Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae , Yielding a C ₂ -Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast

Abstract

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc ؊ ) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc ؊ S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C 2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc ؊ strain without these growth defects. An acetateindependent Pdc ؊ mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C 2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc ؊ S. cerevisiae mutant (TAM) that could grow in batch cultures ( max ‫؍‬ 0.20 h ؊1 ) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucoserepressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter ؊1 was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass ؊1 h ؊1 during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose ؊1 .

Fermentation performance of engineered and evolved xylose‐fermenting Saccharomyces cerevisiae strains

“…The successful improvement of desirable process properties from various pentose-utilizing strains by different random approaches (Becker and Boles, 2003;Sonderegger and Sauer, 2003;van Maris et al, 2004;Wahlbom et al, 2003) suggests that further improved process strains will be generated in this fashion. By altering the expression pattern of several genes involved in redox metabolism, evolutionary strategies have significantly reduced xylitol formation in the TMB3001-derivatives C1 and C5 (Sonderegger and Sauer, 2003;Sonderegger et al, 2004).…”

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae

“…10,14 This requirement for C2 compounds was attributed to a deficiency of this Pdc-negative strain to synthesize cytosolic acetyl-CoA. 17,18 On the defined medium, the Pdc-negative MK5376 strain exhibited no growth in the presence of glucose as reported, 10,14 but showed growth in the presence of glycerol, glycerol plus ethanol, pyruvate, and, in particular, with mannitol alone (Fig.…”

“…This was a higher yield, but lower productivity, compared to the TAM strain (yield of 0.54 g of pyruvate per g of glucose, 135 g/L pyruvate production, consumption of 250 g/L glucose for 4 days). 10 The difference in pyruvate productivity between the Pdcnegative strain MK5376 and the TAM strain could be attributed to fact that MK5376 metabolized mannitol less efficiently than TAM metabolized glucose, as indicated by the amount of sugar consumption (1.12 g/L mannitol vs 250 g/L glucose) and biomass formation (A 600 of 1.7 [MK5376] vs. A 600 of 50 [TAM strain]) after 4 d of cultivation (Fig. 2).…”

“…17 Pyruvate production from mannitol In a previous attempt to produce pyruvate from glucose using S. cerevisiae, a Pdc-negative strain in a CEN.PK113-7D background was evolved to a TAM strain. 10 The TAM strain was independent of C2 compounds and tolerant to glucose, and produced 135 g/L pyruvate with an overall yield of 0.54 g of pyruvate per g of glucose.…”

“…8,9 Pyruvate production from glucose has been achieved using a pyruvate decarboxylase (Pdc)-negative S. cerevisiae TAM strain. 10 The goal of this study was to produce pyruvate from mannitol using our mannitol-assimilating S. cerevisiae strain. 6 …”