| Carotenoids are a class of natural pigments with antioxidant properties and are widely distributed in nature.Many studies have shown that a variety of environmental stresses can promote the synthesis of carotenoids in microbial cells to reduce reactive oxygen species(ROS)damage caused by environmental stresses.In our previous study,we found that Rhodosporidium kratochvilovae strain YM25235 increased intracellular reactive oxygen species(ROS)levels and promoted carotenoid synthesis under glucose starvation.Pyruvate dehydrogenase bypass(PDH bypass)is the main pathway for the synthesis of intracellular acetyl-Co A.Our previous transcriptome sequencing analysis also showed that pyruvate metabolic pathway was significantly enriched under glucose starvation stress.Overexpression of the acetyl coenzyme A synthetase(ACS)gene Rk ACS1 in the PDH bypass pathway significantly promoted the the synthesis of carotenoids and oils in YM25235 strain under glucose starvation.These results suggest that PDH bypass may be related to the increased synthesis of carotenoids in YM25235 strain under glucose starvation,but the correlation between the two remains to be further studied.ACS is the key enzymes in the PDH bypass.Generally,yeasts contains two ACS isoenzymes: ACS1 and ACS2,which differ in subcellular localization,mode of regulation,and kinetic properties and may have different functions during metabolism.Therefore,in this study,Rk ACS1 gene knockout and Rk ACS2 gene overexpression were combined to further elucidate the relationship between PDH bypass and the increased synthesis of carotenoids in YM25235 strain under glucose starvation.First,based on the established CRISPR/Cas9 gene editing system,the Rk ACS1 gene knockout mutant strain YM25235 Rk ACS1Δ was successfully obtained,and subsequently the Rk ACS1 gene complementary strain YM25235 Rk ACS1Δ/p RZRk ACS1 was obtained.The analysis results showed that Rk ACS1 knockout in YM25235 caused the mutant strain YM25235 Rk ACS1Δ inability to utilize acetic acid as the sole carbon source and weak growth on ethanol or glycerol as the sole carbon source as comparison with the control strain,and the Rk ACS1 gene complementation significantly restored the mutant strain’s ability to utilize these carbon sources.These results indicated that the Rk ACS1 gene plays an important role in the utilization of non-fermentative carbon sources for the YM25235 strain,especially for acetic acid utilization,which is consistent with previous similar reports.Further analysis showed that the Rk ACS1 knockout led to the accumulation of acetate and the decrease of intracellular acetyl-Co A in the mutant strain,and the content of carotenoids consequently decreased by 20.17%,but the lipid synthesis increased by 140.62%.However,it was also observed that these metabolic changes in the mutant strain were completely restored by gene complementation with the Rk ACS1 gene.Some studies have indicated that ACS2 might mainly be involved in the intracellular fatty acid synthesis and oil accumulation.Therefore,the increase in lipid synthesis in the mutant strain YM25235 Rk ACS1Δ might be resulted from the significant up-regulation of Rk ACS2 gene expression after Rk ACS1 gene knockout,but further analysis is needed to verify.To further investigate the relationship between the Rk ACS2 gene and the synthesis of carotenoids and lipid in strain YM25235 under glucose starvation stress,the Rk ACS2 gene was further amplified by PCR from the c DNA of strain YM25235.Sequence analysis showed that Rk ACS2 was 2094 bp in size and encoded 697 amino acids.The encoded Rk ACS2 protein was characteristic of conserved residues in the adenylate forming enzyme superfamily and had higher sequence similarity with several published ACS2 proteins.These results suggest that Rk ACS2 gene is a new potential ACS gene.Consistent with similar studies,the expression of Rk ACS1 was inhibited in the presence of glucose,while the expression of Rk ACS2 was stable during the culture process.The expression level of Rk ACS2 was significantly lower than that of Rk ACS1,especially under glucose starvation.Further analysis results showed that,under glucose starvation,the overexpression of Rk ACS2 gene in YM25235 also resulted in a decrease in intracellular acetic acid and a significant increase in intracellular acetyl-Co A,and the content of carotenoids in YM25235 was not significantly changed,but the lipid accumulation was increased by 33.73%,suggesting that Rk ACS2 gene might be more connected with lipid synthesis in YM25235 strain.In addition,subcellular localization analysis also showed that,unlike Rk ACS1 which was distributed in the cytoplasm,Rk ACS2 was located in both the nucleus and cytoplasm.Based on the results of other similar studies,we hypothesized that Rk ACS2,like its homologous protein,might be involved in histone acetylation in the nucleus,regulating the relevant protein expression of endoplasmic reticulum,Golgi apparatus and vacuole-to affect the lipid synthesis.It also located in the endoplasmic reticulum to participate in lipid synthesis.However,further verifications are needed for the identification of its specific functionsIn summary,this study,through Rk ACS1 gene deletion and complementation and Rk ACS2 gene overexpression analysis,revealed that Rk ACS1 was connected with the increased synthesis of carotenoids in YM25235 strain under glucose starvation,and Rk ACS2 was connected with the lipid accumulation during culture process,which might be due to the differences in subcellular localization,mode of regulation,and kinetic properties for these two isoenzymes.This study preliminarily revealed the functional differences of different acetyl-Co A synthetases in Rhodosporidium kratochvilovae YM25235 and their involvement in the regulation of carotenoid and lipid synthesis,which laid a foundation for the optimization of strategies for the carotenoid and lipid synthesis by YM25235.This study also provides a reference for the further research of acetyl-Co A synthetases in Rhodosporidium kratochvilovae and other red yeasts. |
