Briefly, MAC-T cells exposed or free to H2O2 were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions was measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. The results revealed that leucine and isoleucine increased cell proliferation, total antioxidant status (TAS), and the relative mRNA expression of occludin, as well as decreased malondialdehyde (MDA), total oxidant status (TOS)/TAS, IL-6, IL-1β, and TOS. When leucine and isoleucine were combined, MDA, TOS/TAS, and the relative mRNA expression levels of claudin-1, occludin, and zonula occludens-1 increased when compared to leucine or isoleucine alone. Proteomics analyses revealed that leucine significantly upregulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways, whereas isoleucine significantly upregulated the peroxisome and propanoate metabolism pathways. In conclusion, leucine protected MAC-T cells from H2O2-induced oxidative stress by generating more ATP to supplement energy demands, and isoleucine improved the deficit in peroxisome transport and promoted acetyl-CoA production. The findings of this study enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.Leucine and isoleucine possess antioxidative and anti-inflammatory properties. However, their underlying protective mechanisms against oxidative damage remain unknown. Therefore, in this study, the protective mechanism of leucine and isoleucine against H The transition period from pregnancy to lactation is critical for dairy cow health, production, and profitability, because alternations in energy metabolism can negatively affect their health, including conditions such as mastitis and retained placenta . The con2O2 concentration in the plasma. Yin et al. [Amino acids, especially branched-chain amino acids (BCAAs), play important roles in cellular metabolism and stress responses –13. BCAAn et al.  found th2O2-induced oxidative damage in MAC-T cells was investigated. Briefly, MAC-T cells exposed or free to H2O2 were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions were measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. This finding will enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.Therefore, in this study, the protective mechanism of leucine and isoleucine against H2 humidity at 37°C. Culturing was performed in a 25 cm2 culture flask  containing 5 mL medium and a seeding number of 5 × 105 cells/flask. The medium was changed every other day.The MAC-T cells used in this study were donated by Professors Liu Jianxin and Liu Hongyun of Zhejiang University . The cells were cultured for 15 passages and grown in Dulbecco's modified eagle medium F12 (DMEM/F12)  and supplemented with 10% fetal bovine serum (FBS)  and a 1% final concentration of antibiotics (penicillin and streptomycin) under 5% COμmol/L H2O2 (working concentration) for 2, 4, 6, 8, or 10 h, after which the cellular viability of the MAC-T cells was measured.At a confluence of 70–80%, the cells were exposed to completed medium containing 0, 200, 400, 600, 800, 1000, or 1200 μmol/L H2O2 (working concentration) for 6 h (based on the results of 2.2.), after which oxidative stress indicators of the MAC-T cells were measured.At a confluence of 70–80%, the cells were exposed to completed medium containing 0, 200, 400, 600, 800, 1000, or 1200 At a confluence of 70–80%, the cells were treated with 0.450, 0.675, 0.900, 1.125, or 1.350 mmol/L leucine  diluted in DMEM/F12 with antibiotics for 6, 12, 24, 48, or 72 h. The experimental treatments correspond to 1, 1.5, 2, 2.5, and 3 times the leucine concentration in DMEM/F12, which was 0.450 mmol/L. The relative proliferation rate of the MAC-T cells was measured.At a confluence of 70–80%, the cells were treated with 0.420, 0.630, 0.840, 1.050, or 1.260 mmol/L isoleucine  diluted in DMEM/F12 with antibiotics for 6, 12, 24, 48, or 72 h. The experimental treatments correspond to 1, 1.5, 2, 2.5, and 3 times the isoleucine concentration in DMEM/F12, which was 0.420 mmol/L. The relative proliferation rate of the MAC-T cells was measured.Based on the leucine (2.4.) and isoleucine (2.5.) results, 24 h was selected as the treatment time and 9 concentrations of leucine and isoleucine combined were designed as follows: (1) 0.450 mmol/L leucine and 0.420 mmol/L isoleucine; (2) 0.450 mmol/L leucine and 0.630 mmol/L isoleucine; (3) 0.450 mmol/L leucine and 0.840 mmol/L isoleucine; (4) 0.675 mmol/L leucine and 0.420 mmol/L isoleucine; (5) 0.675 mmol/L leucine and 0.630 mmol/L isoleucine; (6) 0.675 mmol/L leucine and 0.840 mmol/L isoleucine; (7) 0.900 mmol/L leucine and 0.420 mmol/L isoleucine; (8) 0.900 mmol/L leucine and 0.630 mmol/L isoleucine; and (9) 0.900 mmol/L leucine and 0.840 mmol/L isoleucine. At a confluence of 70–80%, the cells were placed in DMEM/F12 with antibiotics containing different combinations of leucine and isoleucine for 24 h to determine the relative proliferation rate.μmol/L H2O2 for 6 h and then incubated in DMEM/F12 medium (1% antibiotics) with different combinations of leucine and isoleucine for 24 h  was seeded into the wells of a 96-well plate. After the cell confluence reached 70–80%, the completed medium was removed, and the cells were washed twice with cold PBS. Then, the cells were incubated in treated medium. The cellular viability was measured using a CCK-8 commercial kit . The absorbance  was determined using an enzyme-labeling instrument  at a wavelength of 450 nm. The relative proliferation rate and cellular viability were calculated using the following formulas:A 200 4 cells/mL) was seeded into the wells of a 6-well plate. After the cell confluence reached 70–80%, the completed medium was removed, and the cells were washed twice with cold PBS. Then, the cells were incubated with treated medium. The MDA content of the MAC-T cells was determined using a microplate method kit . The total oxidant status , total antioxidant status , TNF-α (Cat No. BPE92091), IL-1β (Cat No. BPE92157), IL-6 (Cat No. BPE92153), and IL-10 (Cat No. BPE92159) of the MAC-T cells were detected using ELISA kits  following the manufacturer's instructions. Briefly, MAC-T cells were digested by trypsin and placed in centrifuge tubes. Then, the tubes were centrifuged at 2,000 rpm for 20 min, and the supernatant was collected. After the cells were counted, the cell suspension was adjusted with PBS to reach a cell concentration of 1 × 106 cells/mL. Absorbance was detected at 530 (MDA) and 450 nm  using an enzyme-labeling instrument .A 2 mL MAC-T cells suspension  was used to extract the total RNA from the MAC-T cells following the manufacturer's instructions. Then, the quantity and quality of the total RNA were assessed using an ultraviolet-visible fluorescence spectrometer . The samples with an A260/A280 ratio of 2.0 : 2.2 were used for subsequent PCR reactions. Finally, the total RNA was reverse transcribed into cDNA using a PrimeScript RT Master Mix kit . The cDNA samples were stored at −20°C for further analysis.β-actin) were synthesized by Sangon Biotechnology Co., Ltd.  , 2 μL ROX Reference Dye (50×), 4.8 μL RNase-free ddH2O, 0.6 μL of reverse primer (100 μmol), 0.6 μL of forward primer (100 μmol), and 2 μL cDNA. The reaction conditions were as follows: 95°C for 3 min; 40 cycles at 95°C for 5 s; and 60°C for 15 s. The cycle threshold values were normalized to β-actin. The relative mRNA expression levels of the selected genes were calculated using the 2ΔΔCt− method [The mRNA expressing certain genes, including claudin-1, occludin, and zonula occludens-1 (ZO-1), was evaluated by RT-qPCR. The primers of these genes and the housekeeping gene  . RT-qPCRt method .Four volumes of lysis buffer containing 8 mol/L urea and 1% protease inhibitor cocktail were added to MAC-T cells samples from each treatment group and then sonicated three times on ice using a high intensity ultrasonic processor . The supernatant was collected after centrifugation at 12,000 g for 10 min at 4°C. Then, the protein concentration was determined using a BCA kit  following the manufacturer's instructions.The protein concentration of each sample was enzymatically hydrolyzed in equal quantities, and the volume was adjusted to be consistent with the lysate. Then, 20% trichloroacetic acid  was gradually added. The solution was mixed, vortexed, and incubated at 4°C for 2 h and then centrifuged at 4,500 g for 5 min. The supernatant was discarded, and the precipitate was washed with prechilled acetone three times. The acetone-precipitated protein pellets were suspended in 200 mmol/L TEAB . Then, trypsin (trypsin : proteins =1 : 50)  was added for digestion overnight. The solution was reduced with 5 mmol/L dithiothreitol  for 30 min at 56°C. Finally, iodoacetamide  was added to a final concentration of 11 mmol/L followed by incubation at room temperature in the dark for 15 min.Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed. Digested peptides were dissolved by LC mobile phase A containing 0.1% formic acid and 2% acetonitrile and then separated by a NanoElute ultraperformance LC system . The elution gradient was as follows: 6–24% solvent B containing 0.1% formic acid in 100% acetonitrile for 70 min, 24–32% for 14 min, 32–80% for 3 min, and finally kept at 80% for 3 min. All operations were performed on a NanoElute ultraperformance LC system at a constant flow rate of 450 nL/min. Peptides were separated by an ultrahigh performance liquid phase system subjected to a capillary ion source and then analyzed by timsTOF Pro MSP ; the electrospray voltage was 1.75 kV. The peptide parent ions and their secondary fragments were detected and analyzed using high-resolution TOF. The secondary MS scanning range was 100–1700 m/z. Data acquisition on the timsTOF Pro was collected using the parallel accumulation serial fragmentation (PASEF) acquisition mode. After the first MS stage, the second MS stage (charge number of the parent ions was 0–5) was recorded using the 10 PASEF mode. A dynamic exclusion time of 30 s was used for the MS/MS scan. When the threshold of the protein relative expression ratio increased by ≥1.5-fold or decreased by ≤0.6-fold, the result was considered significant.p value < 0.05, which was considered significant. The ggplot2 package in R software was used to construct a volcano map of the DE proteins with a 1.5-times fold change. Hierarchical clustering based on different protein pathways was conducted using the collated categories obtained after enrichment and after p values were filtered, which were enriched in at least 1 cluster with p < 0.05. Then, the filtered p value matrix was transformed using the function:A bioinformatics analysis using 4 comparison groups  was performed. Functional annotations of the quantified proteins were acquired using the Gene Ontology Annotation (GOA) Database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis databases . For thex values were z-transformed for each functional category. Then, the z-scores were clustered by one-way hierarchical clustering  using Genesis [The  Genesis .The expression levels of the candidate proteins screened by 4D label-free quantitative proteomic analyses were verified by quantification by parallel reaction monitoring (PRM) technique . The spep < 0.05 was used.The data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS version 22.0 . The data between different groups were compared by a one-way analysis of variance (ANOVA) followed by Duncan's post hoc multiple comparisons test. For the different statistical tests, a significance threshold of 2O2 concentration increased over time (μmol/L H2O2 decreased significantly (p < 0.05) after 4 h  but decreased significantly (p < 0.05) in 200, 400, 800, and 1000 μmol/L H2O2 after 6 h . Thus, the H2O2 treatment time of 6 h was selected for the oxidative stress model.The cellular viability of cells gradually decreased as the H2O2 significantly increased (p < 0.05) the TOS/TAS, MDA, and TOS but significantly decreased (p < 0.05) the TAS in MAC-T cells treated with ≥600 μmol/L H2O2 when compared to 0 μmol/L H2O2   and peaked at 1.33 ± 0.07 under 0.900 mmol/L leucine (p < 0.05) to 0.84 ± 0.14 under 1.350 mmol/L leucine. Similarly, after 24 h, the relative proliferation rate significantly increased (p < 0.05) to 1.30 ± 0.14 under 0.900 mmol/L leucine (p < 0.05) to 0.83 ± 0.03 under 1.350 mmol/L leucine. Likewise, after 48 h, despite an initial significant increase (p < 0.05), the relative proliferation rate significantly decreased (p < 0.05) from its peak under 0.900 mmol/L leucine at 1.15 ± 0.04 to 0.78 ± 0.15 under 1.350 mmol/L leucine  to 0.77 ± 0.02 after 6 h and 0.60 ± 0.08 after 48 h under 1.260 mmol/L isoleucine. However, after 12 h (p < 0.05) to 0.80 ± 0.04 under 1.260 mmol/L isoleucine. Similarly, after 24 h (p < 0.05) from 1.00 under 0.420 mmol/L isoleucine and peaked at 1.10 ± 0.02 under 0.840 mmol/L isoleucine before significantly decreasing (p < 0.05) to 0.57 ± 0.01 under 1.260 mmol/L isoleucine. After 72 h, the relative proliferation rate decreased significantly (p < 0.05) from 1.00 under 0.420 mmol/L isoleucine to 0.44 ± 0.02 under 1.260 mmol/L isoleucine  than 0.450 mmol/L leucine and 0.420 mmol/L isoleucine , while TOS, MDA, and TOS/TAS significantly increased (p < 0.05) in the H1L1I group when compared to the control group, indicating that H2O2-induced oxidative damage had occurred in MAC-T cells (p < 0.05) in the H1L2I, H2L1I, and H2L1.5I groups by 23.08%, 34.62%, and 24.36%, respectively (p < 0.05) in the H1L2I and H2L1I groups by 46.15% and 38.46%, respectively (p < 0.05) than the H1L1I group  than in the H2L1.5I group. Compared to the H1L1I group, TOS/TAS significantly decreased (p < 0.05) in the H1L2I, H2L1I, and H2L1.5I groups (p < 0.05) by 25.20% in the H2L1.5I group when compared to the H1L2I and H2L1I groups.The relative proliferation rate and TAS significantly decreased  in the H1L1I group when compared to the control group (p < 0.05) by 48.24% in the H1L2I, H2L1I, and H2L1.5I groups, while 1 L-1β significantly decreased (p < 0.05) in the H1L2I group by 65.64%  than that in the H2LI and control groups  in MAC-T cells due to oxidative stress  the mRNA expression levels of claudin-1 and occludin and isoleucine significantly increased (p < 0.05) the mRNA expression level of occludin (p < 0.05). Additionally, combined leucine and isoleucine significantly increased (p < 0.05) the mRNA expression levels of claudin-1, occludin, and ZO-1 , cellular component (CC), and molecular function (MF), a cluster analysis was conducted to compare the functional correlations between DE proteins in the experimental groups; the results are displayed in a heat map . When ce2O2-induced oxidative damage in MAC-T cells, a KEGG pathway enrichment analysis was conducted. Most of the pathways were significantly altered after MAC-T cells were subjected to oxidative stress (p < 0.05), while the map 00280 valine, leucine, and isoleucine degradation, map 00640 propanoate metabolism, and map 04714 thermogenesis pathways were significantly downregulated (p < 0.05). When compared to the H1L1I group, the upregulation of a common pathway (map 00640 propanoate metabolism) occurred in the H1L2I and H2L1I groups, while unique pathways in the H1L2I (map 04146 peroxisome) and H2L1I groups  were upregulated; these pathways were not identified in the H2L1.5I group. Despite six upregulated pathways in the H2L1.5I group, these pathways were not detected when compared to the H1L1I and control groups. No significantly downregulated pathways were detected in the H1L2I or H2L1.5I groups. Although five downregulated pathways existed in the H2L1I group, these were not associated with H2O2-induced oxidative damage.To further explore the regulation pathways of leucine and isoleucine against He stress . For exap < 0.05) in the propanoate metabolism; peroxisome; valine, leucine and isoleucine degradation; and thermogenesis pathways, respectively (Tables p < 0.05) after leucine intervention (p < 0.05)  .The 8 target proteins shown in Tables 2O2-induced oxidative damage in bovine mammary epithelial cells was outlined in 2O2-induced oxidative damage (Therefore, based on all above data, the protective mechanism of leucine and isoleucine against He damage .2O2 is the main product of oxidative stress [in vitro by treating MAC-T cells with H2O2. Our findings suggested that leucine and isoleucine alleviated oxidative stress through certain pathways, which furthers our understanding of the antioxidant effects of certain nutrients.Free radical production plays an essential role in normal metabolism, including hydrogen peroxide, superoxide radicals, and hydroxyl radicals . Excessie stress . Therefo2O2 resulted in cumulative oxidative damage to cellular components, altered cellular functions, and apoptotic cell death. We also observed an increase in TOS and MDA, as well as a decrease in the relative proliferation rate and TAS when MAC-T cells were treated with H2O2, indicating that these cells were subjected to oxidative stress. Leucine and isoleucine acting as antioxidants have been reported [Oxidative stress and apoptosis are considered effective immune defense mechanisms when the body is subjected to various harmful stimuli . Sordillreported –26. Simi2O2-induced oxidative damage in MAC-T cells. Additionally, isoleucine positively regulated the propanoate metabolism and peroxisome pathways in MAC-T cells after oxidative damage. Peroxisomes are small membrane-bound organelles that play diverse roles in the cellular metabolism, including the conversion of hydrogen peroxide to nontoxic forms [2O2-induced oxidative damage in MAC-T cells. Interestingly, no DE proteins were enriched in the KEGG pathways associated with oxidative stress in MAC-T cells after oxidative damage when leucine and isoleucine concentrations were simultaneous increased. In addition, the H2L1.5I group had a higher TOS/TAS than the H1L2I and H2L1I groups. These results may be related to the concentrations and proportions of leucine and isoleucine or their interactions, as BCAAs share the same transporter on the cell membrane [The KEGG pathway enrichment analysis results indicated that leucine intervention positively regulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways in MAC-T cells after oxidative damage. The upregulation of ACAD8 and ALDH6A1 expression in the valine, leucine, and isoleucine degradation pathway was observed, which promotes valine metabolism and generates succinyl-CoA , 31. Sucic forms , 36. Mammembrane , 38. Howα, IL-1β, and IL-6 [Oxidative stress can activate the inflammation signaling pathway by activating pro-inflammatory cytokines, such as TNF-and IL-6 , therebyand IL-6 , 41. J. and IL-6  reportedand IL-6 . In the and IL-6 .Oxidative stress can affect barrier functions, leading to increased cell permeability . Tight jInterestingly, we found that leucine and isoleucine could act as antioxidants in MAC-T cells, which was expected. Additionally, we found that the protective mechanisms of leucine and isoleucine against oxidative stress were related to multiple regulatory pathways. Notably, the antagonism between leucine and isoleucine existed in antioxidative stress, but not in barrier functions or as anti-inflammatory activities. Based on this experimental data alone, the exact mechanism between the interaction of leucine and isoleucine was difficult to determine and remains unknown. Therefore, further research should be conducted to further analyze, expound, and prove this mechanism.2O2-induced oxidative stress by regulating the propanoate metabolism; valine, leucine and isoleucine degradation; and thermogenesis pathways, thereby generating more ATP to supplement energy demands. Moreover, the protective mechanisms of isoleucine against oxidative stress improved the deficit in peroxisome transport by regulating the peroxisome pathway and promoting acetyl-CoA production by regulating the propanoate metabolism pathway. The findings of this study enhance our understanding of the underlying protective mechanisms of leucine and isoleucine against oxidative damage.In conclusion, leucine was found to protect MAC-T cells from H


Chromochloris zofingiensis is capable of producing high levels of triacylglycerol rich in C18 unsaturated fatty acids (UFAs). FA desaturation degree is regulated by FA desaturases (FADs). Nevertheless, it remains largely unknown regarding what FADs are involved in FA desaturations and how these FADs collaborate to contribute to the high abundance of C18 UFAs in triacylglycerol in C. zofingiensis.The green microalga CzFADs) and updated their gene models. Functional validation of these CzFADs in yeast and cyanobacterial cells revealed that seven are bona fide FAD enzymes with distinct substrates. Combining the validated functions and predicted subcellular compartments of CzFADs and the FA profiles of C. zofingiensis, the FA desaturation pathways in this alga were reconstructed. Furthermore, a multifaceted lipidomic analysis by systematically integrating thin-layer chromatography, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry techniques was conducted, unraveling profiles of polar membrane lipids in C. zofingiensis and major desaturation steps occurring in these lipids. By correlating transcriptional patterns of CzFAD genes and changes of lipids upon abiotic stress conditions, our results highlighted collaboration of CzFADs for C18 UFA synthesis and supported that both de novo FA synthesis and membrane lipid remodeling contributed C18 UFAs to triacylglycerol for storage.To address these issues, we firstly determined the transcription start sites of 11 putative membrane-bound FAD-coding genes (C. zofingiensis and shed light on collaboration of CzFADs for the synthesis and enrichment of C18 UFAs in triacylglycerol.Taken together, our study for the first time elucidated the pathways of C18 FA desaturations and comprehensive profiles of polar membrane lipids in The online version contains supplementary material available at 10.1186/s13068-021-02037-2. PMLs, serving as cell boundaries, not only maintain the integrity and identity of subcellular compartments, but also play roles in the initiation of intracellular signals –3. On thoperties . PUFAs eArabidopsis thaliana, which harbors over ten FAD-coding genes [∆9 from C18:0 [∆9 can be either incorporated into the chloroplast membrane lipids or exported out of the chloroplast and then incorporated into the endoplasmic reticulum (ER) membrane lipids for further desaturation. AtFAD4 and AtFAD5, both residing in the chloroplast, act on C16:0 in phosphatidylglycerol (PG) to form C16:1∆3t (a trans double bond on the ∆3 position) [∆7 [∆9 to C18:2∆9,12 [∆9 to C18:2∆9,12 and C16:1∆7 to C16:2∆7,10 in the chloroplast membrane lipids [∆7,10,13 and C18:3∆9,12,15, ω3 desaturases are needed. Arabidopsis has three isozymes of ω3 desaturase, AtFAD3, AtFAD7 and AtFAD8. AtFAD3 is localized in the ER and desaturates C18:2∆9,12 in ER membrane lipids to C18:3∆9,12,15 [∆9,12 and C16:2∆7,10 in the chloroplast membrane lipids to form C18:3∆9,12,15 and C16:3∆7,10,13, respectively [The functional roles of FADs have been well studied in land plants particularly the model plant ng genes , 10. In om C18:0 , 12. C16osition)  and on Cion) [∆7 , 15, res8:2∆9,12 . AtFAD6,e lipids . To prod∆9,12,15 . By contectively , 22.Chlamydomonas reinhardtii, with annotated chromosome-level genome sequence and well-developed genetic tools, has long been used to study lipid metabolism including FA desaturation [C. reinhardtii to form C18:1∆9-ACP, which is either incorporated into chloroplast membrane lipids or released as free FA and then exported outside the chloroplast for ER lipids. CrFAD2 is responsible for the desaturation of C18:1∆9 in ER membrane lipids while CrFAD6 likely functions as a ω6 desaturase in the chloroplast. Nevertheless, C. reinhardtii, unlike Arabidopsis, contains only one ω3 desaturase , which is located in the chloroplast; and upon CrFAD7 suppression, more ω6 FAs (both C16 and C18) accumulate at the expense of ω3 FAs [C. reinhardtii synthesizes FAs  that are not present in Arabidopsis, additional FADs are involved, including Cr∆4FAD that is chloroplast-located and introduces a double bond on the ∆4 position of C16:3∆7,10,13 in MGDG [Similar to Arabidopsis, algae particularly green microalgae perform the de novo FA synthesis in the chloroplast and carries out FA desaturations in both compartments of the chloroplast and ER , 23. Thef ω3 FAs . Probabl in MGDG , and CrD in MGDG .C. reinhardtii is generally not considered as a good production strain as the biomass density that can be achieved for this alga is unsatisfactory. By contrast, Chromochloris zofingiensis, a unicellular green alga closely related to C. reinhardtii, is able to grow robustly under multiple trophic conditions and achieve ultrahigh biomass densities under the heterotrophic fed-batch mode using glucose as the carbon source [C. zofingiensis has the ability to synthesize high levels of triacylglycerol (TAG) rich in C18 UFAs [C. zofingiensis [C. zofingiensis has been cited as an emerging model alga for studying lipid metabolism [Nevertheless, n source –30. MoreC18 UFAs , 31, 32,C18 UFAs , a valueC18 UFAs –36. The ngiensis –39 allowngiensis , workablngiensis –43, and ngiensis –47, C. ztabolism .C. zofingiensis has long been profiled, what FADs are involved in FA desaturations, what substrates these FADs use, and how they respond to environmental fluctuations and contribute to high-abundance C18 UFAs in TAG remain largely unknown. C. zofingiensis genome is predicted to encode 13 putative membrane-bound FADs [FAD genes and confirmed that the 13 genes in fact belong to 11 gene loci that encode membrane-bound FADs of six subgroups. To better understand their functional roles, we then conducted a multifaceted study by integrating the functional validation in yeast and cyanobacterial cells, transcriptional profiling of FAD genes and lipidomics of C. zofingiensis responding to various abiotic stress conditions. Our study reconstructed the pathways of C18 FA desaturations, profiled the polar membrane lipids and provided implications into the collaboration of FADs for C18 FA synthesis and enrichment in TAG in C. zofingiensis.Although the FA composition of und FADs . Here, wC. reinhardtii and Arabidopsis as the query sequences, Blast against the C. zofingiensis non-redundant protein sequences database revealed the presence of 14 putative FAD-coding genes  to 442 bp (CzFAD6B) 2-3HH and H/Q(X)1-2HH  that functions in converting C18:1∆9 to C18:2∆9,12 [∆9 and introduces a double bond on the ∆12 position [∆9 to C18:2∆9,12 but also C16:1∆7 to C16:2∆7,10 [n-3 positions of C18 and C16 PUFAs [sn-2 position of MGDG and probably digalactosyldiacylglycerol (DGDG) [C. reinhardtii FAD3 (CrFAD3 or Cr∆4FAD) that introduces a ∆4 double bond on MGDG-linked C16 PUFAs with a preexisting ∆7 double bond [trans double bond on ∆3 of C16:0 in sn-2 position of PG [To understand the evolutionary position of CzFADs, a phylogenetic analysis was conducted using FADs of different functions from various organisms  , 15; CzFble bond ; CzFAD4 on of PG .CzFAD genes, their coding sequences were each sub-cloned into the yeast expression vector pYES2-CT and introduced to the baker yeast S. cerevisiae for heterologous expression. The presence of CzFAD genes was confirmed by colony PCR  relative to the total FAs (TFA)  to the cyanobacterium S. elongatus for functional validation. The presence of CzFAD genes was also confirmed by colony PCR  based on the quantification of FA profiles . Expression of CzFAD6B in S. elongatus, on the other hand, did not produce new FA  including C16:1∆9, C18:1∆9 and C18:1∆11. When supplied with C16:1∆9, higher abundance of C18:1∆11 was observed in S. elongatus transformed with the empty vector  and its elongated product C18:2∆11,14 (1.8- or 1.9-fold), while C18:1∆11 addition did not promote C18:2∆11,14 abundance  instead of C18:1∆11 desaturation. Together with that C18:1∆9 addition caused considerably increased abundance of C18:2∆9,12 , C18:2∆9,15 (2.8%) and C18:2∆11,14 (3.6%)  desaturation activity on C18 UFAs being the major and the ∆12 desaturation activity on C16:1aturases –64, but cterized , whethercterized , 62.S. elongatus transformant carrying CzFAD5A contained the newly synthesized C16:1∆7  than on C18:3∆9,12,15 (75.1%), indicating that the desaturase prefers the latter (ω3 type) over the former (ω6 type) as the substrate.The expression of uct Fig. a. By conCzFAD3A but not CzFAD3B in S. elongatus led to formation of C16:1∆4, regardless of FFA feeding or not , also possesses ∆4 activity yet on C16:3∆7,10,13 to form C16:4∆4,7,10,13 [C. zofingiensis consists of similar C16 UFAs as C. reinhardtii [∆7,10,13 as well. Unfortunately, this FFA is not commercially available and thus was not examined in the present study. It is worth noting that C. reinhardtii lacks the ∆6 UFAs C18:3∆6,9,12 and C18:4∆6,9,12,15 [Interestingly, the expression of ,7,10,13 . Considenhardtii , CzFAD3A,9,12,15 , 24, ind,9,12,15 .S. elongatus expressing CzFAD4 produced a new FA, C16:1∆3t (a trans double bond on the ∆3 position), at the expense of C16:0  under stress conditions such as nitrogen deprivation (ND), sulfur deprivation (SD) and salt stress (SS), etc. [CzFAD genes in C. zofingiensis respond to these stress conditions at the transcriptional levels, RT-qPCR was performed in a time-resolved manner. The gene expression was presented as the Log2 transformed fold changes relative to 0 h , CzFAD6A, CzFAD7A, CzFAD5A, CzFAD5B and CzFAD3B were moderately up-regulated (two- to fourfold increase), while CzFAD6B, CzFAD5C, CzFAD3A and CzFAD4 were moderately down-regulated. When exposed to SD, the expression patterns of most genes were similar to that under ND conditions with several differences: CzFAD6A, CzFAD7A and CzFAD5B were down-regulated by SD but up-regulated by ND, while CzFAD6B was up-regulated by SD but down-regulated by ND. Under SS conditions, the gene expression patterns resembled that under ND conditions except for CzFAD6B, CzFAD5B and CzFAD4: CzFAD6B was up-regulated and CzFAD5B was down-regulated by SS, contrary to that under ND conditions; CzFAD4 showed little transcriptional difference upon SS but was down-regulated by ND. Taken the three stress conditions for comparison, CzSAD, CzFAD2, CzFAD7B, CzFAD5A and CzFAD3B were up-regulated, while CzFAD5C and CzFAD3A were down-regulated under all the three conditions.S), etc. , 45, 65.0 h Fig. . Upon NDC. zofingiensis under different stress conditions, quantification of FAs from both TFA and TAG was carried out. For TFA, the FAs consist of C16:0, C16:1∆7, C16:1∆3t, C16:2∆7,10, C16:3∆7,10,13, C16:4∆4,7,10,13, C18:0, C18:1∆9, C18:2∆9,12, C18:3∆6,9,12, C18:3∆9,12,15 and C18:4∆6,9,12,15, with C16:0, C18:1∆9, C18:2∆9,12 and C18:3∆9,12,15 being the major ones , thin-layer chromatography (TLC) and gas chromatography–mass spectrometry (GC–MS). LC–ESI–MS analysis of PMLs under favorable growth conditions revealed the lipid species of MGDG, DGDG, sulfoquinovosyl diacylglycerol (SQDG), PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) , indicating that turnover of the preexisting PMLs is not enough to support the massive TAG accumulation. When examining the changes of FAs in TAG, the major increased ones were C16:0, C18:1∆9, C18:2∆9,12 and C18:3∆9,12,15, and accounted for 98.1% of total increases; of which, the three C18 UFAs represented 81.8% of total increases  in a continuing manner to meet the requirement of TAG synthesis for C18:2∆9,12.In response to ND, TAG level showed a considerable increase, accompanied by severe decreases of PMLs particularly the glycolipids such as MGDG, DGDG and SQDG Fig. a. The amC. zofingiensis performs its de novo fatty acid synthesis in the chloroplast [∆9-ACP [C. reinhardtii [Using acetyl-CoA as the precursor and building block, oroplast . The plaoroplast , 45. CzS [∆9-ACP . These tnhardtii . These Fnhardtii , 68, arenhardtii  and thenC. zofingiensis genome encodes 11 putative membrane-bound FADs yet only seven of them were demonstrated as active desaturases when expressed in yeast and/or cyanobacterial cells. These include CzFAD2, CzFAD6A, CzFAD7B, CzFAD5A, CzFAD3A, CzFAD3B and CzFAD4 . Similarly, CzFAD2 shows no substrate dependence, but is ER-targeted with access to the extraplastidial membrane lipids. The ω3 desaturase CzFAD7B functions independently on substrates either, yet it likely resides at the plastid outer envelope and can access both plastidial and extraplastidial membrane lipids for desaturation, as proposed for CrFAD7 in C. reinhardtii [sn-2 position of MGDG and DGDG, while the latter is restricted to C16:0 in the sn-2 position of PG.Taken together, likely, CzFAD6A, localized in the chloroplast, functions as an ω6 desaturase with no dependence on substrates such as acyl chains (C16 and C18), lipid head group types  or stereo positions  rose from 18.8% to 43.2% on day 2 of ND  are controlled by both plastidial and extraplastidial CzFADs at least partly at the transcriptional level. This may partially explain why FAs particularly C18:1∆9, C18:2∆9,12 and C18:3∆9,12,15 had higher levels under ND than under SD or SS  exhibited little change and C18:3∆9,12,15 (product of CzFAD7B) had an over twofold decrease  [C. zofingiensis such as CzDGAT1A and CzDGTT5 accept C18:1∆9-CoA and C18:1∆9-containing DAGs as substrate for TAG synthesis [∆9 that is incorporated into TAG for storage would not be further desaturated by CzFAD2 and CzFAD7B, thus leading to enhanced relative abundance of C18:1∆9 in C. zofingiensis. By contrast, in C. reinhardtii under stress conditions such as ND, CrSAD is just stimulated mildly while the desaturases responsible for downstream desaturations of C18:1∆9 such as CrFAD2 and CrFAD7 are highly induced [C. reinhardtii has a considerably lower abundance of C18:1∆9 yet a higher abundance of C18 PUFAs than C. zofingiensis.Interestingly, although CzSAD, CzFAD2 and CzFAD7B were up-regulated in comparable degrees in response to ND  . In suppynthesis . As desa induced . This maC. zofingiensis, TAG is synthesized only at a basal level under favorable growth conditions, with FAs being predominantly esterified with membrane lipids; upon stress conditions such as ND, TAG accumulates substantially and dominates over other lipid classes accompanied by a severe decrease in membrane lipids [In e lipids , 44, 48.e lipids –84, indiC. zofingiensis. Firstly, when TAG increased in response to ND, we observed a decrease in membrane lipids as a whole and each of the individual membrane lipids as well, though the decrease levels varied greatly depending on the membrane lipid classes  that have to be synthesized in membrane lipids via the action of membrane-bound CzFADs prior to incorporation into TAG. The FAs in membrane lipids can be directly transesterfied to TAG mediated by phospholipid:diacylglycerol acyltransferase (PDAT) or released as FFAs from sn-1/2 positions and then incorporated into TAG catalyzed by DGAT [C. reinhardtii (CrPDAT) has been characterized and shown to transfer FAs from the sn-2 position of a broad range of membrane lipids for TAG synthesis [C. reinhardtii, C. zofingiensis harbors a single PDAT [sn-2 position of membrane lipids  may be needed to release C18:2∆9,12 and C18:3∆9,12,15 from membrane lipids to support DGAT-mediated incorporation. As a support, the DGAT enzymes in C. zofingiensis such as CzDGAT1A and CzDGTT5 have high in vitro activities in using C18:2∆9,12-CoA and C18:3∆9,12,15-CoA as acyl donors for TAG synthesis [Our quantitative data of lipids here further support that FAs particularly C18 UFAs recycled from the remodeling of membrane lipids are used for TAG biosynthesis in ses Fig. a. Second by DGAT . The PDAynthesis . Neverthgle PDAT , which igle PDAT , 45. It position . C. zofinditions –46. If iynthesis .C. zofingiensis under this condition. Moreover, the C16:0, C18:0 and C18:1∆9 in TAG may also be recycled from membrane lipids, as is the case for C18:1∆9 in C. reinhardtii where C18:1∆9 from MGDG turnover contributes to ca. 40% of C18:1∆9 in TAG [C. zofingiensis. The C. zofingiensis TAG is rich in C18:1∆9 (~ 50%) , purchased from the American Type Culture Collection , was maintained on an agar plate of modified BG-11 medium at 16 °C with dim light in our lab. When necessary, a single colony from the agar plate was inoculated into 10 mL of liquid medium (in a 100-mL flask) and grown aerobically at 25 °C for 6 days with orbital shaking at 150 rpm and continuous illumination of 30 µmol photons m−2 s−1. The algal cells were then inoculated at 10% (v/v) into glass columns (3.0 cm in diameter) with continuous illumination of 70 µmol photons m−2 s−1 and aeration of 1.5% CO2 enriched air and grown at 25 °C to late exponential phase (4 days), which were used as seed cultures for subsequent experiments.C. zofingiensis, namely, nitrogen deprivation (ND), sulfur deprivation (SD), and salinity stress (SS). Briefly, the seed cells were centrifuged for 5 min at 5000 g, washed with deionized water and re-suspended at an initial cell density of 0.5 g L−1 in fresh BG-11 medium without nitrogen  or sulfur (MgSO4 was replaced with MgCl2) or containing 200 mM NaCl (SS). All cultures were grown in 250-mL glass columns with the same parameters mentioned above. The samples from 0, 6, 12, 24 and 48 h of treatments were used for RNA extraction and RT-qPCR, while samples from 0, 2, 4 and 6 days were used for lipid extraction and analysis.Three treatments were applied to C. zofingiensis FAD genes, their transcription start sites were determined by 5′ rapid amplification of cDNA ends (RACE)-PCR using the SMARTer RACE 5′ Kit  and the 5′ gene-specific primers  and the phylogenetic tree was generated under MEGA6 using the neighbor-joining method [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), respectively. Subcellular localization prediction was performed using PredAlgo, a multi-subcellular localization prediction tool dedicated to green algae , TargetP (http://www.cbs.dtu.dk/services/TargetP/), and WoLF PSORT (https://wolfpsort.hgc.jp/).Sequence alignment of FAD proteins from various organisms was conducted using ClustalX2.1 with default parameters  (Invitrogen) and selected on SD-URA (synthetic defined medium with uracil omitted) agar plates containing 1% glucose, according to our previously procedures [For heterologous expression in yeast, ocedures . The empCzFAD genes were each introduced into Synechococcus elongatus PCC7942  and selected on BG-11 agar plates containing 10 μg mL−1 of spectinomycin under continuous illumination of 50 μE m−2 s−1 and temperature of 30 °C, following the manual of GeneArt™ Synechococcus Protein Expression Vector. The empty vector pSyn6 was also introduced into S. elongatus and used as the control. The putative colonies were validated by genomic PCR. To test the function of CzFADs in S. elongatus, positive transformants were grown in liquid BG-11 medium containing 10 μg mL−1 of spectinomycin for 7 days, with orbital shaking of 120 rpm, continuous illumination of 50 μE m−2 s−1 and temperature of 30 °C. When needed, free fatty acids were fed to the S. elongatus cultures at a concentration of 250 µM.For heterologous expression in cyanobacteria, CzFAD genes were calculated relative to the internal control gene β-actin.Total RNA extraction from algae samples and removal of contaminated DNA were performed using the plant RNA extraction kit  according to the manufacturer's instructions. After purification, the RNA samples were subjected to NanoDrop 2000c  for concentration determination and electrophoresis for quality checking. The cDNA synthesis and quantitative PCR were conducted on a 7500 Fast Real-Time PCR System  with SYBR® Premix Ex Taq™ II (Tli RNase H Plus) (TaKaRa), as described previously . PrimersTotal lipids were extracted from dried cell samples  using a solvent mixture of chloroform/methanol/0.75% NaCl solution , according to our previous methods . The chlC. zofingiensis samples: neutral lipids were developed on a silica gel TLC plate  with a mobile phase of hexane/tert-butyl methyl ether/acetic acid  and polar lipids were developed with a mobile phase of chloroform/methanol/acetic acid/water  [sn-2 position of membrane lipids, each of the recovered membrane lipids was treated with Rhizopus arrhizus lipase  and the resultant lyso-membrane lipids were recovered again by TLC separation [Thin-layer chromatography (TLC) was used to separate lipids extracted from  volume) . The spoparation .Total lipids and lipids recovered from TLC plates were transesterified with sulfuric acid in methanol. The resulting fatty acid methyl esters (FAMEs), after extraction from the reaction mixture, were analyzed by using an Agilent 7890 capillary gas chromatograph (GC) equipped with a 5975 C mass spectrometry (MS) detector and a HP-88 capillary column  for quantification . IndividC. zofingiensis samples were dissolved chloroform/methanol  and then subjected to analyses on a triple quadrupole MS/MS  with electrospray ionization (ESI) source coupled with an Acquity Ultra-Performance Liquid Chromatography (UPLC) system (Waters). MGDG, DGDG, DGTS and PC were analyzed in the positive mode, while PG, SQDG, PE and PI were in the negative mode. Multiple reaction monitoring (MRM) was used for quantitative analysis. The internal standards included MGDG C18:0/C18:0, DGDG C18:0/C18:0, DGTS C16:0/C16:0 d9, PE C17:0/C14:1, PG C17:0/C20:4 and PI C17:0/C20:4. The external standards used for calibration, on the other hand, included MGDG , DGDG , DGTS (C16:0/C16:0), PG (C16:0/C18:1 and C18:0/C18:1), PE (C18:0/C18:1), PI (C18:1/C18:1) and SQDG (C16:0/C18:3). The DGTS standard was used for PC quantification as well. Except that MGDG (C18:0/C18:0) and DGDG (C18:0/C18:0) were purchased from Matreya, LLC , other lipid standards were from Avanti Polar Lipids .The quantification of polar membrane lipids by liquid chromatography–mass spectrometry (LC–MS) was preformed according to the previously described methods , 91. BriCzFAD genes are deposited in NCBI GenBank with accession numbers as followed: MT323105 (CzFAD2), MT323106 (CzFAD6A), MT323107 (CzFAD6B), MT323108 (CzFAD6C), MT323109 (CzFAD7A), MT323110 (CzFAD7B), MT323111 (CzFAD5A), MT323112 (CzFAD5B), MT323113 (CzFAD5C), MT323114 (CzFAD3A), MT323115 (CzFAD3B) and MT323116 (CzFAD4).The full-length coding sequences of Additional file 1: Figure S1. Comparison between the gene models of CzFADs predicted from Roth et al. [Figure S2. Characterization of 5′ UTR sequence and cloning of full-length CDS of CzFAD genes. Figure S3. Conserved domains detected in CzFADs by NCBI Conserved Domains Search. Figure S4. Sequence logo and alignment of functional motifs of Δ12, ω6 and ω3 FADs (a), Δ7/Δ9 FADs (b), Δ3trans FADs (c), and front-end FADs (d). Figure S5. Predicated transmembrane domains for CzFADs by TMHMM. Figure S6. Cladogram of fatty acid desaturases of difference functions from various organisms. Figure S7. PCR characterization of the S. cerevisiae transformants (a) and S. elongatus transformants (b) harboring individual CzFAD genes. Figure S8. The mass spectra of unusual fatty acids (in the form of methyl ester) produced in transformed S. elongatus.. Figure S9. GC–MS chromatography of fatty acids from S. elongatus expressing the empty vector pSy6, CzFAD3A, or CzFAD3B. Newly synthesized fatty acid is designated in red. Figure S10. Relative abundance of species of membrane lipid classes in C. zofingiensis under favorable growth conditions. Figure S11. Fatty acid relative abundance of individual membrane lipid classes in C. zofingiensis under favorable growth conditions. Figure S12. Fatty acid relative abundance of sn-2 position of individual membrane lipid classes in C. zofingiensis under favorable growth conditions.h et al.  and oursAdditional file 2: Table S1. Sequence features of FAD genes in C. zofingiensis.Table S2. Primers used for constructing CzFADs-containing yeast expression vectors. Table S3. Primers used for 5′ RACE experiments of CzFAD genes. Table S4. Primers used for constructing CzFADs-containing S. elongatus expression vectors. Table S5. Primers used for RT-qPCR of CzFAD genes. Table S6. Fatty acid composition of TFA in C. zofingiensis as affected by various stress conditions of ND, SD and SS. Table S7. Fatty acid composition of TAG in C. zofingiensis as affected by various stress conditions of ND, SD and SS.


T and CM04-06T, were isolated from faeces of two healthy Chinese donors and characterized using a polyphasic approach. The two strains were observed to be gram-negative, non-motile, and rod-shaped. Both strains grew optimally at 37 °C and pH 7.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains clustered with species of the genus Faecalibacterium and were most closely related to Faecalibacterium prausnitzii ATCC 27768T with sequence similarity of 97.18% and 96.87%, respectively. The two isolates shared a 16S rRNA gene sequence identity of 98.69%. Draft genome sequencing was performed for strains AF52-21T and CM04-06T, generating genome sizes of 2.85 Mbp and 3.01 Mbp. The calculated average nucleotide identity values between the genomes of the strains AF52-21T and CM04-06T compared to Faecalibacterium prausnitzii ATCC 27768T were 83.20% and 82.54%, respectively, and 90.09% when comparing AF52-21T and CM04-06T. Both values were below the previously proposed species threshold (95–96%), supporting their recognition as novel species in the genus Faecalibacterium. The genomic DNA G + C contents of strains AF52-21T and CM04-06T calculated from genome sequences were 57.77 mol% and 57.51 mol%, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, we conclude that both strains represent two new Faecalibacterium species, for which the names Faecalibacterium butyricigenerans sp. nov. (type strain AF52-21T = CGMCC 1.5206T = DSM 103434T) and Faecalibacterium longum sp. nov. (type strain CM04-06T = CGMCC 1.5208T = DSM 103432T) are proposed.Exploiting a pure culture strategy to investigate the composition of the human gut microbiota, two novel anaerobes, designated strains AF52-21 The composition and diversity of the gut microbiota are affected by numerous factors, including host genetics4, long-term diet6, drugs8, and several other environmental factors9. Evidence suggests that the composition of the microbiota is associated with the development of obesity12, diabetes14, inflammatory bowel disease16, colorectal cancer18, and non-alcoholic fatty liver disease20. Therefore, the composition and function of the microbial species living in our gut are of crucial importance for maintenance of health. Short-chain fatty acids (SCFAs) produced by fermentation of dietary fibre by several abundant genera of the intestinal microbiota, including Roseburia, Eubacterium, and Faecalibacterium21, have been reported to elicit beneficial effects on energy metabolism and prevent colonization of pathogens22. Bacteria of the genus Faecalibacterium, abundant butyric acid-producing bacteria colonizing the human gut, display anti-inflammatory effects and may be used as potential probiotics for treatment of gut inflammation24.The human gastrointestinal tract harbours complex microbial communitiesFaecalibacterium, belonging to the family Ruminococcaceae within the order Clostridiales, comprises only one validated species, Faecalibacterium prausnitzii25, and two non-validated published species, ‘Faecalibacterium moorei’26 and ‘Faecalibacterium hominis’27, all originally isolated from human faeces. F. prausnitzii is a gram-negative, non-spore-forming, and strictly anaerobic rod-shaped bacterium. The genomic G + C content of the genus Faecalibacterium ranges from 47 to 57%28. The fermentation products from glucose are butyrate, D-lactate, and formate. In the present study, we describe two novel species of the genus Faecalibacterium by using a polyphasic taxonomy approach along with whole genome sequence analysis.The genus T and CM04-06T were isolated from the faeces of two healthy Chinese donors. Both strains were observed to be obligate anaerobic, gram-negative, non-spore-forming, non-motile, and rod-shaped bacteria  for AF52-21T and 30–45 °C (optimum 37 °C) for CM04-06T. Growth was observed at pH 6.0–7.5 (optimum 7.0–7.5) for AF52-21T and pH 5.0–8.0 (optimum 7.0–7.5) for CM04-06T. Strains AF52-21T and CM04-06T grew with 0–1% and 0–3% NaCl, respectively. Both strains were found to be catalase-negative. The major metabolic end products for strains AF52-21T and CM04-06T were acetic acid, formic acid, butyric acid, and lactic acid. Differential physiological and biochemical characteristics of strains AF52-21T and CM04-06T with the closest related species of genus Faecalibacterium are listed in the species description and in Table Strains AF52-21T and CM04-06T and related species are shown in Table T were found to be C18:1ω9c (39.0%), C16:0 (16.3%), iso-C19:0 (12.9%), C18:1ω7c (8.1%), and C14:0 (5.9%). The profiles including C18:1ω9c (32.5%), C16:0 (25.5%), iso-C17:1 I/anteiso B (9.7%), C18:1ω7c (7.5%), and iso-C19:0 (5.9%) were detected as the predominant fatty acids for strain CM04-06T. The highest levels of fatty acids, including C16:0 and C18:1ω9c, were found to be similar, but not identical comparing strains AF52-21T, CM04-06T, and ATCC 27768T. Furthermore, strains AF52-21T, CM04-06T, and ATCC 27768T could be differentiated by less abundant fatty acids, such as C18:1 2OH, anteiso-C15:0, anteiso-C17:0, C13:0 3OH/Iso-C15:1 I, C16:1ω7c/C16:1ω6c, and antei-C18:0 /C18:2ω6, 9c , phosphatidylglycerol (PG), and several unidentified glycolipids  being present in both strains. However, the presence/absence of three unidentified lipid , unidentified phospholipid (PL), unidentified phosphoglycolipids (PGL) and an unidentified glycolipid (GL2) can be used to distinguish strains AF52-21T and CM04-06T from the closest relative. Quinones were not detected in either strain 25. CheckM analysis of the genomes showed high completeness (> 90%) and low contamination (< 5%) , teichoic and lipoteichoic acids, lipopolysaccharides, and metabolism of polar lipids and polyamines by RAST annotation, comparing strains AF52-21T and CM04-06T with ATCC 27768T are shown in Table T, 11 genes/proteins were observed to be associated with biosynthesis of DAP, 18 genes/proteins with biosynthesis of polar lipids, 12 genes/proteins with biosynthesis of polyamines, 3 genes/proteins with biosynthesis of teichoic and lipoteichoic acids, and 14 genes/proteins with biosynthesis of quinones. For strain CM04-06T, 12 genes/proteins were found to be associated with biosynthesis of DAP, 19 genes/proteins with biosynthesis of polar lipids, 13 genes/proteins with biosynthesis of polyamines, 2 genes/proteins with biosynthesis of teichoic and lipoteichoic acids, and 16 genes/proteins with biosynthesis of quinones. We detected no genes involved in the biosynthesis of lipopolysaccharides or mycolic acids in strains AF52-21T and CM04-06T.For genome annotation, the distributions of the genes into clusters of orthologous groups (COGs) functional categories are depicted in Supplementary Fig. T, CM04-06T, and ATCC 27768T contain a complete acetyl-CoA to butyrate synthesis pathway, but possess butyryl-CoA:acetate CoA-transferase activity only in the final step  showed that strains AF52-21T and CM04-06T both contain two potential BGCs, which encode bacteriocin and sactipeptide, respectively, while ATCC 27768T contains BGCs encoding microcin and sactipeptide, respectively  /ribbon-helix-helix protein, helix-turn-helix, and predicted transcriptional regulators. Moreover, the antibiotic resistance analysis indicated that strain AF52-21T contains macrolide antibiotic, lincosamide antibiotic, and streptogramin antibiotic genes, while strains CM04-06T and ATCC 27768T contain aminoglycoside antibiotic genes , which may be used for disease treatment37.Most strains in the genus F. prausnitzii26. A recent study analysing the Faecalibacterium-like MAGs, proposed that Faecalibacterium from the human gut can be divided into 12 clades37. These studies have expanded the diversity of the genus Faecalibacterium and proposed that different phylotypes have different functions with potentially different contributions in relation to health or diseases.Previous studies based on comparative genomics of isolates suggested a wide diversity of this genus, with the presence of at least two phylotypes in Faecalibacterium can be used for in vitro functional verification and animal model experiments to further explore possible probiotic functions, and ultimately, used in clinical disease intervention trials.Moreover, as a candidate taxa for the NGPs, the bacteria of the genus et al25 with the following changes. Cells are able to produce formic acid, acetic acid, and butyric acid. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and several unidentified glycolipids. Genomic DNA G + C content is 47–63 mol%. Genome size is 2.68–3.32 Mb.The genus description is as given by Duncan 16:0, C18:1ω7c, and C18:1ω9c. The rest of the species characteristics are as described by Cato et al38, Duncan et al25, and Fitzgerald et al26. The type strain is Faecalibacterium prausnitzii ATCC 27768T (= NCIMB 13872T).Cells are able to produce formic acid, acetic acid, butyric acid, and lactic acid. The major fatty acids  are CFaecalibacterium butyricigenerans .14:0, C16:0, C18:1ω7c, C18:1ω9c, and iso-C19:0.Cells are gram-negative, non-motile, non-spore-forming and rod-shaped. Strictly anaerobic and catalase negative. Colonies on PYG agar are round, creamy white to yellowish, convex, and opaque with entire margins, and colony size is approximately 1.0–2.0 mm in diameter after incubation at 37 °C for 2 days. Cells are able to grow at 20–42 °C with optimum temperature at 37 °C. The pH range for growth is 6.0–7.5 (optimum at 7.0–7.5). Growth occurs at NaCl concentrations 0–1%. Indole is not produced. Positive for hydrolysis of esculin and negative for gelatin. Formic acid, acetic acid, butyric acid, and lactic acid are the fermentation products. The major fatty acids are CT (= CGMCC 1.5206T = DSM 103434T), was isolated from human faeces. The G + C content of the genomic DNA is 57.77 mol% as calculated from whole genome sequencing.The type strain, AF52-21Faecalibacterium longum .16:0, C18:1ω7c, C18:1ω9c, iso-C19:0, and iso-C17:1 I/anteiso B.Cells are gram-negative, non-motile, non-spore forming, long rod in shape. Strictly anaerobic. Catalase and urease are negative. Colonies are round, yellowish, slightly convex, and opaque with entire margins with 2.0 mm in diameter on PYG agar for incubation at 37 °C for 48 h under anaerobic condition. The strain shows growth at 30–45 °C (optimum temperature is 37 °C). Growth is observed at pH 5.0–8.0 (optimum pH is 7.0–7.5). NaCl is tolerated with concentrations up to 3%. Indole is not produced. Gelatin is hydrolysed, but aesculin is not. Major end products are acetic acid, formic acid, butyric acid, and lactic acid. The major fatty acids  are CT (= CGMCC 1.5208T = DSM 103432T), was isolated from human faeces. The G + C content of the genomic DNA is 57.51 mol% as calculated from whole genome sequencing.The type strain, CM04-062, 5% CO2, and 5% H2, v/v) for 3–5 days. Single colonies were randomly picked and purified by repetitive subculturing on the new plates containing the same medium and incubated under the same conditions as described above. Among the pure cultures, two isolates, designated as AF52-21T and CM04-06T, respectively, were obtained and subsequently maintained in 20% (v/v) glycerol and frozen at -80 °C.Faeces samples were collected from two healthy donors living in Shenzhen, Guangdong province, China, one donor is an adult female (AF), and the other is a male child (CM). The samples were stored refrigerated and kept anaerobically until processed. The collection of the samples was approved by the Institutional Review Board on Bioethics and Biosafety of BGI under number BGI-IRB17005-T2. All protocols were in compliance with the Declaration of Helsinki and explicit informed consent was obtained from the participant and the parents of the male child. 1 g of faecal sample was diluted with 0.1 M PBS  and spread onto modified peptone-yeast extract-glucose  agar plates in an anaerobic box . The plates were incubated at 37 °C under anaerobic conditions  during the exponential phase of growth. Cell motility was examined using semi-solid MPYG medium containing 0.5% agar39. The Gram reaction was carried out using a Gram-staining kit . Spore formation and presence of flagella were determined by staining using spore stain kit and flagella stain kit supplied by Solarbio (China) following the manufacturer’s instructions. Colony morphology was observed following growth of the cultures on PYG agar for 2 days at 37 °C. Optimal temperature for growth was determined using growth in MPYG medium at 4, 10, 20, 25, 30, 35, 37, 45, and 50 °C for 7 days. The pH range for growth was also measured in MPYG medium covering the range of pH 3.0–10.0  at 37 °C for 7 days, and the pH test medium stabilized with the appropriate buffers as described by Sorokin40. Growth at various NaCl concentrations  was performed for determining tolerance to NaCl. Catalase activity was assessed by gas formation after dropping the fresh cells in 3% H2O2 solution. Biochemical properties, including utilization of substrates, acid production from carbohydrates, enzyme activities, hydrolytic activities, were determined using the API 20A, API 50CHL, and API ZYM systems  according to the manufacturer’s instructions with modification by adding sodium acetate at concentration of 0.5% in all tests. The reference type strain was tested under the same condition as used for strains AF52-21T and CM04-06T. In all tests, the strains were incubated under anaerobic conditions.The morphological characteristics of strains AF52-21T, CM04-06T, and ATCC 27768T were harvested from cells growing in MPYG at 37 °C under anaerobic conditions for 2 days. Whole cell fatty acid methyl esters (FAMEs) were extracted, separated and identified according to the MIDI Microbial Identifications System and performed by CGMGG  identification service. The diagnostic isomer of diaminopimelic acid in whole-cell hydrolysates was identified by TLC as described by Zou et al.41. The polar lipids of strain AF52-21T, CM04-06T, and ATCC 27768T were extracted from lyophilized bacterial cells and analysed using two-dimensional TLC as described42. Menaquinone components were extracted and identified by HPLC  coupled with a single quadrupole mass spectrometer  as described42.Chemotaxonomic features were investigated by analysing of cellular fatty acids, cell wall composition, polar lipids, and quinones. Biomasses of strains AF52-21For analysis the metabolic end products from glucose fermentation, including SCFAs and organic acids, cells were cultured in MPYG broth at 37 °C under anaerobic conditions for 2 days. Supernatant harvested from the cultures centrifuged at 10,000 g for 10 min was used for determining SCFAs and organic acids. SCFAs detection was performed using a gas chromatograph  equipped with a flame ionization detector (FID) and capillary column packed with Agilent 19091 N-133HP-INNOWax porapak HP-INNOWax (30 m × 0.25 mm × 0.25 μm). Organic acids were analysed by equipping capillary column packed with Agilent 122-5532G DB-5 ms (40 m × 0.25 mm × 0.25 μm).T and CM04-06T, genomic DNA was extracted following the method described above. The draft genome was sequenced on an Ion Proton Technology (Life Technologies) platform at BGI-Shenzhen  after constructing a paired-end DNA library with insert size of 500 bp. The resulting reads were assembled using the SOAPdenovo 2 package43. CheckM (v1.1.2) was used to estimate genome completeness and contamination44. Genome assemblies were visualized using CGView Server45 . Annotation of the assembled genome was performed using the Rapid Annotation Using Subsystem Technology (RAST) server46 and COG database47. The G + C content in genomic DNA was calculated from the whole genome sequence. The genes in known pathways from acetyl-CoA to butyrate were annotated by BLAST 30. AntiSMASH 5.0 was used to predict BGCs. A search for prophages was performed by PHAST (http://phast.wishartlab.com/)48. Antibiotic resistance was analysed using the CARD database49. The carbohydrate active enzymes genes were annotated by dbCAN250. The dbCAN-PUL51 database was used to determine genes related to important carbohydrate metabolism pathways.For genome sequences of strains AF52-2152, with a value of 95–96% proposed for delineating bacterial species, corresponding to the traditional 70% DNA-DNA reassociation standard54. The ANI values between strains AF52-21T, CM04-06T, and closely related species were determined using the FastANI55.Genome relatedness was investigated by calculating average nucleotide identity (ANI)56. The obtained 16S rRNA gene sequences of strains AF52-21T and CM04-06T were compared with the sequences of type strains retrieved from the EzBioCloud database (https://www.ezbiocloud.net/)57 and an unrecognized species ‘Faecalibacterium hominis’ 4P-1527 using the BLAST program to determine the nearest phylogenetic neighbours and 16S rRNA gene sequence similarity values. Phylogenetic trees were reconstructed by using the neighbour-joining method58 (K2 + G model of substitution), maximum-likelihood method59 (GTR + G + I model of substitution) and minimum-evolution method60 (K2 + G model of substitution) with the MEGA X program package61, after Clustal W multiple alignment of the sequences. 1548 nucleotide positions were finally used for tree constructions. Robustness of the phylogenetic trees was evaluated by using the bootstrap resampling method (1000 resamplings) of Felsenstein62.16S rRNA gene sequences were extracted from the genomes using RNAmmerSupplementary Information.


Poly(ε-L-lysine) and poly(L-diaminopropionic acid) are valuable homopoly (amino acids) with antimicrobial properties and mainly produced in submerged fermentation. In this study, we investigated their co-production using waste biomass and spent mushroom substrate in solid-state fermentation. Simultaneous production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) was achieved in a single fermentation process using pearl oyster mushroom residues as substrate, with the supplement of glycerol and corn steep liquor. After optimization of the fermentation parameters, the maximum yield of poly(ε-L-lysine) and poly(L-diaminopropionic acid) reached 51.4 mg/g substrate and 25.4 mg/g substrate, respectively. The optimal fermentation conditions were 70% initial moisture content, pH of 6.5, 30°C and an inoculum size of 14%. Furthermore, the fermentation time was reduced from 8 days to 6 days using repeated-batch solid-state fermentation. Finally, the antimicrobial effects of poly(L-diaminopropionic acid) and poly(ε-L-lysine) were evaluated in freshly pressed grape juice, which indicated tremendous potential of this mixture in its use as biological preservative.  Despite their similarity, poly(L-diaminopropionic acid) has an antimicrobial spectrum that complements poly(ε-L-lysine), as it exhibits a stronger inhibitory effect on eukaryotic cells. Therefore, the combined use of poly(L-diaminopropionic acid) and poly(ε-L-lysine) could have tremendous potential in various fields. Although strategies involved in breeding of high-yield strains, pH control and dissolved oxygen regulation had been developed in submerged fermentation [Homopoly (amino acids) are unique biopolymers consisting of a single type of amino acids. In nature, only four homopoly (amino acids) are found poly(γ-glutamic acid) (γ-PGA), poly(L-diaminopropionic acid) (PDAP), poly(γ-L-diaminobutanoic acid) (γ-PAB) and poly(ε-L-lysine) (ε-PL) . Homopol albulus . Poly is regarded as a promising technology for the production of microbial secondary metabolites . It prodon chain . And bioon chain .China produces more than 75% of worldwide edible fungus, yearly generating at least two million tons of spent mushroom substrate (SMS), a by-product of the mushroom industry that mainly consists of lignin, cellulose, hemicellulose and carbohydrates. Since spent mushroom substrate is not suitable for animal feed, treatment and disposal of this waste biomass is a major environmental problem. Therefore, the potential economic impact of recycling spent mushroom substrate is significant , and onePleurotus ostreatus), we hypothesize that this spent mushroom substrate could be used for co-production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) in solid state fermentation of S. albulus. The aim of this work was to recycle waste biomass spent mushroom substrates into high value-added homopoly (amino acids). In order to increase the yield of the two products, carbon and nitrogen sources were supplemented, and fermentation conditions were optimized. A repeated-batch process was also investigated to shorten the fermentation cycle. Finally, the extracted products were applied to freshly pressed grape juice as biological preservatives to evaluate their effects on food spoilage.After evaluating the composition of residues from pearl oyster mushroom . The bevel medium contained 8 g/L glucose, 2 g/L polypeptone, 1 g/L yeast extract, 1 g/L beef meat extract, and 18 g/L agar. The medium used for the inoculum was composed of 50 g/L glucose, 10 g/L (NH4)2SO4, 5 g/L yeast extract, 0.5 g/L MgSO4 7H2O, 0.04 g/L ZnSO4 7H2O, 0.03 g/L FeSO4 7H2O, 1.36 g/L KH2PO4 and 0.8 g/L K2HPO4. The pH of the medium was adjusted to 6.8 using NH4OH solution . After complete sporulation on agar bevel medium, 100 mL medium in a 500 mL flask was inoculated with spore suspensions of S. albulus and cultured at 30°C with agitation at 200 rpm. This culture was used to inoculate the substrates in solid-state fermentation.2.2The mushroom substrate residue was obtained from a local farm. The lignin content of the spent mushroom substrate was determined from the total solids and acid-insoluble lignin in biomass . The cel3, filtered, and made up to 20 mL using distilled water. The phosphorous content was determined with a molybdate blue colorimetric method [Dried spent mushroom substrates were heated in a Muffle furnace at 550°C, resulting in weight loss due to elimination of organic matter. To determine the mineral content, the ash from the Muffle furnace was dissolved in 50% HCl and 50% HNOc method . Elementc method , and all−1. Firstly, poly(L-diaminopropionic acid) was eluted with equilibrating solution for 10 minutes and poly(ε-L-lysine) remained in the column. When the ratio of 0.05% trifluoroacetic acid to acetonitrile ranged from 85% to 95% in a linear gradient, ε-PL was eluted over 5 minutes. The yield of the two products was expressed as milligrams per gram of dry substrate (mg/g substrate). Residual glycerol in the supernatant was determined using HPLC with a differential refractometer  at 35°C, with flow rate of 0.5 mL/min and where the mobile phase was acetonitrile–water . Concentrations of glycerol was calculated based on calibration curves built using standard chemicals [For the analysis of fermentation products, a mixture of 2 g spent mushroom substrate and 20 mL distilled water was incubated for 1 h with stirring. The filtrate after cotton gauze was centrifuged at 9,000 g for 15 min at 4°C. Poly(L-diaminopropionic acid) and poly(ε-L-lysine) concentrations were determined from the supernatant by HPLC  using a hemicals .2.3S. albulus liquid culture suspension and thoroughly mixed under aseptic conditions. For direct fermentation of spent mushroom substrate, fermentation was conducted at 30°C for 12 days. The yield of poly(L-diaminopropionic acid) and poly(ε-L-lysine) was determined by daily sampling. To screen suitable supplemental carbon sources in solid-state fermentation, wheat grain, rice bran, cane molasses, glycerol and corn flour were tested, and 4% glucose was used as a control. As nitrogen sources, we used 3% (w/w) of yeast extract, soybean meal, corn steep liquor and malt extract, whereas (NH4)2SO4 was used as control. All these supplemented raw materials were milled and sieved before addition. The fermentation temperature and time were 30°C and 8 days, respectively.For the solid-state fermentation, 40 g of spent mushroom substrate was used in 500 mL flasks. After autoclaving at 121°C for 20 min, the substrates were inoculated with 8% of After optimizing the supplementary sources for solid-state fermentation, fermentation conditions were stepwisely optimized, and the optimization process was illustrated in Figure S1: initial pH, moisture content, fermentation temperature and initial inoculum size. The dried spent mushroom substrate was rehydrated to 55%, 60%, 65%, 70%, 75% or 80% moisture content. The pH was varied from 5.5 to 8.0. The incubation temperature was varied from 26°C to 36°C, and inoculum sizes were 5%, 8%, 11%, 14%, 17% or 20%.2.4Amplified fermentation was conducted using 400 g of spent mushroom substrate in 5 L flasks using the optimized parameters: initial pH 6.5, 70% humidity, 14% inoculum size and 30°C fermentation temperature for 12 days. In repeated-batch fermentation, 10% substrate was used as an inoculum for subsequent batches and 8 cycles were conducted. For the determination of product concentration during the fermentation cycle, substrates were mixed, and 2 g samples were collected at 24 h intervals.2.5A mixture of fermented spent mushroom substrate (50 g) and 500 mL distilled water was incubated for 1 h by stirring. The supernatant was filtered through cotton gauze and used for extraction of products. The pH of culture filtrate was first adjusted to 2.5 with HCl solution. For the extraction of poly(ε-L-lysine), the culture filtrate was saturated with methanol/acetone (3:1) from 40% to 67% and then purified according to methods previously described . To obtaGood-quality grapes were purchased from the local market. After washing, cleaning and stemming, juice was extracted with an electric juicer and filtered with a muslin cloth. Freshly pressed grape juice was divided into different groups treated with 0.2 g/kg ε-PL, 0.16 g/kg ε-PL plus 0.04 g/kg PDAP, or 0.12 g/kg ε-PL plus 0.08 g/kg PDAP, respectively. Samples without treatment were used as a control. All the samples were stored at 4°C before organoleptic evaluation ,30. The 3.Pleurotus ostreatus) was analyzed, and we hypothesize that it was suitable for the growth of S. albulus and co-production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) in solid state fermentation. To achieve this supplemental carbon and nitrogen sources were screened, fermentation conditions were optimized, and a repeated-batch process was investigated. Finally, the potential use of extracted products as food preservatives was evaluated in grape juice.In this study, to recycle the waste biomass spent mushroom substrates into high value-added products, the composition of residues from pearl oyster mushroom  and lignin (19.7%) in total weight. These structural macromolecules may provide an inert matrix within which carbon and nitrogen sources can be adsorbed and support mass and oxygen transfer [P. ostreatus could serve as a suitable solid substrate for the growth of S. albulus. The spent mushroom substrate contained 68.6% (w/w) moisture and the dried substrate contained 1.5% total nitrogen, 1.2% total soluble sugar and mineral elements (Table S1). Previous studies have shown that S. albulus required 50 g/L carbon and 10 g/L nitrogen for growth and metabolism, indicating that carbon and nitrogen content in spent mushroom substrates may not be sufficient to support the growth of S. albulus and the production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) [The composition of transfer , suggestignin 19.% in totaS. albulus. In this study, eight abundant mineral elements were detected in spent mushroom substrate from P. ostreatus (Table S1), the direct use of this substrate could be possible without additional mineral supplements in the fermentation. Thus, the spent mushroom substrate was initially tested without any additives, and the production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) were recorded during 12 days of fermentation. The products poly(ε-L-lysine) and poly(L-diaminopropionic acid) were detected on the second and third day, respectively  and poly(ε-L-lysine). However, the direct use of spent mushroom substrate in solid-state fermentation is not advantageous in obtaining large quantities of products even though cellulase or other enzymes may be produced to increase the utilization of the substrate. Supplemental carbon and nitrogen sources are necessary since intracellular ATP levels are important for the biosynthesis of secondary metabolites [Mineral elements play an important role in cell growth and metabolism  such as ectively . When thabolites .Figure 3.2 Screening of carbon sources and nitrogen sources for co-production of PDAP and ε-PL in solid-state fermentation using spent mushroom substrateResults of direct solid-state fermentation of spent mushroom substrate showed that the two homopoly (amino acids) could be co-produced in a single fermentation, but the yield of poly(L-diaminopropionic acid) was significantly lower compared with poly(ε-L-lysine). Thus, several low-cost carbon and nitrogen sources were screened as supplements. We tested glucose, wheat grain, rice bran, cane molasses, glycerol and corn flour as potential carbon sources ). The re42- was indispensable for the production of poly(ε-L-lysine) in Streptomyces, where (NH4)2SO4 was used as a primary nitrogen source [4)2SO4  as positive control. Corn steep liquor produced comparable amounts of poly(L-diaminopropionic acid) and poly(ε-L-lysine)  ), wherea3.33.3.1Streptomyces sp. was only achieved when the initial moisture was 60% [In addition to nutritional factors, fermentation conditions also have a significant impact on the production of secondary metabolites, and therefore the effects of moisture content, initial pH, fermentation temperature and inoculum size were investigated. Moisture content of materials plays an important role in solid-state fermentation, with previous studies reporting that a high yield of the antibiotic meroparamycin in  was 60%  and maxi was 60% . The cri was 60% . In the 3.3.2S. albulus grows optimally at pH 6.0. Thus, a two-stage pH control strategy seems preferable for fermentation. When the spent mushroom substrate was used for solid-state fermentation, the initial pH varied from 5.5 to 8.0 and the fermentation was not subject to pH control. Interestingly, the pH in the stationary phase was constant around 4.0, and the products were efficiently accumulated. Maximum yields of poly(ε-L-lysine) (44.8 mg/g substrate) and poly(L-diaminopropionic acid) (22.4 mg/g substrate) were achieved at pH 6.5 (Production of these homopoly (amino acids) was affected by pH. An acidic pH of 4.0 is required, possibly because of the regulation of enzyme catalysis in ε-PL or PDAP synthetase , but S. t pH 6.5 ). This w3.3.3After optimizing the pH in the fermentation, the effect of temperature was investigated. Extremes of temperature adversely affect microbial metabolic activity . We foun3.3.4The inoculum level was also important in fermentation, therefore various inoculum levels were analyzed to evaluate their effects. The highest yields of poly(ε-L-lysine) (49.7 mg/g substrate) and poly(L-diaminopropionic acid) (24.8 mg/g substrate) were obtained when the inoculum level was 14% (v/w) ). At lowOverall, the optimal solid-state fermentation conditions for the co-production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) using spent mushroom substrate were 70% initial moisture content, pH 6.5, 30°C and an initial inoculum size of 14%.3.4After optimization of the fermentation conditions using spent mushroom substrate, amplified fermentation in 5 L flasks was investigated. The accumulation of poly(L-diaminopropionic acid) and poly(ε-L-lysine) was proportional to the consumption of glycerol ). The maE. coli were significantly increased by repeated batch fermentation compared with fed-batch fermentation [S. albulus growth took 2–3 days, and repeated-batch experiments were carried out to shorten the fermentation time and increase the specific time for biosynthesis of products. Poly(L-diaminopropionic acid) and poly(ε-L-lysine) were produced in eight fermentation cycles, and the fermentation time was reduced from 8 days to 6 days . During3.5The antimicrobial effects of the extracted poly(L-diaminopropionic acid) and poly(ε-L-lysine) were evaluated in food preservation experiments. Freshly pressed grape juice was used since its low pH and high sugar content favors the growth of yeast. In the control experiment, where no preservative was added, both flavor and color scores decreased dramatically after 3 weeks of storage. In contrast, grape juice treated with 0.12 g/kg ε-PL and 0.08 g/kg of PDAP increased its preservation time to 6 weeks and the addition of homopoly (amino acids) at this amount had no obvious effect on the taste, whereas treatment with ε-PL only extended the shelf life one week more than the control ). Polyε). Previo4.In this study, the co-production of poly(L-diaminopropionic acid) and poly(ε-L-lysine) was achieved using spent mushroom substrate in solid-state fermentation. Through supplementation of glycerol and corn steep liquor and optimization of fermentation parameters, the yield of poly(ε-L-lysine) and poly(L-diaminopropionic acid) reached 51.4 mg/g substrate and 25.4 mg/g substrate, respectively. Repeated-batches of solid-state fermentation indicate that these products could be constantly co-produced with high efficiency. In addition, the combined application of poly(L-diaminopropionic acid) and poly(ε-L-lysine) exhibited a good potential as biological preservative. Thus, this fermentation strategy not only improves production but also enables the recycling of waste biomass and product diversification.Click here for additional data file.