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Intracellular product recycling in high succinic acid producing yeast at low pH.

ABSTRACT: The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.The metabolic flux distributions at two time-points of the fermentation process were analyzed. 13C labeled succinic acid was added to the extracellular space and intracellular enrichments were measured and subsequently used for the estimation of metabolic fluxes. The labeling was introduced by a labeling switch experiment, leading to an immediate labeling of about 85% of the acid while keeping the total acid concentration constant. Within 100 s significant labeling enrichment of the TCA cycle intermediates fumarate, iso-citrate and ?-ketoglutarate was observed, while no labeling was detected for malate and citrate. These findings suggest that succinic acid is rapidly exchanged over the cellular membrane and enters the oxidative TCA cycle. Remarkably, in the oxidative direction malate 13C enrichment was not detected, indicating that there is no flux going through this metabolite pool. Using flux modeling and thermodynamic assumptions on compartmentation it was concluded that malate must be predominantly cytosolic while fumarate and iso-citrate were more dominant in the mitochondria.Adding labeled product without changing the extracellular environment allowed to quantify intracellular metabolic fluxes under high producing conditions and identify product degradation cycles. In the specific case of succinic acid production, compartmentation was found to play a major role, i.e. the presence of metabolic activity in two different cellular compartments lead to intracellular product degradation reducing the yield. We also observed that the flux from glucose to succinic acid branches at two points in metabolism: (1) At the level of pyruvate, and (2) at cytosolic malate which was not expected.

SUBMITTER: Wahl SA

PROVIDER: S-EPMC5442661 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Intracellular product recycling in high succinic acid producing yeast at low pH.

Wahl S Aljoscha SA Bernal Martinez Cristina C Zhao Zheng Z van Gulik Walter M WM Jansen Mickel L A MLA

Microbial cell factories 20170523 1

<h4>Background</h4>The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.<h4>Results</h4>The metabolic flux distributions ...[more]

PMID: 28535757

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Project description:To understand cell responses against high succinate stress, a continuous culture was performed for 9 months using a wild Escherichia coli under gradually increasing succinate stress. The final adapted strain against high succinate stress, named DST160, was able to grow with no lag phase in medium containing 1.35 M of succinate stress while wild W3110 showed more than 38 h lag phase. The maximum growth rate and growth yield of DST160 under the stress condition were 0.20 h-1 and 0.192 g-cell/g-glucose, which were 53.8 % and 12.3 % higher than those of W3110 (0.13 h-1 and 0.171 g-cell/g-glucose, respectively). A single colony of E. coli W3110 was inoculated into a 15-mL tube containing 4 mL of LB medium. After 12 h cultivation in a shaking incubator at 37℃ and 220 rpm, pre-culture (1 mL) was transferred to a 250 mL-fermentor (Mini-chemostat fermentor; Biotron Inc., Bucheon, Korea) containing 100 mL medium consisted of M9-glucose minimal medium (3 g glucose , 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4•2H2O, 3 g KH2PO4 with separately added trace elements of 0.2 g MgSO4•7H2O, 0.1 g CaCl2, 1 mg thiamine) per liter supplemented with 254 mmole of succinate (30 g succinic acid) per liter. The initial pH of the medium was set to be pH 7.5 by adding 6N NaOH. The fermentor was operated at 37℃, 350 rpm with air flushing (100 mL/min.). After 12 h batch mode growth, feeding and outlet pumps were turned on at flow rate of 0.167 mL/min (Dilution rate=0.1 h-1). The fresh medium reservoir (10 L) contained the same contents and pH with the initial medium. Every 10 turnover time (100 h), the reservoir was changed with the same medium composition except succinate concentration. The succinate concentration of the reservoir was gradually increased by 25.4 ~ 84.7 mM (3 ~ 10 g/L of succinic acid) at a time than previous states depending on the cell concentrations at the outlet. When the cell concentration was reduced below O.D.=0.2, succinate feeding pump was temporarily stopped to go back to the slightly lower succinate concentration. The continuous culture was performed until the steady state when succinate in the reservoir reached 1.35 M (160 g/L in succinic acid form). The final adapted cells in 100 mL culture were harvested by centrifuge and frozen rapidly in a liquid nitrogen tank for further analyses including DNA microarray. To understand the change of up/down regulations in the high-succinate adapted E. coli, total mRNAs of control cells and the adapted E. coli cells were extracted using a RNA extraction kit (RNeasy mini kit; Quiagen, Hilden, Germany), and the transcriptomes were compared using DNA microarray. Hybridization and washing were accomplished by a facility of Digital Genomics Co (Seoul, Korea). DNA microarray were scanned using an Axon GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed with GenePix Pro 3.0 software to obtain gene expression ratios.

Project description:Background:Xylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results:In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD600: 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations. Conclusion:The recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

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Intracellular product recycling in high succinic acid producing yeast at low pH.

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Intracellular product recycling in high succinic acid producing yeast at low pH.

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