Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast.
Proc Natl Acad Sci U S A
; 117(37): 22974-22983, 2020 09 15.
Article
in En
| MEDLINE
| ID: mdl-32873649
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.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Oxidoreductases
/
Fatty Alcohols
Language:
En
Journal:
Proc Natl Acad Sci U S A
Year:
2020
Type:
Article
Affiliation country:
Sweden