Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain

Abstract

Background: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol.

Results: In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h(-1), respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH1 (81D) allele restored its growth on glucose at a maximum specific rate of 0.053 h(-1) in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h(-1).

Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.

Fig. 1

Growth profiles of the evolved Pdc negative strains and reverse engineered strains M81-11 and M81C. E1A, E1B and E1C are three evolved Pdc negative strains. M81-11 is a Pdc negative strain with a point mutation in MTH1 (ura3-52 his3-Δ1 pdc1∆ pdc5∆ pdc6∆ mth1::MTH1 ^81D). M81C is a M81-11 mutant with the CIT1 deletion. The cultivations were performed in minimal medium with 2% glucose in replicate. Error bars represent ±standard errors.

Fig. 2

Mapping and analysis of Mth1 mutations. Magenta text indicates positions for non-synonymous mutations identified in this study. a Result of a multiple alignment using homologous sequences (n = 22). The Mth1 sequence of S. cerevisiae is shown with colored conservation levels. Yellow indicates low conservation, white intermediate, and blue high. Gray and italic text indicates the phosphorylation region of casein kinase Yck1. b Analysis of the 22 amino acids from the conserved region at position 70–91 and secondary structure predictions using six different prediction programs [25, 26]. c A helix wheel representation of amino acids predicted to form an alpha-helix (codon 75–89). The amino acid types are colored as in b.

Fig. 3

Transcription analysis of HXTs (HXT1-7) in the M81-11 and wild type strain CEN.PK 113-11C. Cells for transcription analysis were harvested at exponential phase (OD₆₀₀ ~ 1). The expression levels of HXTs in wild type strain were set as 1.

Fig. 4

A simple illustration for possible roles of mutated proteins in the evolved Pdc negative strains. The blue solid arrows represent the reactions catalyzed by the enzymes, which are indicted in blue text. The blue dash line represents the transportation between different subcellular organelles. The black lines with a bar at one end represent the repression or inhibition. The red circles represent the block due to Pdc deletions. a Simplified acetyl-CoA metabolism in the parental Pdc negative strain. The PDH complex and TCA cycle enzymes is repressed by high glucose uptake via hexose transporters (HXT). b Simplified acetyl-CoA metabolism in evolved Pdc negative strain with point mutated Mth1 (Mth1*) and Cit1 (Cit1*). Glucose uptake via HXT decreases in the presence of Mth1*, resulting in derepression of the PDH complex and TCA cycle enzymes. Cit1* with predicted decreased activity allows more mitochondrial acetyl-CoA convert to acetate by Ach1, which can be transported to the cytosol and converted to acetyl-CoA there.