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Series GSE59814 Query DataSets for GSE59814
Status Public on Nov 04, 2014
Title Engineering acetyl-CoA supply: Functional expression of a bacterial pyruvate-dehydrogenase complex in the cytosol of Saccharomyces cerevisiae
Organism Saccharomyces cerevisiae
Experiment type Expression profiling by high throughput sequencing
Summary The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl-CoA is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of a pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required the simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2 and E3 subunits, as well as genes involved in lipoylation of E2 and addition of lipoate to growth media. A strain lacking ACS, that expressed these E. faecalis genes, grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial micro-organisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways.
Overall design For both strains - mutant strain IMY104 and reference strain CEN.PK113-7D' three independent chemostat cultures were performed. Each of the chemosta was sampled for transcriptome analysis. Samples were processed as described below.
Contributor(s) Kozak BU, van Rossum HM, Luttik MA, Benjamin KR, Wu L, de Vries S, Daran J, Pronk JT, van Maris AJ
Citation(s) 25336454
Submission date Jul 28, 2014
Last update date May 15, 2019
Contact name Jean-Marc Daran
Phone +31 15 278 2412
Organization name Delft University of Technology
Department Department of Biotechnology
Lab Kluyver centre for genomics of industrial organisms
Street address Julianalaan 67
City Delft
ZIP/Postal code 2628BC
Country Netherlands
Platforms (1)
GPL17342 Illumina HiSeq 2500 (Saccharomyces cerevisiae)
Samples (6)
GSM1446826 BK34
GSM1446827 BK35
GSM1446828 BK36
BioProject PRJNA256305
SRA SRP044920

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Supplementary file Size Download File type/resource
GSE59814_annotation_map.txt.gz 163.8 Kb (ftp)(http) TXT
GSE59814_processed_data.txt.gz 507.7 Kb (ftp)(http) TXT
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Raw data are available in SRA
Processed data are available on Series record

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