Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae

Significance The export of organic acids is typically proton or sodium coupled and requires energetic expenditure. Consequently, the cell factories producing organic acids must use part of the carbon feedstock on generating the energy for export, which decreases the overall process yield. Here, we show that organic acids can be exported from yeast cells by voltage-gated anion channels without the use of proton, sodium, or ATP motive force, resulting in more efficient fermentation processes.


Engineering energetically efficient transport of dicarboxylic acids in
Steven Axel van der Hoek, Irina Borodina

Media and yeast cultivation conditions
Yeast cells were grown in standard YPD medium at 30°C. For selection, SC plates (20 g/L agar) without leucine, uracil or histidine or a combination of these were used. Production of dicarboxylic acids was demonstrated in 250 ml baffled shake flasks and 50-ml working volume of a defined mineral medium with 50 g/L glucose unless indicated. The agitation was 250-rpm. The mineral medium consisted of 1.5 g/L urea, 3 g/L KH 2 PO 4 , 6.6 g/L K 2 SO 4 , 0.5 g/L MgSO 4 ·7H 2 O, 1 ml/l of vitamin solution and 2 ml/l trace elements as described in (1) and pH was set to 4.8. Cells from the seed culture were washed with sterile water, resuspended in the medium and added to deep-well plates or shake flasks containing CaCO 3 (2) in a final concentration of 25 g/L. The cells were inoculated to initial OD 600 of 2 (ca. 0.3 g/L cell dry weight). For seed culture medium, 7.5 g/L (NH 4 ) 2 SO 4 was used instead of urea and K 2 SO 4 was omitted. Further, 14.4 g/L KH 2 PO 4 and 20 g/L glucose was used and pH set to 6.

Strain construction
All yeast strains constructed are derived from CEN.PK TAM strain (3) and listed in Table S1. Native and heterologous genes under the control of strong constitutive promoters were integrated into the genome of the parental yeast strain. Before yeast transformation, the integrative vectors were linearized by FastDigest NotI (ThermoFisher Scientific) restriction enzyme. Yeast cells were transformed by the standard PEG/LiAc method (4). The cells were plated on selective plates with the appropriate selection. The plates were typically incubated for 3-5 days. Verification of correct integrations was done by colony PCR using OneTaq® Hot Start Quick-Load® 2X Master Mix (New England Biolabs) using the manufacturer's protocol and primers listed in Table S2.

DNA Constructs
The integrative plasmids (Table S3) were constructed by USER fusion (5). The particular gene and promoter BioBricks (Table S4) were amplified by PCR with Phusion U polymerase (ThermoFisher Scientific). Used primers and templates are listed in Table S2  and Table S4. Native genes were amplified from CEN.PK genomic DNA. Heterologous genes were synthetized by GeneArt. The exception was SpMae1, which was amplified from S. pombe genomic DNA. Empty integrative vectors were digested with FastDigest SfaAI (ThermoFisher Scientific) restriction endonuclease, nicked with Nb.BsmI (New England BioLabs) and assembled with a PCR amplified gene and a promoter of choice. To express the transporter coding genes in oocytes, genes were cloned downstream of the T7 promoter in the USER compatible Xenopus expression vector pUSER016 (6). The empty vector was digested by PacI and Nt.BbvCI (New England BioLabs). The amplified DNA fragments were gel purified and together with the linearized plasmids incubated with USER enzyme (New England BioLabs) for 25 min at 37°C, followed by incubation at 25°C for 25 min. The reactions were transformed into chemically competent E. coli cells. All the cloned plasmids were verified by Sanger sequencing.

Transport assays in Xenopus oocytes
The Xenopus laevis oocytes were obtained from Ecocyte Bioscience (Germany) and kept at 18°C. Linear cassettes (including T7 promoter, the gene of interest, and 3UTR) were amplified with Phusion Hot Start polymerase (ThermoFisher Scientific) and used as template for in-vitro transcription. Capped cRNAs was synthesized using the mMESSAGE mMACHINE® T7 Transcription Kit (AM1344; ThermoFisher). The quality and quantity of RNAs were determined by Agilent 2100 Bioanalyzer. For expression in oocytes, 25 ng of in-vitro produced cRNAs for the transporters was microinjected into oocytes 3 days prior to transport assays (6). For microinjection of cRNAs and compounds, we used the RoboInject (Multi Channel Systems, Germany) automatic injection system (7,8). Injection needles with opening of 25 µm were used (Multi Channel Systems). The stock solution of 50 nl containing 40 mM citrate and 30 mM fumarate was used for microinjection into the oocytes to obtain estimated internal concentrations of 2 mM and 1.5 mM, respectively, assuming an after-injection dilution factor of 20 (6). Following four washing steps, each batch of 20 oocytes was incubated for 180 min in 90 µl Kulori buffer at pH 5. After incubation, 70 µl of the medium was collected from each batch with intact oocytes and added onto 70 µl 60% MeOH before LC-MS analysis. Statistical significant differences were determined through one-way ANOVA followed by Duncan's Multiple Range Test.

Chemicals and HPLC/LC-MS analyses
Residual calcium carbonate in the cultivation broth was dissolved by adding HCl to a final concentration up to 0.5 M before the samples of fermentation broth were centrifuged. Glucose and other metabolites levels in the culture supernatants were determined by HPLC. Aminex HPX87H ion exclusion column (300x7.8 mm, 9µm) at 60°C with 5 mM H 2 SO 4 as the mobile phase was used. Glucose, acetate and glycerol were detected by RI-detector. Succinic and fumaric acid were detected by UV detector at 205 nm. The data was acquired and analyzed with Chromeleon software. Malic acid quantification was performed spectrophotometrically using a malic acid assay kit (K-LMAL-58A; Megazyme). For oocyte transport assays, metabolite levels were measured using LC-MS. The LC-MS data was collected on EVOQ EliteTriple Quadrupole Mass Spectrometer system coupled with an Advance UHPLC pump (Bruker, Fremont Ca). Samples were held in the CTC HTS PAL autosampler at a temperature of 5.0 °C during the analysis. Injections of the samples with 1uL in volume were made onto a Waters ACQUITY HSS T3 C18 UHPLC column, with a 1.8 um particle size, 2.1 mm i.d. and 100 mm long. The column was held at 30.0 °C. The solvent consisted of solvent A (milliQ water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The flow rate was 0.400 ml/min with an initial solvent composition of %A = 100, %B = 0 held until 0.50 min, the solvent composition was then changed following a linear gradient until it reached %A = 5.0 and %B = 95.0 at 1.00 min. This was held until 1.79 min when the solvent was returned to the initial conditions and the column was re-equilibrated until 4.00 min. The column eluent flowed directly into the Heated ESI probe of the MS which was held at 250 °C and a voltage of 2500 V. MRM data was collected in negative ion mode. The target masses are shown in Table S5. The other MS settings were as follows: Sheath Gas Flow Rate of 50 units, Nebuliser Gas Flow Rate of 50 units, Cone Gas Flow Rate of 20 units, collision gas pressure of 1 mTorr, and Cone Temp at 350 °C.

Confocal microscopy and expression analysis
Transient expression in oocytes was performed by injecting cRNAs into the oocytes. Visualization of the GFP and its fusions derivatives in Xenopus oocytes was carried out using a Leica TCS SP5-II confocal microscope. To minimize autofluorescence of oocytes, excitation was performed with a low intensity of 16% at 488 nm and using Argon-ion laser at 20% intensity. Emission was also limited to the narrow range of 504-515 nm. GFP fused to the C-terminal of the wild-type and F/A mutated transporters were used to detect GFP signal of strains using microtiter plate reader (excitation was at 485 nm and emission at 515 nm).

Structural and functional-motif analyses
Protein domain predictions were performed by HMMER version 3.1b2 (9). This was done based on an in-house indexed target population of 16712 protein superfamily domains, which was built from Pfam library version 31 (available at ftp://ftp.ebi.ac.uk/pub/databases/Pfam/releases/) and also 65016 motif-domains from Gene3d version 16 (http://download.cathdb.info/gene3d/v16.0.0/gene3d_hmmsearch/). Transmembrane domains were predicted by TMHMM server v. 2.0 (10) and visualized by TMRPres2D (11). Three dimensional structures of HiTehA(p) and SpMae1(p) were built using the Phyre2 (12) and based on the crystal structure of HiTehA(p) (13). Structural alignment was performed by CLC Main Workbench version 7.6.4 (QIAGEN, Aarhus A/S). The degree of structural accordance quantified as TM score (14), a tradeoff between the alignment length and the alignment accuracy ranged from 0 to 1 (1.0 for identical proteins, > 0.7 for highly similar models, and < 0.5 for different folds). The maximum likelihood phylogenetic trees were built on the WAG substitutional matrixbased model for amino acid sequences and a bootstrap value of 1000 (15)