Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids

  • Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.

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Author:Laura PöschelORCiDGND, Mónica Guevara-MartínezORCiD, David HörnströmORCiD, Antonius J. A. van MarisORCiD, Markus BuchhauptORCiDGND
Parent Title (English):Applied microbiology and biotechnology
Place of publication:Berlin ; Heidelberg [u.a.]
Document Type:Article
Date of Publication (online):2023/08/12
Date of first Publication:2023/08/12
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/11/13
Tag:3-Hydroxybutyric acid; Dicarboxylic acids; Enzyme engineering; Methylorubrum extorquens; Thioesterase
Page Number:18
First Page:6219
Last Page:6236
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International