Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast

Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast

ACS Synthetic Biology

10.1021/acssynbio.5c00143

https://doi.org/10.1021/acssynbio.5c00143

Nazreen V. M. Abdul Muthaliff, Nur Eka Fitriani, Derek Smith, Jing Sen Ong, Lay Kien Yang, Coleen Toledo Busran, Aaron Thong, Prakash Arumugam, Naazneen Sofeo

03 September 2025

Abdul Muthaliff, N. V. M., Fitriani, N. E., Smith, D., Ong, J. S., Yang, L. K., Busran, C. T., Thong, A., Arumugam, P., & Sofeo, N. (2025). Mutations in the Substrate-Binding Pocket of DiacylGlycerol Acyltransferase Alter the Fatty Acid Composition of Triacylglycerides in Yeast. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.5c00143

Triacylglycerols (TAGs) are the main components of food oils and fats. The fatty acid composition of TAGs varies for different oils and fats. Specific enzymes sequentially add three fatty acids to the glycerol backbone of TAGs. Diacylglycerol acyltransferase or DGAT adds the third and ultimate fatty acid to the glycerol backbone at the sn-3 position. In this study, we characterized the substrate-binding pocket of enzyme DGAT1 from Arabidopsis thaliana through heterologous expression in the DGAT mutant of Saccharomyces cerevisiae. We performed site saturation mutagenesis on 10 amino acid residues in the catalytic site and examined their effects on the fatty acid profile of yeast cells. Our results indicate that mutations F373G, T240I, M289F, and V248I impact the yeast TAG profile either in terms of the total saturation level or the carbon chain length of the fatty acids, suggesting that they change the DGAT’s substrate preference. This offers insights into crucial amino acid residues in the DGAT binding pocket which can be engineered for fine tuning the lipid profile. In summary, we have harnessed the power of enzyme engineering to modify the fatty acyl makeup of triglycerides and created a sustainable platform for the production of customized alternative lipids.

Publisher Copyright

This research / project is supported by the Agency for Science, Technology, and Research (A*STAR), Republic of Singapore - Career Development Fund (CDF 2022)
Grant Reference no. : C222812021

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see 10.1021/acssynbio.5c00143.

https://oar.a-star.edu.sg/communities-collections/articles/22212

2161-5063
2161-5063

Singapore Institute of Food and Biotechnology Innovation