| Rhodosporidium kratochvilovae YM25235 is an oil-producing red yeast strain with low-temperature growth adaptation isolated from Chenghai Lake,Lijiang,Yunnan.Previous studies by our group found that ROS levels were elevated in strain YM25235 under glucose starvation stress,and carotenoid synthesis levels were also significantly increased.Transcriptome sequencing analysis showed that the fatty acid degradation pathway was significantly enriched under glucose starvation stress,and the transcript level of RkACAA1,an acetyl-CoA acyltransferase 1(ACAA1)gene in the pathway was significantly upregulated.Therefore,the present study was proposed to investigate the relationship between fatty acid degradation and changes in carotenoid content in strain YM25235 under glucose starvation stress through functional studies of the RkACAA1 gene.Currently,few studies have been reported on the relationship between fatty acidβ-oxidation and increased carotenoid synthesis under glucose starvation stress.Based on the sequence information of RkACAA1 gene revealed by the transcriptome sequencing of strain YM25235,specific primers of RkACAA1 gene were designed and a1275 bp c DNA fragment was finally obtained by cloning the RkACAA1 gene using the c DNA of strain YM25235 as a template.The c DNA sequence analysis of RkACAA1 gene showed that it included an open reading frame(ORF)encoding 424 amino acids.Further analysis of the amino acid sequence encoded by it showed that the RkACAA1 protein has the type Ⅱ peroxisomal targeting signal(PTS2)RLSGLVSHL(amino acid sites13-21),which is consistent with the common sequence(R/K)-(L/V/I)-x5-(H/Q)-(L/A)of the PTS2,and the protein also has an amino acid sequence containing two N-terminal domains(amino acid sites 37-151 and 281-303),a loop-domain(amino acid sites152-280),a C-terminal domain(amino acid sites 304-421),and three highly conserved amino acid residues critical in the catalytic thiolysis reaction: Cys122,His379,and Cys409.The amino acid sequence of RkACAA1 is different from the known ACAA1.These results suggest that RkACAA1 is a potential new ACAA1 gene.To investigate the effect of RkACAA1 gene on carotenoid synthesis in R.kratochvilovae YM25235 under glucose starvation stress,we obtained strains YM25235/p RHRkACAA1,YM25235 RkACAA1Δ and YM25235 RkACAA1Δ/p RZRkACAA1 by overexpression of RkACAA1 gene,gene deletion using CRISPR/Cas9 technology and gene complementation,respectively.The analysis showed that compared with YM25235,the carotenoid content in overexpression strain YM25235/p RHRkACAA1 increased by 35.61%,the oil and total fatty acid contents decreased by 11.79% and 17.73%,respectively,and the contents of palmitic acid,stearic acid,oleic acid and linoleic acid in different fractions of fatty acids also decreased significantly,and the intracellular acetyl-CoA content significantly increased by 38.18%.However,while the content of lipids,total fatty acids and most fatty acid fractions were significantly increased in the knockout mutant strain YM25235 RkACAA1Δ and the intracellular acetyl-CoA content was significantly decreased,the carotenoid content was instead significantly increased by 44.28%.Further quantitative real-time fluorescence PCR and analysis of acetic acid content indicated that the increased carotenoid content in the knockout strain might be related to the further activation of the Pyruvate dehydrogenase bypass(PDH bypass),which promotes more conversion of pyruvate to acetic acid and thus acetyl-CoA synthesis.At the same time,a significant increase in intracellular ROS levels(22.48%)due to the inhibition of fatty acid degradation may also promote more intracellular acetyl-CoA into the carotenoid synthesis pathway,leading to increased carotenoid synthesis in the knockout strain.This was also tentatively verified by RkACAA1 gene complementation.The lipids content of YM25235 RkACAA1Δ/p RZRkACAA1 was significantly lower than that of RkACAA1 knockout strain,moreover,the expression level of Rk ACS1 gene in PDH bypass was restored to the same level as that in YM25235,and the expression level of Rk PDC gene was significantly lower than that of RkACAA1 knockout strain,but further activated the expression of Rk ACS2 gene,the lipids content of YM25235RKACAA1Δ/p RZRkACAA1 was significantly higher than that of YM25235.Although the acetyl-CoA and carotenoid contents were increased by 28.04% and 21.21%,respectively.However,it may be due to the higher transcript level of RkACAA1 gene in the complementation strain than YM25235 strain,resulting in higher acetyl-CoA and carotenoid content than YM25235 strain as well.The exact mechanism still needs to be further analyzed and studied.In summary,the results of functional analysis of RkACAA1 gene indicated that acetyl-CoA production by fatty acid β-oxidation in R.kratochvilovae YM25235 under glucose starvation stress promoted carotenoid synthesis.In addition,the analysis results also indicated that PDH bypass plays an important role in the synthesis of acetyl-CoA and carotenoids in strain YM25235 under glucose starvation stress.This study initially revealed the relationship between fatty acid β-oxidation and carotenoid content changes in strain YM25235 under glucose starvation stress,which laid the foundation for further fully revealing the regulatory mechanism of carotenoid synthesis in strain YM25235,and also provided a reference for the research and application of using related mechanisms to construct carotenoid high-yielding strains and process carotenoid production... |
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