Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

Abstract

Phosphoketolases catalyze an energy- and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.

Keywords: Acetyl-CoA; Acetyl-phosphate; Carbon efficiency; Phosphoketolase; Saccharomyces cerevisiae.

Fig. 1

Native and heterologous metabolic pathways for improved cytosolic acetyl-CoA production in S. cerevisiae. The native enzymatic pathways are indicated with light blue arrows, while the colored arrows indicate the alternative pathways that have been evaluated for cytosolic acetyl-CoA production in yeast. A-Ald acetylating acetaldehyde dehydrogenase, Ac-CoA acetyl-CoA, AcAld acetaldehyde, Ace acetate, Acl ATP citrate lyase, AcP acetyl-phosphate, Cit citrate, E4P erythrose-4-phosphate, F6P fructose-6-phosphate, G3P glycerol-3-phosphate, G6P glucose-6-phosphate, Pdh _cyto cytosolic pyruvate dehydrogenase complex, Pfl pyruvate formate lyase, Pta phosphotransacetylase, Pyr pyruvate, R5P ribose-5-phosphate, Ri5P ribulose-5-phosphate, S7P seduheptulose-7-phosphate, X5P xylulose-5-phosphate, Xfpk phosphoketolase

Fig. 2

Phylogeny of investigated phosphoketolase candidates. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree (with the sum of branch length = 2.424) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree

Fig. 3

Specific activities of the nine tested Xfpk enzymes with respect to substrates X5P and F6P. The assay was performed using crude cell free extracts. The legends refer to the different xfpk genes expressed in strains AB1–AB9. A strain harboring pSP-GM1 (AB10) served as negative control. All xfpk candidates except for xfpk(AN) and xfpk(LPP) displayed significantly higher activity (p < 0.005) when compared to the negative control with respect to both substrates. The results shown are averages from two biological replicates each performed in duplicates; error bars indicate the standard deviation

Fig. 4

AcP degradation in crude extracts of CEN.PK113-5D and corresponding GPP1 and/or GPP2 deletions strains. The legends refer to the strains used to prepare crude cell free extracts. Protein extraction buffer (Buffer) served as a negative control. 20 mM AcP was added in the reaction mixture. Strains carrying deletions show significantly reduced AcP levels when compared to the control strain CEN.PK 113-5D. The results shown are averages from two biological replicates each performed in duplicates; error bars indicate the standard deviation

Fig. 5

Phosphoketolase activity correlates with increased acetate accumulation and reduced growth rate and biomass formation. a Maximal acetate accumulation and maximum specific growth rate measured in cultivations of xfpk expressing strains. b Acetate accumulation and optical density measurements during whole growth phase. The legends refer to the xfpk genes expressed in strains AB1, AB3, AB5 and AB6. pSP-GM1 harboring strain AB10 served as negative control. Triangles correspond to acetate measurements and circles optical density. Strains were grown in shake flasks with minimal medium containing 2% glucose. The results shown are averages from three biological replicates; error barss indicate the standard deviation