Conferring thermotolerant phenotype to wild‐type Yarrowia lipolytica improves cell growth and erythritol production

Abstract

In microbial engineering, heat stress is an important environmental factor modulating cell growth, metabolic flux distribution and the synthesis of target products.Yarrowia lipolytica, as a GARS (generally recognized as safe) nonconventional yeast, has been widely used in the food industry, especially as the host of erythritol production. Biomanufacturing economics is limited by the high operational cost of cooling energy in large-scale fermentation. It is of great significance to select thermotolerant Y. lipolytica to reduce the cooling cost and elucidate the heatresistant mechanism at molecular level. For this purpose, we performed adaptive evolution and obtained a thermotolerant strain named Y. lipolytica BBE-18. Transcriptome analysis allows us to identify four genes in thiamine metabolism pathway that are responsible for the complicated thermotolerant phenotype. The heatresistant phenotype was validated with the model strain Y. lipolytica Po1f by overexpression of single and combined genes. Then, conferring the thermotolerant phenotype to the wild-type Y. lipolytica BBE-17 enable the strain to produce threetimes more erythritol of the control strain with 3°C higher than optimal cultivation temperature. To our knowledge, this is the first report on engineering heat-resistant phenotype to improve the erythritol production in Y. lipolytica. However, due to the increase of culture temperature, a large amount of adenosine triphosphate is consumed to ensure the life activities of Y. lipolytica which limits the potential of cell synthetic products to a certain extent. Even so, this study provides a reference for Y. lipolytica to produce other products under high temperature.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement

“…Starting with a strain with an optimum temperature of 30 • C, they applied a progressive adaptive evolution scheme that allowed them to obtain, after 11 months of continuous cultivation and selection, an improved strain with an optimum temperature of 35 • C. However, the erythritol yield was considerably lower in this selected strain, which prompted the use of genetic engineering as a last resort: the authors performed a transcriptome analysis on their thermotolerant strain in order to identify the genes linked to this phenotype, reconstructed the thermotolerant phenotype using a surrogate Po1f strain and finally transferred the target gene modifications into a BBE-17∆Ku70∆URA3 strain. This GM strain was able to produce a three-fold higher erythritol yield, without detrimental effects on cell growth, at a temperature of 33 • C compared to the parent BBE-17T strain [308]. Thus, such an example illustrates the limits of traditional mutagenesis, even when combined to adaptative evolution strategies, when used alone as an alternative to genetic engineering.…”

“…It appeared that single cell FACS was favoring intracellular riboflavin accumulation when droplet FACS was favoring extracellular product accumulation [305]. In addition to this example, adaptative laboratory evolution strategies have recently been applied in Y. lipolytica for various purposes: restoring the glucose metabolism of a Po1f derivative engineered for succinic acid production [306]; enhancing lipid storage from ACA-DC 50109 strain [135]; selecting the ionic liquid-tolerant YlCW001 strain [161]; improving limonene tolerance during its production by GM strains [307]; enhancing thermotolerance during industrial fermentation for erythritol production [308]; increasing tolerance to aromatic aldehydes or to ferulic acid, both for a more efficient lignocellulose valorization [309,310].…”

“…Interestingly, some genetic engineering technics were implemented in their project, but not for the design of the production strain: these tools were used to build a genetically-encoded erythritol biosensor strain (using an erythritol-responsive transcription factor to activate expression of an eGFP-encoding gene, leading to a fluorescence signal) that was used in the high-throughput screening test applied to the mutant strains generated through a combined UV/ARTP mutagenesis step [326]. The same authors pursued the optimization of their erythritol production bioprocess by applying an adaptative evolution strategy to the producing strain BBE-17 in order to enhance its thermotolerance during industrial fermentation, as evoked above in Section 4.5.3 [308]. Starting with a strain with an optimum temperature of 30 • C, they applied a progressive adaptive evolution scheme that allowed them to obtain, after 11 months of continuous cultivation and selection, an improved strain with an optimum temperature of 35 • C. However, the erythritol yield was considerably lower in this selected strain, which prompted the use of genetic engineering as a last resort: the authors performed a transcriptome analysis on their thermotolerant strain in order to identify the genes linked to this phenotype, reconstructed the thermotolerant phenotype using a surrogate Po1f strain and finally transferred the target gene modifications into a BBE-17∆Ku70∆URA3 strain.…”

Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement

“…It appeared that single cell FACS was favoring intracellular riboflavin accumulation when droplet FACS was favoring extracellular product accumulation [266]. Beside this example, adaptative laboratory evolution strategies have recently been applied in Y. lipolytica for various purposes: restoring the glucose metabolism of a strain engineered for succinic acid production [267]; selecting the ionic liquid-tolerant YlCW001 strain [137]; improving limonene tolerance during its production by GM strains [268]; enhancing thermotolerance during industrial fermentation for erythritol production [269]; increasing tolerance to aromatic aldehydes or to ferulic acid, both for a more efficient lignocellulose valorization [270,271].…”

“Fight-flight-or-freeze” – how Yarrowia lipolytica responds to stress at molecular level?

“…Yarrowia lipolytica , which is commonly used as a protein over-production platform, is often claimed to be an extremophile yeast species (Bankar et al 2009 ). It is frequently isolated from a range of challenging natural environments and shows resistance to extreme conditions simulated in the laboratory, such as ambient pH from 2.5 up to 9.5, high salinity/osmolality, elevated temperatures up to 38 ℃, or presence of toxic compounds (Andreishcheva et al 1999 ; Walker et al 2019 ; Madzak 2021 ; Qiu et al 2021 ; Sekova et al 2021 ). High resistance to severe environmental conditions has its practical consequences in biotechnological processes, especially in relation to the occurrence of unavoidable gradients of temperature, pH, oxygen, osmolality, and concentration of chemical compounds, of different nature, intensity, duration, and/or frequency.…”