Engineering the activity and specificity of Saccharomyces cerevisiae Acetate Transporter Ady2/Ato1

Organic acids are industrially relevant chemicals with application in polymer, food, agricultural and pharmaceutical sectors. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Since organic acid export over the plasma membrane represents one of the key steps in microbial production of these compounds, organic acid transporters started receiving greater attention in metabolic engineering strategies. Ato1 is the main transporter responsible for uptake of acetic acid into the cytosol in S. cerevisiae, while also being able to mediate organic acid transport in the opposite direction, as it was shown to be involved in the export of lactic acid from S. cerevisiae cells engineered for lactic acid production. Ato1 is a member of the Acetate Uptake Transporter Family (AceTR), with several functionally characterized homologues in yeast, fungi, and bacteria. Recently solved crystal structure of its bacterial homologue, SatP, depicts a hexameric anion channel. In this work, we studied the relationship between structure and function of Ato1 via rational mutagenesis and identified residues critical for Ato1 substrate specificity and transport activity. By utilizing computer-assisted three-dimensional modelling tools, we provide possible explanations of acquired features. Our final goal is to test applicability of these transporters in yeast cell factories that produce organic acids.Supported by strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and TransAcids (PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood (NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement. TC acknowledges Yeastdoc European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 764927

Quantitative evaluation of yeast's requirement for glycerol formation in very high ethanol performance fed-batch process

<p>Abstract</p> <p>Background</p> <p>Glycerol is the major by-product accounting for up to 5% of the carbon in <it>Saccharomyces cerevisiae </it>ethanolic fermentation. Decreasing glycerol formation may redirect part of the carbon toward ethanol production. However, abolishment of glycerol formation strongly affects yeast's robustness towards different types of stress occurring in an industrial process. In order to assess whether glycerol production can be reduced to a certain extent without jeopardising growth and stress tolerance, the yeast's capacity to synthesize glycerol was adjusted by fine-tuning the activity of the rate-controlling enzyme glycerol 3-phosphate dehydrogenase (GPDH). Two engineered strains whose specific GPDH activity was significantly reduced by two different degrees were comprehensively characterized in a previously developed Very High Ethanol Performance (VHEP) fed-batch process.</p> <p>Results</p> <p>The prototrophic strain CEN.PK113-7D was chosen for decreasing glycerol formation capacity. The fine-tuned reduction of specific GPDH activity was achieved by replacing the native <it>GPD1 </it>promoter in the yeast genome by previously generated well-characterized <it>TEF </it>promoter mutant versions in a <it>gpd2</it>Δ background. Two <it>TEF </it>promoter mutant versions were selected for this study, resulting in a residual GPDH activity of 55 and 6%, respectively. The corresponding strains were referred to here as <it>TEFmut7 </it>and <it>TEFmut2</it>. The genetic modifications were accompanied to a strong reduction in glycerol yield on glucose; the level of reduction compared to the wild-type was 61% in <it>TEFmut7 </it>and 88% in <it>TEFmut2</it>. The overall ethanol production yield on glucose was improved from 0.43 g g^-1in the wild type to 0.44 g g^-1measured in <it>TEFmut7 </it>and 0.45 g g^-1in <it>TEFmut2</it>. Although maximal growth rate in the engineered strains was reduced by 20 and 30%, for <it>TEFmut7 </it>and <it>TEFmut2 </it>respectively, strains' ethanol stress robustness was hardly affected; i.e. values for final ethanol concentration (117 ± 4 g L^-1), growth-inhibiting ethanol concentration (87 ± 3 g L^-1) and volumetric ethanol productivity (2.1 ± 0.15 g l^-1h^-1) measured in wild-type remained virtually unchanged in the engineered strains.</p> <p>Conclusions</p> <p>This work demonstrates the power of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Under the conditions used in this study (VHEP fed-batch), the two strains with "fine-tuned" <it>GPD1 </it>expression in a <it>gpd2</it>Δ background showed slightly better ethanol yield improvement than previously achieved with the single deletion strains <it>gpd1</it>Δ or <it>gpd2</it>Δ. Although glycerol reduction is known to be even higher in a <it>gpd1</it>Δ <it>gpd2</it>Δ double deletion strain, our strains could much better cope with process stress as reflected by better growth and viability.</p

The dicarboxylate transporters from the AceTr family and Dct-02 oppositely affect succinic acid production in S. cerevisiae

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.This research was funded by European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Yeastdoc grant agreement No 764927, by the strategic program UID/BIA/04050/2020 and the project LA/P/0069/2020 granted to the Associate Laboratory ARNET, funded by Portuguese funds through the FCT I.P., and by River2Ocean NORTE-01-0145- FEDER-000068, co-financed by the European Regional Development Fund (ERDF) through Programa Operacional Regional do Norte (NORTE 2020). I.S-S. was supported by the program contract FCTUMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC)

The expression of glycerol facilitators from various yeast species improves growth on glycerol of <i>Saccharomyces cerevisiae</i>

Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18 h−1 and a biomass yield coefficient of 0.56 gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker's yeast. Keywords: Yeast, Saccharomyces cerevisiae, Glycerol, Transport, Glycerol facilitator, Fps1, Stl

Engineering of AceTr membrane transporters to improve organic acid production in yeast

Organic acids are industrially relevant chemicals obtainable from renewable feedstocks via microbial cell factories. Microbially produced organic acids have a wide variety of applications, including bioplastic synthesis. Thus, they possess the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for microbial production of organic acids, namely due to their tolerance of low pH environments. Such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in low pH conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Our current objective is to deepen the knowledge on organic acid transporters from the AceTR family (1,2,3). We performed functional characterization by studying transporter kinetics, energetics and specificity as well as site-directed mutagenesis to acquire insight into the structural features of transporters. Finally, we aim to improve organic acid production in S. cerevisiae cell factories via expression of engineered AceTR transporters with altered activity and substrate specificity.UID/BIA/04050/2013(POCI-01-0145-FEDER-007569) and TransAcids(PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood(NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement.TCacknowledgesYeastdocEuropean Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curie grant agreement No 76492

Exploring plasma membrane transporters to improve organic acid production in yeast – Characterization and engineering

Organic acids are industrially relevant building-block chemicals obtainable from renewable feedstocks by utilization of microbial cell factories. With a wide variety of applications, including bioplastics synthesis, microbially produced organic acids have the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments, since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in such conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Here, we are characterizing and engineering plasma membrane transporters with the final aim to improve production of dicarboxylic acids, namely succinic acid, in yeast. This includes transporters that have already been described as efficient dicarboxylate transporters, as well as promising transporters from the AceTr family. First, we perform functional characterization by studying transporter kinetics, energetics and specificity, as well as site-directed mutagenesis, to acquire insight into functional-structural relationship of transporters. This insight further uncovers engineering targets that can lead to improved transporter activity as well as altered substrate specificity. Finally, the performance of these transporters can be assessed via their expression in S. cerevisiae that is engineered for succinic acid production.This work was supported by the strategic programme UID/BIA/ 04050 2019 funded by Portuguese funds through the FCT IP, the project TransAcids (PTDC/ 5184 2014 funded by FCT IP and ERDF by COMPETE 2020 POCI and the project EcoAgriFood (NORTE 01 0145 FEDER 000009 supported by NORTE 2020 under the PORTUGAL 2020 Partnership Agreemen