Supplementary Figures from 3-hydroxyisobutyryl-CoA hydrolase involved in isoleucine catabolism regulates triacylglycerol accumulation in <i>Phaeodactylum tricornutum</i>

Supplemental Figure 1. Sequence alignment of the human PCC (HsPCC_PCC1), Ruegeria pomeroyi PCC (RpPCC_PCC1) a subunits, and the predicated PCC a subunits of Phaeodactylum tricornutum (PtPCC_PCC1) and Thalassiosira pseudonana (Tp_PCC1). Residues in the active site are indicated by arrows. Supplemental Figure 2. Sequence alignment of the human PCC (HsPCC_PCC2), Ruegeria pomeroyi PCC (RpPCC_PCC2) b subunits, and the predicated PCC b subunits of Phaeodactylum tricornutum (PtPCC_PCC2) and Thalassiosira pseudonana (Tp_PCC2). Supplemental Figure 3. Alignment of the amino acid sequences HIBCH from Homo sapiens (HsHIBCH), Arabidopsis thaliana (AtCHY1), Saccharomyces cerevisiae (ScHIBCH) and the predicted HIBCH in Chlamydomonas reinhardtii (CrHIBCH) and Phaeodactylum tricornutum (PtHIBCH). Amino acid residues identical in five, four, and three of the sequences are in red, green and blue respectively. Cleavage sites of mitochondria transit predicted by TargetP and the calculated score mTP are respectively shown by arrows and the numbers below the sequences. C-terminal peroxisome targeting signal are boxed. Asterisks above the sequence indicate proposed catalytic residues. Supplemental Figure 4. Phylogenetic relationship of the HIBCH protein family. The ML tree was generated using the MrBayes program. HIBCH are grouped into three distinct clades. Numbers above branches represent the support values (Bayesian posterior probabilities). The scale bar represents the number of nucleotide replacements per site. Supplemental Figure 5. Principal component analysis (PCA) of metabolic profiles in wild-type (WT), two HIBCH overexpression lines (hibch-OE1 and hibch-OE3) and one PCC RNAi silenced line (pcc1p). (a) Scores plot shows separation of the samples among the four groups; (b) loading plot indicated the main metabolites contributing to the separation

Supplementary Data Sets from 3-hydroxyisobutyryl-CoA hydrolase involved in isoleucine catabolism regulates triacylglycerol accumulation in <i>Phaeodactylum tricornutum</i>

Supplemental Data S1: MCEE gene distribution in different organisms. Supplemental Data S2: Primers used in this study for qPCR and the construction of RNAi expression vector, overexpression vector and GFP fusion vector. Supplemental Data S3: Metabolite contents (mg g-1 dry biomass) in wild-type, two overexpression lines (hibch-OE1 and hibch-OE3) and one silenced strain (pcc1p). Values are presented as the mean of three biological replicates. Supplemental Data S4: The values of fold change in transcript levels of genes encoding componens involved in BCAA degradation

Supplementary Figures from 3-hydroxyisobutyryl-CoA hydrolase involved in isoleucine catabolism regulates triacylglycerol accumulation in <i>Phaeodactylum tricornutum</i>

Supplemental Figure 1. Sequence alignment of the human PCC (HsPCC_PCC1), Ruegeria pomeroyi PCC (RpPCC_PCC1) a subunits, and the predicated PCC a subunits of Phaeodactylum tricornutum (PtPCC_PCC1) and Thalassiosira pseudonana (Tp_PCC1). Residues in the active site are indicated by arrows. Supplemental Figure 2. Sequence alignment of the human PCC (HsPCC_PCC2), Ruegeria pomeroyi PCC (RpPCC_PCC2) b subunits, and the predicated PCC b subunits of Phaeodactylum tricornutum (PtPCC_PCC2) and Thalassiosira pseudonana (Tp_PCC2). Supplemental Figure 3. Alignment of the amino acid sequences HIBCH from Homo sapiens (HsHIBCH), Arabidopsis thaliana (AtCHY1), Saccharomyces cerevisiae (ScHIBCH) and the predicted HIBCH in Chlamydomonas reinhardtii (CrHIBCH) and Phaeodactylum tricornutum (PtHIBCH). Amino acid residues identical in five, four, and three of the sequences are in red, green and blue respectively. Cleavage sites of mitochondria transit predicted by TargetP and the calculated score mTP are respectively shown by arrows and the numbers below the sequences. C-terminal peroxisome targeting signal are boxed. Asterisks above the sequence indicate proposed catalytic residues. Supplemental Figure 4. Phylogenetic relationship of the HIBCH protein family. The ML tree was generated using the MrBayes program. HIBCH are grouped into three distinct clades. Numbers above branches represent the support values (Bayesian posterior probabilities). The scale bar represents the number of nucleotide replacements per site. Supplemental Figure 5. Principal component analysis (PCA) of metabolic profiles in wild-type (WT), two HIBCH overexpression lines (hibch-OE1 and hibch-OE3) and one PCC RNAi silenced line (pcc1p). (a) Scores plot shows separation of the samples among the four groups; (b) loading plot indicated the main metabolites contributing to the separation

Table_1_Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.XLSX

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22–28°C), pH (6–8), and inoculum size (2–8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 μg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 μg/mL and 9358.08 ± 122.96 μg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis–Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.</p

Table_3_Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.XLSX

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22–28°C), pH (6–8), and inoculum size (2–8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 μg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 μg/mL and 9358.08 ± 122.96 μg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis–Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.</p

MOESM2 of A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica

Additional file 2: Table S1. DGAT protein sequences used for the construction of phylogenetic tree in additional file 1: Figure S3. Table S2. Primers used in the present study. Underlined sequences designate the restriction enzyme sites. The sequences in box indicate the linker fragment introduced before GFP coding sequence

Image_1_Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.TIF

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22–28°C), pH (6–8), and inoculum size (2–8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 μg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 μg/mL and 9358.08 ± 122.96 μg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis–Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.</p

Table_2_Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.DOCX

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22–28°C), pH (6–8), and inoculum size (2–8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 μg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 μg/mL and 9358.08 ± 122.96 μg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis–Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.</p

Image_2_Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.TIF

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22–28°C), pH (6–8), and inoculum size (2–8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 μg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 μg/mL and 9358.08 ± 122.96 μg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis–Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.</p