Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast
Journal article, 2020

Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme’s substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

High-throughput screening

Medium-chain fatty alcohols

Protein engineering

Carboxylic acid reductase

Saccharomyces cerevisiae

Author

Yating Hu

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Zhu Zhiwei

Dalian University of Technology

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

David Gradischnig

Technische Universität Graz

Margit Winkler

Austrian Centre of Industrial Biotechnology GmbH

Technische Universität Graz

Jens B Nielsen

Technical University of Denmark (DTU)

BioInnovation Institute

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Verena Siewers

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Proceedings of the National Academy of Sciences of the United States of America

0027-8424 (ISSN) 1091-6490 (eISSN)

Vol. 117 37 22974-22983

Subject Categories

Biochemistry and Molecular Biology

Microbiology

Biocatalysis and Enzyme Technology

DOI

10.1073/pnas.2010521117

PubMed

32873649

More information

Latest update

10/12/2020