Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation inSaccharomyces cerevisiae

Abstract

An increase in ethanol yield by yeast from the fermentation of conventional sugars such as glucose and sucrose is possible by reducing the production of a key byproduct such as cellular biomass. Previously we have reported that overexpression of PHO8 gene encoding non-specific ATPhydrolyzing alkaline phosphatase can lead to a decrease in cellular ATP content and to an increase in ethanol yield during glucose fermentation by Saccharomyces cerevisiae. In this work we further report on 2 new successful approaches to reduce cellular levels of ATP that increase ethanol yield and productivity. The first approach is based on the overexpression of the heterologous Escherichia coli apy gene encoding apyrase or SSB1 part of the chaperon that exhibit ATPase activity in yeast. In the second approach we constructed a futile cycle by the overexpression of S. cerevisiae genes encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase in S. cerevisiae. These genetically engineered strains accumulated more ethanol compared to the wild-type strain during alcoholic fermentation.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli

“…Chao et al [19] Koebmann et al [9] Holm et al [18] Hädicke et al [8] Liu et al [10] Semkiv et al [15] This study Direct ATP wasting via F 1 -ATPase…”

“…For pathways with ATP limitations, increasing the pool of available ATP may improve the production of desired compounds, e.g., of succinic acid or of recombinant proteins . A contrary strategy for metabolic engineering has been proposed more recently based on enforced ATP consumption (or enforced ATP wasting) . The main idea behind this approach is as follows: if product synthesis is coupled to net ATP synthesis, an increased ATP drain should—not only for thermodynamic reasons, but also due to evolutionary pressure—lead to an increased flux along the product pathway.…”

“…[14] A contrary strategy for metabolic engineering has been proposed more recently based on enforced ATP consumption (or enforced ATP wasting). [8,10,15,16] The main idea behind this approach is as follows: if product synthesis is coupled to net ATP synthesis, an increased ATP drain should-not only for thermodynamic reasons, but also due to evolutionary pressure-lead to an increased flux along the product pathway. Enforced ATP hydrolysis has been implemented either by the introduction of short futile cycles [8,15,17] or by a more "direct" approach via expressing ATP-hydrolyzing enzymes such as the (uncoupled) cytosolic F 1 -subunit of the ATPase from Escherichia coli.…”

“…[8,10,15,16] The main idea behind this approach is as follows: if product synthesis is coupled to net ATP synthesis, an increased ATP drain should-not only for thermodynamic reasons, but also due to evolutionary pressure-lead to an increased flux along the product pathway. Enforced ATP hydrolysis has been implemented either by the introduction of short futile cycles [8,15,17] or by a more "direct" approach via expressing ATP-hydrolyzing enzymes such as the (uncoupled) cytosolic F 1 -subunit of the ATPase from Escherichia coli. [9,10,18] Early studies [9,18,19] focused mainly on the physiological response of the cells upon exposure to elevated ATP drain and did not aim to increase the synthesis of certain products ( Table 1).…”

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)

“…Therefore, intracellular FBPase activity level is maintained at basal level in cells grown in media containing fermentable carbon sources. To overcome the tight regulation of the yeast FBPase, the bacterial FBPase from Escherichia coli that is insensitive to fructose-2,6-biphosphate inhibition was constitutively expressed in yeast [127,164]. The resulting recombinant strain exhibited a threefold increase in FBPase activity, a 30% reduced intracellular ATP level and up to 9% increase of ethanol production relative to that of parental strain [164].…”