Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase

Abstract

Background: The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity.

Results: Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production.

Conclusion: Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.

Figure 1

Specific alkaline phosphatase activity in nmoles of product/mg of prot.*min (A) and estimation of integrated expression cassette copy number by dot-blot hybridization (B) in the S. cerevisiae transformants and control strain. BY4742 – recipient strain; BY4742/Pho8vac – recombinant strains bearing plasmid pUC57-delta1_2-ADHpr-PHO8-CYCt-kanMX. Alkaline phosphatase activity is shown in nmoles of product/mg of prot.*min. For dot-blot hybridization genomic DNA was isolated from WT and recombinant strains, diluted and loaded onto nitrocellulose film. Amounts of loaded DNA is shown under the photo. PHO8 gene was used as a probe.

Figure 2

Analysis of expression cassette integration pattern. (A) Southern hybridization. PHO8 gene was used as a probe. HindIII was used for genomic DNA restriction. 1, 12 – plasmid pUC57-delta1_2-ADHpr-PHO8-CYCt-kanMX; 2 – ladder; 3 - wild type strain, BY4742; 4–11 – BY4742/Pho8vac – recombinant strains containing vector pUC57-delta1_2-ADHpr-PHO8-CYCt-kanMX; B) “head-to-tail” conformation of vector integration.

Figure 3

Growth, glucose consumption and ethanol production during alcoholic fermentation of recombinant strains overexpressing vacuolar form of alkaline phosphatase. Strains were cultured under semianaerobic conditions in YNB medium with 200 g/L D-glucose. Cultures were incubated at 30°C, shaking at 120 rpm using. AS400 – WT, recipient strain. AS400/Pho8vac – strain containing vector pUC57-delta1_2-ADHpr-PHO8-CYCt-kanMX. A – biomass accumulation (g of biomass/L of medium); B – glucose consumption (g of glucose/L of medium); C – ethanol production (g of ethanol/L of medium).

Figure 4

Linear schemes of plasmids used in this study: pUC57-delta1_2-ADHpr-CYCt-kanMX (A), pUC57-delta1_2-ADHpr-PHO8-CYCt-kanMX (B), pUC57-delta1_2-ADHpr-PHO8_trunc-CYCt-kanMX (C). δ elements are shown as thick black arrows. ADH1 promoter, CYC1 terminator and kanMX4 gene are shown as chessboard-alike, doted and diagonal-hatched boxes, respectively. Unmodified PHO8 gene ORF is shown as white box and truncated PHO8 gene ORF is shown as black doted box. Origin of replication ORI and ampicillin resistance gene (bla) are shown as thin arrows.