Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase

Abstract

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald(-) strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald(-) A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.

Keywords: acetyl-CoA; energetics; evolutionary engineering; intracellular metabolites; precursor supply; yeast.

Figure 1.

Maximum specific growth rates in synthetic medium shake-flask cultures with 20 g L⁻¹ ethanol as the sole carbon source (black bars) and the comparison with maximum specific growth rates on synthetic medium with 20 g L⁻¹ ethanol and 0.68 g L⁻¹ sodium acetate trihydrate (white bars, panel A); or synthetic medium with 20 g L⁻¹ ethanol as a main carbon source and 6.7 g L⁻¹ alanine as the nitrogen source (white bars, panel B) of the A-ALD-dependent evolved strains: IMS456 (evolved Ald⁻dmpF^T137S), IMS457 (evolved Ald⁻dmpF^I196L), IMS458 (evolved Ald⁻lin1129), IMS459 (evolved Ald⁻ Acs⁻eutE^V013D) and IMS460 (evolved Ald⁻ Acs⁻eutE ^Q4_E7del); the A-ALDs dependent reverse engineered strains: IMZ510 (Ald⁻acs1Δ dmpF), IMZ511 (Ald⁻acs1Δ eutE), IMZ512 (Ald⁻acs1Δ lin1129), IMZ513 (Ald⁻acs1Δ dmpF^T137S), IMZ514 (Ald⁻acs1Δ eutE ^Q4_E7del), IMZ528 (Ald⁻acs1Δ dmpF^I196L) and IMZ529 (Ald⁻acs1Δ eutE^V013D). For the evolved strains, partial genotype was indicated in the brackets; additional mutations identified in those strains are described in the text. Averages and mean deviations were obtained from duplicate experiments.

Figure 2.

Schematic representation of the selected reactions of central metabolism of S. cerevisiae growing on ethanol as the only carbon source. Abbreviations: ACS—acetyl-CoA synthetase, ALD—acetaldehyde dehydrogenase, A-ALD—acetylating acetaldehyde dehydrogenase, Ach1—CoA-transferase, Idp2—isocitrate dehydrogenase, PPP—pentose phosphate pathway and TCA—tricarboxylic acid cycle.