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Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica.

ABSTRACT: BACKGROUND:Integrating waste management with fuels and chemical production is considered to address the food waste problem and oil crisis. Approximately, 600 million tonnes crude glycerol is produced from the biodiesel industry annually, which is a top renewable feedstock for succinic acid production. To meet the increasing demand for succinic acid production, the development of more efficient and cost-effective production methods is urgently needed. Herein, we have proposed a new strategy for integration of both biodiesel and SA production in a biorefinery unit by construction of an aerobic yeast Yarrowia lipolytica with a deletion in the gene coding succinate dehydrogenase subunit 5. RESULTS:Robust succinic acid production by an engineered yeast Y. lipolytica from crude glycerol without pre-treatment was demonstrated. Diversion of metabolic flow from tricarboxylic acid cycle led to the success in generating a succinic acid producer Y. lipolytica PGC01003. The fermentation media and conditions were optimized, which resulted in 43 g L(-1) succinic acid production from crude glycerol. Using the fed-batch strategy in 2.5 L fermenter, up to 160 g L(-1) SA was yielded, indicating the great industrial potential. CONCLUSIONS:Inactivation of SDH5 in Y. lipolytica Po1f led to succinic acid accumulation and secretion significantly. To our best knowledge, this is the highest titer obtained in fermentation on succinic acid production. In addition, the performance of batch and fed-batch fermentation showed high tolerance and yield on biodiesel by-product crude glycerol. All these results indicated that PGC01003 is a promising microbial factorial cell for the highly efficient strategy solving the environmental problem in connection with the production of value-added product.

SUBMITTER: Gao C

PROVIDER: S-EPMC5004273 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica.

Gao Cuijuan C Yang Xiaofeng X Wang Huaimin H Rivero Cristina Perez CP Li Chong C Cui Zhiyong Z Qi Qingsheng Q Lin Carol Sze Ki CSK

Biotechnology for biofuels 20160830 1

<h4>Background</h4>Integrating waste management with fuels and chemical production is considered to address the food waste problem and oil crisis. Approximately, 600 million tonnes crude glycerol is produced from the biodiesel industry annually, which is a top renewable feedstock for succinic acid production. To meet the increasing demand for succinic acid production, the development of more efficient and cost-effective production methods is urgently needed. Herein, we have proposed a new strate ...[more]

PMID: 27579143

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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.

Project description:BackgroundAlkali used for pH control during fermentation and acidification for downstream recovery of succinic acid (SA) are the two largest cost contributors for bio-based SA production. To promote the commercialization process of fermentative SA, the development of industrially important microorganisms that can tolerate low pH has emerged as a crucial issue.ResultsIn this study, an in situ fibrous bed bioreactor (isFBB) was employed for the metabolic evolution for selection of Y. lipolytica strain that can produce SA at low pH using glucose-based medium. An evolved strain named Y. lipolytica PSA3.0 that could produce SA with a titer of 19.3 g/L, productivity of 0.52 g/L/h, and yield of 0.29 g/g at pH 3.0 from YPD was achieved. The enzyme activity analysis demonstrated that the pathway from pyruvate to acetate was partially blocked in Y. lipolytica PSA3.0 after the evolution, which is beneficial to cell growth and SA production at low pH. When free-cell batch fermentations were performed using the parent and evolved strains separately, the evolved strain PSA3.0 produced 18.4 g/L SA with a yield of 0.23 g/g at pH 3.0. Although these values were lower than that obtained by the parent strain PSA02004 at its optimal pH 6.0, which were 25.2 g/L and 0.31 g/g, respectively, they were 4.8 and 4.6 times higher than that achieved by PSA02004 at pH 3.0. By fed-batch fermentation, the resultant SA titer of 76.8 g/L was obtained, which is the highest value that ever achieved from glucose-based medium at low pH, to date. When using mixed food waste (MFW) hydrolysate as substrate, 18.9 g/L SA was produced with an SA yield of 0.38 g/g, which demonstrates the feasibility of using low-cost glucose-based hydrolysate for SA production by Y. lipolytica in a low-pH environment.ConclusionsThis study presents an effective and efficient strategy for the evolution of Y. lipolytica for SA production under low-pH condition for the first time. The isFBB was demonstrated to improve the metabolic evolution efficiency of Y. lipolytica to the acidic condition. Moreover, the acetate accumulation was found to be the major reason for the inhibition of SA production at low pH by Y. lipolytica, which suggested the direction for further metabolic modification of the strain for improved SA production. Furthermore, the evolved strain Y. lipolytica PSA3.0 was demonstrated to utilize glucose-rich hydrolysate from MFW for fermentative SA production at low pH. Similarly, Y. lipolytica PSA3.0 is expected to utilize the glucose-rich hydrolysate generated from other carbohydrate-rich waste streams for SA production. This study paves the way for the commercialization of bio-based SA and contributes to the sustainable development of a green economy.

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

Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica.

Publications

Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica.

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