunyAzV9ghzkQxc9BtBMb2pWIrgsFoZar-dB9AjgkNNrZlefFyywLn9P8EswGiGpTtxcTMDJ6Rh23TSeFDjPsoInj33FDX4vZk4d6bq2kG59KA9U3EVM5c6RClQOXi5pK3bsBjfcbmpDrXuhL-k5zsY8esHg2SGcr5CUV3nSny80FKQT2Q__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"slug":"Better_library_design_data_driven_protein_engineering","translated_slug":"","page_count":12,"language":"en","content_type":"Work","owner":{"id":124399282,"first_name":"Lorenzo","middle_initials":null,"last_name":"Camisi","page_name":"LorenzoCamisi","domain_name":"unibo","created_at":"2019-08-30T13:14:37.617-07:00","display_name":"Lorenzo Camisi","url":"https://unibo.academia.edu/LorenzoCamisi"},"attachments":[{"id":62459815,"title":"","file_type":"pdf","scribd_thumbnail_url":"https://attachments.academia-assets.com/62459815/thumbnails/1.jpg","file_name":"2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e.pdf","download_url":"https://www.academia.edu/attachments/62459815/download_file?st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&st=MTczMjc3NTIyOCw4LjIyMi4yMDguMTQ2&","bulk_download_file_name":"Better_library_design_data_driven_protei.pdf","bulk_download_url":"https://d1wqtxts1xzle7.cloudfront.net/62459815/2007_Better_library_design__data-driven_protein_engineering_20200324-79295-3ov48e-libre.pdf?1585128372=\u0026response-content-disposition=attachment%3B+filename%3DBetter_library_design_data_driven_protei.pdf\u0026Expires=1732778828\u0026Signature=fIb5OPPmx5tlYxZt~XONQ8AM2wrmYfdbPUGhVj3JomN7vexCpbnu9FBZGfCFmDo3mhWfyHSbRd0TbivUiXtWTIhEISnhDaUp2aRVkPruj7LaKZubfRTz5h94PPi3Z4yF1KYR1P-Zh7NtWWZ-VWsOO6fxZet3pW55gHkQBunyAzV9ghzkQxc9BtBMb2pWIrgsFoZar-dB9AjgkNNrZlefFyywLn9P8EswGiGpTtxcTMDJ6Rh23TSeFDjPsoInj33FDX4vZk4d6bq2kG59KA9U3EVM5c6RClQOXi5pK3bsBjfcbmpDrXuhL-k5zsY8esHg2SGcr5CUV3nSny80FKQT2Q__\u0026Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA"}],"research_interests":[],"urls":[]}, dispatcherData: dispatcherData }); 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The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, stereo-, and regioselectivity), and/or stability (temperature, organic solvents, pH) is just limited by the throughput of the genetic selection, display, or screening system that is available for a given protein. Sometimes it is possible to analyze millions of protein variants from combinatorial libraries per day. In other cases, however, only a few hundred variants can be screened in a single day, and thus the creation of smaller yet smarter libraries is needed. Different strategies have been developed to create these libraries. One approach is to perform mutational scanning or to construct \"mutability landscapes\" in order to understand sequence-function relationships that can guide the actual directed evolution process. Herein we provide a protocol for economically constructing scanning mutagenesis libraries using a cytochrome P450 enzyme in a high-throughput manner. The goal is to engineer activity, regioselectivity, and stereoselectivity in the oxidative hydroxylation of a steroid, a challenging reaction in synthetic organic chemistry. Libraries based on mutability landscapes can be used to engineer any fitness trait of interest. 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