Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

Abstract

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.

Keywords: Acetyl-CoA; Coenzyme A; Krebs cycle; Mass spectrometry; Stable isotope labeling; Yeast.

Figure 1. Pantothenate is an essential nutrient in organisms lacking pantothenate synthetase, encoded for by the PAN6 gene in S. cerevisiae

Enzymes are shown in bold: Valine-pyruvate transaminase (VPT); ketopantoate hydroxymethyl transferase (KPHMT); ketopantoate reductase (KPR); aspartate alpha-decarboxylase (ADC); pantothenate synthetase (PS); pantothenate kinase (PanK); phosphopantothenoylcysteine synthetase (PPCS); phosphopantothenoylcysteine decarboxylase (PPCDC); phosphopantetheine adenylyltransferase (PPAT); dephospho-CoA kinase (DPCK). (B) Incorporation of [¹³C₃ ¹⁵N₁]-isotopic label into CoA via biosynthesis of CoA from pantothenate.

Figure 2. Incorporation of [¹³C₃ ¹⁵N₁]-isotopic label into CoA via biosynthesis of CoA from pantothenate

Chemical structures for conversion of pantothenate to 4’-phosphopantothenate (1), (R)-4’-phosphopantothenoyl-L-cysteine (2), 4’-phosphopantotheine (3), 3’-dephosphocoenzyme A (4), and coenzyme A (5).

Figure 3. Biosynthesis and incorporation of isotopic labels from pantothenate (vitamin B5) into Coenzyme A

(A) Comparative workflow for SILEC with mammalian versus yeast culture. (B) Confirmation of pan6Δ yeast auxotrophy for vitamin B5 (VB5) by growth on agar with (+) or without (-) pantothenate.

Figure 4. Yeast SILEC rapidly produces efficiently labeled [¹³C₃ ¹⁵N₁]-acyl-CoAs from [¹³C₃ ¹⁵N₁]-pantothenate

Pan6Δ yeast incubated over 31 h at 30°C show an increase in (A) optical density and (B) amount of isotopically labeled acetyl-CoA. (C) Lysis by sonication (black fill) or with glass beads (white fill) did not significantly alter yields. Maximum yield of [¹³C₃ ¹⁵N₁]-acetyl-CoA was obtained at 26-29 h, corresponding to the end of log phase growth.

Figure 5. Formation and stability of labeled acyl-CoAs by yeast SILEC

Pan6Δ yeast was incubated over 31 h at 30°C and extracted using the short-chain acyl-CoA method. (A) Levels of [¹³C₃ ¹⁵N₁]-labeled acetyl-CoA and HMG-CoA. (B) Levels of [¹³C₃ ¹⁵N₁]-labeled succinyl-CoA, CoA, and βHB-CoA. (C) Stability of yeast SILEC-derived acetyl-CoA through five freeze-thaw cycles.

Figure 6. MH⁺ signals from constant neutral loss scans of 507 Da for the acyl-CoA extract from

(A) Hepa1c1c7 SILEC (B) yeast SILEC, and mixed chain acyl-CoA extract from (C) Hepa1c1c7 SILEC (D) yeast SILEC (E) yeast SILEC induced with fatty acid treatment.