Dataset Information

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae.

ABSTRACT: Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO₂ in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study, we reported a new group of succinic acid transporters from the AceTr family, which were also described as anion channels. Here, we expressed these transporters in a succinic acid overproducing strain and compared their impact on extracellular succinic acid accumulation with that of the Dct-02 transporter. The results show that the tested transporters of the AceTr family hinder succinic acid accumulation in the extracellular medium at low pH, which is in strong contrast to Dct-02. Data suggests that the AceTr transporters prefer monovalent succinate, whereas Dct-02 prefers divalent succinate anions. In addition, the results provided deeper insights into the characteristics of Dct-02, showing its ability to act as a succinic acid importer (thus being bidirectional) and verifying its capability of exporting malate.

SUBMITTER: Rendulic T

PROVIDER: S-EPMC9409672 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae.

Rendulić Toni T Mendonça Bahia Frederico F Soares-Silva Isabel I Nevoigt Elke E Casal Margarida M

Journal of fungi (Basel, Switzerland) 20220806 8

Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO2 in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study ...[more]

PMID: 36012810

Similar Datasets

Project description:The unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di- and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid-tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2-1 and PkJEN2-2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L-malate into the cell. In addition, PkJEN2-1 can transport α-ketoglutarate, while PkJEN2-2 cannot. Since PkJEN2-1 shows higher transcript abundance than PkJEN2-2, its role in dicarboxylate transport is more important than PkJEN2-2. In addition, PKJEN2-2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model-based structure analysis and rational mutagenesis further proved that amino acid residues 392-403 of the tenth transmembrane span (TMS-X) of PkJEN2-2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di-/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis.

Project description:BackgroundThe microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol-1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield.ResultsBy changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol-1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol-1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase.ConclusionsThe data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.

Dataset Information

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae.

Publications

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in <i>S. cerevisiae</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets