Engineering cytosolic acetyl-coenzyme A supply in Saccharomyces cerevisiae: Pathway stoichiometry, free-energy conservation and redox-cofactor balancing

Abstract

Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation

“…In S. cerevisiae , five genes were assigned to encode ACDHs: ALD1 / ALD6 (YPL061w), ALD2 (YMR170c) and ALD3 (YMR169c), which encode cytosolic isoforms, as well as ALD4 (YOR374w) and ALD5 (YER073w), which encode mitochondrial isoforms (Navarro‐Avino, Prasad, Miralles, et al, ). Acetate is mainly produced in the cytoplasm as the product of PDH (pyruvate dehydrogenase) bypass, which works by supplying cytosolic acetyl‐CoA used for anabolic reactions towards biomass production, as well as for regulatory acetylation of proteins and chromatin (reviewed by Van Rossum, Kozak, Pronk, et al, ). This PDH bypass is the same as that involved in the production of ethanol, which starts with the action of pyruvate decarboxylase (PDC) to convert pyruvate from glucose to acetaldehyde.…”

“…Therefore, the balance between ADH and ACDH activities and the redox state of the cell metabolism directs the fate of acetaldehyde and might define the fermentative capacity of the yeast. Besides the PDH bypass, acetate can also be provided by the mitochondria through the action of acetyl‐CoA hydrolase, encoded by the gene ACH1 (Van Rossum et al, ). Physiologically, the Ach1 enzyme of S. cerevisiae works mainly in the direction of transferring CoA from succinyl‐CoA to the acetate, which enters the mitochondria from the cytosol.…”

Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

“…In addition, the natural production route requires two ATP energy equivalents per acetyl-CoA formed, as the activation of acetate by acetyl-CoA synthase (Acs) consumes ATP and releases AMP. Due to these limitations, many strategies have been evaluated to increase cytosolic acetyl-CoA supply in S. cerevisiae (van Rossum et al 2016). In short, the strategies fall into three categories: (1) native pathway upregulation (Shiba et al 2007), (2) bypass of the ATP-dependent Acs-reaction with heterologous pathways, such as pyruvate formate lyase (Pfl), acetylating acetaldehyde dehydrogenase (A-Ald) and cytosolic pyruvate dehydrogenase (Pdh cyto ) (Kozak et al 2014a, b; Zhang et al 2015) or (3) introduction of the additional acetyl-CoA generating pathways, either an ATP-citrate lyase (Acl) (Feng et al 2015; Rodriguez et al 2016; Tang et al 2013; Zhou et al 2016) or a two-step pathway based on a phosphoketolase and a phosphotransacetylase (Xfpk/Pta) (de Jong et al 2014a; Sonderegger et al 2004).…”

“…Furthermore, AcP can be converted to acetyl-CoA by another common bacterial enzyme, phosphotransacetylase (Pta; EC 2.3.1.8), without requiring any energy input (Campos-Bermudez et al 2010). These combined aspects make this metabolic route highly attractive when targeting production of acetyl-CoA derived biochemicals in yeast, and optimal from a carbon-conservation perspective (Bogorad et al 2013; Henard et al 2015; van Rossum et al 2016). …”

Transcriptome Analyses Shed New Insights into Primary Metabolism and Regulation of Blumeria graminis f. sp. tritici during Conidiation

“…et al, 2008) were up-regulated ( Figure 3 ). Moreover, two genes acting in the glyoxylate cycle and two in the carnitine transport, where acetyl CoA is transported from peroxisome to mitochondria (van Rossum et al, 2016), were also activated ( Figure 3 ). These results suggest that lipid degradation is repressed, while unsaturated fatty acid oxidation is enhanced.…”