Alternative Substrate Metabolism in Yarrowia lipolytica

Spagnuolo, Michael; Hussain, Murtaza; Gambill, Lauren; Blenner, Mark

doi:10.3389/fmicb.2018.01077

Cited by 91 publications

(77 citation statements)

References 144 publications

(166 reference statements)

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“…Different from S. cerevisiae, Y. lipolytica lacks Crabtree effects generating no overflowed metabolism toward ethanol under high glucose conditions 29 , which might be more suitable for high gravity fermentation and process control. Y. lipolytica is reported to valorize a broad range of cheap and renewable feedstocks 30, 31 , including sugars, volatile fatty acids, alkanes and municipal organic wastes. This substrate flexibility provides us an environmentally friendly approach to upgrade low-value carbons to high value chemicals with reduced carbon footprint and improved process economics.…”

Section: Introductionmentioning

confidence: 99%

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

Zhu

et al. 2020

Preprint

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AbstractsYarrowia lipolytica is an attractive host to upgrade renewable low-cost carbon feedstocks to high-value commodity chemicals and natural products. In this work, we systematically characterized and removed the rate-limiting steps of the shikimate pathway and achieved de novo synthesis of five aromatic chemicals in Y.lipolytica. We determined that engineering feedback-insensitive DAHP synthases and eliminating amino acids formation are critical steps to mitigate the feedback regulation of shikimate pathway. Further overexpression of heterologous phosphoketolase and deletion of pyruvate kinase provided a sustained metabolic driving force that channels E4P (erythrose 4-phosphate) and PEP (phosphoenolpyruvate) precursors through the shikimate pathway. Precursor competing pathways and byproduct formation pathways were also blocked by inactivating chromosomal genes. To demonstrate the utility of our engineered chassis strain, three natural products, 2-PE, p-coumaric acid and violacein, which were derived from phenylalanine, tyrosine and tryptophan, respectively, were chosen to test the chassis performance. We obtained 2426.22 mg/L of 2-phenylethanol (2-PE), 593.53 mg/L of p-coumaric acid, 366.30 mg/L of violacein and 55.12 mg/L of deoxyviolacein from glucose in shake flask. The 2-PE production represents a 286-fold increase over the initial strain (8.48 mg/L). Specifically, we obtained the highest 2-PE, violacein and deoxyviolacein titer ever reported from the de novo shikimate pathway in yeast. These results set up a new stage of engineering Y. lipolytica as a sustainable biorefinery chassis strain for de novo synthesis of aromatics with economic values.

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Section: Introductionmentioning

confidence: 99%

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

Zhu

et al. 2020

Preprint

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“…Remarkably however, no direct triglyceride import mechanism is known. Access to this carbon source relies on the secretion of extracellular lipases, which hydrolyze the triglycerides to glycerol and free fatty acids . Subsequently, the separate components are taken up by the cell.…”

Section: Uptake Of Lipophilic Compoundsmentioning

confidence: 99%

Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane

2019

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In yeasts, the plasma membrane forms the barrier that protects the cell from the outside world, but also gathers and keeps valuable compounds inside. Although it is often suggested that hydrophobic molecules surpass this checkpoint by simple diffusion, it now becomes evident that protein‐facilitated transport mechanisms allow for selective import and export of triglycerides, fatty acids, alkanes, and sterols in yeasts. During biomass production, hydrophobic carbon sources enter and exit the cell efficiently in a strictly regulated manner that helps avoid toxicity. Furthermore, various molecules, such as yeast pheromones, secondary metabolites and xenobiotics, are exported to ensure cell–cell communication, or increase chances of survival. This review summarizes the current knowledge on how hydrophobic compounds interact with protein‐facilitated transport systems on the plasma membrane and how selective import and export across the yeast plasma membrane is achieved. Both the model organism Saccharomyces cerevisiae, as well as unconventional yeasts are discussed.

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“…Both Y. lipolytica and C. violacein were isolated from marine environment with high GC content (up to 65% in the coding sequence), we argue that Y. lipolytica might be engineered as a novel production platform for violacein production due to its GRAS status. Different from Baker's yeast, Y. lipolytica is abundant in acetyl-CoA due to its cytosolic ATP citrate lyase (ACL) [25]. Y. lipolytica has been reported to grow on a wide range of inexpensive raw materials [26], including glucose, glycerol, xylose [27,28], volatile fatty acids [29,30], alcohols and wax alkanes et al…”

Section: Introductionmentioning

confidence: 99%

Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

Tong

Zhou

Zhang

et al. 2019

Preprint

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1Violacein is a naturally occurring anticancer therapeutic with deep purple color. 2 Yeast fermentation represents an alternative approach to efficiently manufacturing 3 violacein from inexpensive feedstocks. In this work, we optimized the extraction 4 protocol to improve violacein recovery ratio and purity from yeast culture, including 5 the variations of organic solvents, the choice of mechanical shear stress, incubation 6 time and the use of cell wall-degrading enzymes. We also established the quantitative 7 correlation between HPLC and microplate reader method. We demonstrated that both 8 HPLC and microplate reader are technically equivalent to measure violacein from 9 yeast culture. Furthermore, we optimized the yeast cultivation conditions, including 10 carbon/nitrogen ratio and pH conditions. Our results indicated that ethyl acetate is the 11 best extraction solvent with glass beads grinding the cell pellets, the maximum 12 violacein and deoxyviolacein production was 70.04 mg/L and 5.28 mg/L in shake 13 flasks, respectively. Violacein purity reaches 86.92% at C/N ratio of 60, with addition 14 of 10 g/L CaCO 3 to control the media pH. Taken together, the development of 15 efficient extraction protocol, quantitative correlation between HPLC and microplate 16 reader, and the optimization of culture conditions set a new stage for engineering 17 violacein production in Y. lipolytica. This information should be valuable for us to 18 build a renewable and scalable violacein production platform from the novel host 19 oleaginous yeast species. 20 21

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Alternative Substrate Metabolism in Yarrowia lipolytica

Cited by 91 publications

References 144 publications

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane

Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization

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