Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast

Yating Hu, Zhiwei Zhu, David Gradischnig, Margit Winkler, Jens Nielsen*, Verena Siewers

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.

Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number37
Pages (from-to)22974-22983
ISSN0027-8424
DOIs
Publication statusPublished - 2020

Keywords

  • Carboxylic acid reductase
  • High-throughput screening
  • Medium-chain fatty alcohols
  • Protein engineering
  • Saccharomyces cerevisiae

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