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The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the <jats:italic>E. coli<\/jats:italic> DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The <jats:italic>Ec<\/jats:italic> DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. <jats:italic>Ec<\/jats:italic> DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Key points<\/jats:title>\n<jats:p>\u2022 <jats:italic>DERA aldolases are promiscuous enzymes.<\/jats:italic><\/jats:p>\n<jats:p>\u2022 <jats:italic>Synthetic utility of DERA aldolase was improved by protein engineering approaches.<\/jats:italic><\/jats:p>\n<jats:p>\u2022 <jats:italic>Machine learning methods aid the protein engineering of DERA.<\/jats:italic><\/jats:p>\n<\/jats:sec>","DOI":"10.1007\/s00253-020-10960-x","type":"journal-article","created":{"date-parts":[[2020,11,4]],"date-time":"2020-11-04T11:03:01Z","timestamp":1604487781000},"page":"10515-10529","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":34,"title":["Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods"],"prefix":"10.1007","volume":"104","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2303-4214","authenticated-orcid":false,"given":"Sanni","family":"Voutilainen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"Heinonen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martina","family":"Andberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmi","family":"Jokinen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannu","family":"Maaheimo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johan","family":"P\u00e4\u00e4kk\u00f6nen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina","family":"Hakulinen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juha","family":"Rouvinen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Harri","family":"L\u00e4hdesm\u00e4ki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel","family":"Kaski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juho","family":"Rousu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Merja","family":"Penttil\u00e4","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anu","family":"Koivula","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,11,4]]},"reference":[{"key":"10960_CR1","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1107\/S0907444909052925","volume":"66","author":"PD Adams","year":"2010","unstructured":"Adams PD, Afonine PV, Bunk\u00f3czi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX\u00a0: a comprehensive Python-based system for macromolecular structure solution. 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