Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1

Abstract

ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase) plays a central role in carbon metabolism and has been the site of action for the development of therapeutics or herbicides, as its product, malonyl-CoA, is a precursor for production of fatty acids and other compounds. Control of Acc1 activity in the yeast Saccharomyces cerevisiae occurs mainly at two levels, i.e., regulation of transcription and repression by Snf1 protein kinase at the protein level. Here, we demonstrate a strategy for improving the activity of ACCase in S. cerevisiae by abolishing posttranslational regulation of Acc1 via site-directed mutagenesis. It was found that introduction of two site mutations in Acc1, Ser659 and Ser1157, resulted in an enhanced activity of Acc1 and increased total fatty acid content. As Snf1 regulation of Acc1 is particularly active under glucose-limited conditions, we evaluated the effect of the two site mutations in chemostat cultures. Finally, we showed that our modifications of Acc1 could enhance the supply of malonyl-CoA and therefore successfully increase the production of two industrially important products derived from malonyl-CoA, fatty acid ethyl esters and 3-hydroxypropionic acid. IMPORTANCE ACCase is responsible for carboxylation of acetyl-CoA to produce malonyl-CoA, which is a crucial step in the control of fatty acid metabolism. ACCase opened the door for pharmaceutical treatments of obesity and diabetes as well as the development of new herbicides. ACCase is also recognized as a promising target for developing cell factories, as its malonyl-CoA product serves as a universal precursor for a variety of high-value compounds in white biotechnology. Yeast ACCase is a good model in understanding the enzyme's catalysis, regulation, and inhibition. The present study describes the importance of protein phosphorylation in regulation of yeast ACCase and identifies potential regulation sites. This study led to the generation of a more efficient ACCase, which was applied in the production of two high-value compounds derived from malonyl-CoA, i.e., fatty acid ethyl esters that can be used as biodiesel and 3-hydroxypropionic acid that is considered an important platform chemical.

FIG 1

Malonyl-CoA works as a major building block. The primary fate of malonyl-CoA is to serve as a precursor for lipids. However, a wide range of industrially interesting fuels and chemicals are derived from malonyl-CoA. A cell factory with enhanced supply of malonyl-CoA could serve as a platform for malonyl-CoA-derived products. Acc1 is a critical enzyme for malonyl-CoA synthesis, and the global regulator Snf1 is involved in regulation of Acc1 at the posttranslational level.

FIG 2

Reconstruction of FAEEs and 3-HP biosynthesis in Saccharomyces cerevisiae. Single and double arrows represent single and multiple enzymatic steps; dashed arrows represent heterologous pathways. FAEEs, fatty acid ethyl esters; 3-HP, 3-hydroxypropionic acid.