Metabolic engineering in the host Yarrowia lipolytica

Biomass-based biorefineries: An important architype towards a circular economy

Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol

Synthetic biology tools for engineering Yarrowia lipolytica.

Pollution to products: recycling of 'above ground' carbon by gas fermentation.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin–Benson–Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h−1. The specific growth rate was further improved to 0.018 h−1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production. A yeast species used to produce proteins and chemicals is engineered to grow solely on the greenhouse gas CO2.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO 2

The yeast Yarrowia lipolytica is a fascinating microorganism with an amazing metabolic flexibility. This yeast grows very well on a wide variety of carbon sources from alkanes over lipids, to sugars and glycerol. Yarrowia accumulates a wide array of industrially relevant metabolites. It is very tolerant to many environmental factors, above all the pH value. It grows perfectly well over a wide pH range, but it has been described, that the pH has a decisive influence on the metabolite pattern accumulated by this yeast. Here, we set out to characterize the metabolism of different Y. lipolytica strains, isolated from various environments, growing on glycerol at different pH values. The conditions applied for strain characterization are of utmost importance. Shake flask cultures lead to very different results, when compared to controlled conditions in bioreactors regarding pH and aeration. Only one of the tested strains was able to accumulate high amounts of citric acid in shake flask experiments, whereas a group of six strains turned out to accumulate citric acid efficiently under controlled conditions. The present study shows that strains isolated from dairy products predominantly accumulate sugar alcohols at any given pH, when grown on glycerol under nitrogen-limitation.

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Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

Spotlight on biodiversity of microbial cell factories for glycerol conversion.

The yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.

Michael Egermeier

Papers

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO 2

Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

Spotlight on biodiversity of microbial cell factories for glycerol conversion.

Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolytica.

Adaptive laboratory evolution and reverse engineering enhances autotrophic growth in Pichia pastoris.