Mechanism-Driven Metabolic Engineering for Bio-Based Production of Free R-Lipoic Acid in Saccharomyces cerevisiae Mitochondria

Abstract

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R-lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S-lipoic acid or precursors with undesired chirality. Toward sustainable production of R-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R-lipoic acid biosynthesis in S. cerevisiae. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R-lipoic acid production.

Mitochondrial Engineering of Yarrowia lipolytica for Sustainable Production of α-Bisabolene from Waste Cooking Oil

“…To benefit from the substantial acetyl-CoA pool in the mitochondria for α-bisabolene production, the target pathway enzymes were fused with an N-terminus 26-amino-acid MLS that originated from subunit IV of the cytochrome oxidase (CoxIV) in S. cerevisiae, which is typically cleaved off upon entry into the mitochondrial matrix . To verify the subcellular targeting capability of the MLS in Y.…”

“…19 To benefit from the substantial acetyl-CoA pool in the mitochondria for α-bisabolene production, the target pathway enzymes were fused with an N-terminus 26-aminoacid MLS that originated from subunit IV of the cytochrome oxidase (CoxIV) in S. cerevisiae, which is typically cleaved off upon entry into the mitochondrial matrix. 5 To verify the subcellular targeting capability of the MLS in Y. lipolytica, fluorescence microscopy was carried out to observe the fluorescence distribution in the yeast strains. By applying a mitochondrion-specific dye "MitoTracker Red" as a reference, the overlap of the red fluorescence signal from the dye with the green fluorescence signal from the MLS-tagged GFP in Po1g MLS-GFP confirmed that the MLS successfully translocated the GFP to the mitochondria (Figure 2).…”

“…Yeasts have been popular microbes for metabolic engineering due to their robustness and favorable characteristics for fermentation. − Conventionally, yeast metabolic engineering has focused mainly on implementing metabolic pathways in the cytoplasm and has overlooked the tremendous potential of developing subcellular compartments into organocellular factories for chemical production. Isolated from the cytosol, each membrane-enclosed organelle has evolved to possess unique physiological properties, such as physiochemical environments (e.g., pH and redox potential) and biomolecular profiles (e.g., precursors, enzymes, and co-factors), to provide congenial conditions for specific biochemical reactions .…”

Heterologous expression of cyanobacterial gas vesicle proteins in Saccharomyces cerevisiae

“…The membrane was incubated for 1 h in blocking buffer (0.5% non-fat dry milk) in TBS buffer containing 0.1% Tween 20 (TBST), washed three times for 10 min in TBST buffer, and incubated overnight at 4 • C with an HRP conjugated anti-HIS antibody diluted by 1:1000 (Thermo Fisher Scientific, MA, USA) in TBST buffer. [45] The membrane was washed for three times using TBST buffer and developed using the Signal Fire ECL reagent (Cell Signaling Technology, MA, USA).…”

NMR chiral recognition of lipoic acid by cinchonidine CSA: A stereocenter beyond the organic function

“…There are distinct studies demonstrating the effectiveness of using only the natural (active) form of ALA 11–14 . In this sense, the development of asymmetric synthetic protocols is imperative to access this goal 15–17 …”