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Ancestral diterpene cyclases show increased thermostability and substrate acceptance
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.ORCID iD: 0000-0001-8644-3408
KTH, Centres, Science for Life Laboratory, SciLifeLab. ESCOM, 1 Allee Reseau Jean Marie Buckmaster, F-60200 Compiegne, France..
Swedish Orphan Biovitrum AB, Stockholm, Sweden..
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Coating Technology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Swedish Orphan Biovitrum AB, Stockholm, Sweden.ORCID iD: 0000-0002-4066-2776
2018 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 24, p. 4660-4673Article in journal (Refereed) Published
Abstract [en]

Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 degrees C. Ancestral spiroviolene synthases catalyze cyclization of the natural C-20-substrate geranylgeranyl diphosphate (GGPP) and also accept C-15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., k(cat)/K-M for GGPP over k(cat)/K-M for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018. Vol. 285, no 24, p. 4660-4673
Keywords [en]
ancestral sequence reconstruction, diterpene cyclase, spiroviolene synthase, protein stability, promiscuity
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-240741DOI: 10.1111/febs.14686ISI: 000453570200010PubMedID: 30369053Scopus ID: 2-s2.0-85056620593OAI: oai:DiVA.org:kth-240741DiVA, id: diva2:1274841
Funder
VINNOVA, 2016-03344Swedish Foundation for Strategic Research , ID16-0036Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20190103

Available from: 2019-01-03 Created: 2019-01-03 Last updated: 2019-01-07Bibliographically approved

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Hendrikse, NatalieSyrén, Per-Olof

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