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Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

ABSTRACT: Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE164470 | GEO | 2021/01/09

REPOSITORIES: GEO

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Project description:To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of ï¢-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains. Experiment Overall Design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .

Project description:Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase is not present. The phylogenetic distribution of phosphofructokinase indicates that in most obligate aerobic organisms PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions the available energy is limiting and PFK provides an advantage, which explains the presence of PFK in many (facultative) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expresses a heterologous phosphofructokinase showed that the yield of biomass on substrate decreased in comparison with a pfkA deficient control strain, which was associated mainly with an increase in CO2 production, whereas production of by-products was comparable between the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield, might be possible after adaptive evolution of the strain, which remains to be investigated. Two-condition experiment, 3 replicates per condition

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Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

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