Salinivibrio costicola strain was isolated from Suaeda maritima (L.) Dumort. collected in Sinan, Republic of Korea. The endophytic characteristics of the Gram-negative bacterium S. costicola were verified with metagenomics sequencing of S. maritima. S. costicola was cultivated for 3 days in a liquid medium with 3.3% sea salt and analyzed the metabolites produced by the strain cultured in five different bacterial cultivation media. From the bacterial cultures, polyhydroxybutyrate derivatives were detected using high-resolution mass spectrometry, and three major compounds were isolated by high-performance liquid chromatography. The chemical structures of the compounds were elucidated using nuclear magnetic resonance and MS analyses. The relationship between the compounds was confirmed with Global Natural Product Social Molecular Networking, which showed clustering of the compounds. From the S. maritima extract, polyhydroxybutyrate derivatives produced by S. costicola were detected as being accumulated in the host plant.In this study, the  The close relationship between plants and their endophytes indicates that individual host plants and microbial communities are essential for thriving and survival . DespiteS. maritima (Amaranthaceae), has habitat-specific microbial communities to overcome osmotic pressure stress, which may have evolved adaptive traits in response to high-salt environments  Dumort. which is a halophyte, was collected from the Taepyeong saltern (34° 59′ 53.0″ N 126° 10′ 19.4″ E) in Sinan, South Korea in October 2021.The plant material® Spin Kit for soil , and the quantity of DNA was measured with an Epoch™ Spectrometer (BioTek). Hypervariable regions V3-V4 of the bacterial 16S rRNA gene were amplified using the primer pair 341F  and 805R  containing the appropriate Illumina adapters following Illumina’s 16S Metagenomics Protocol for the first amplicon PCR. The second PCR (index PCR) was performed with forward index i5  and reverse index i7 . PCR reagents  were 10X buffer (2.5 μL), dNTP (2.5 μL), forward primer , reverse primer , Taq polymerase , DNA (2 μL), and D.W. (15.75 μL). The quantity and quality of a single library were determined using the Quanti-iT PicoGreen dsDNA Assay Kit (Invitrogen) and Agilent 2,100 Bioanalyzer System . The samples were sequenced paired-end using a MiSeq® Reagent Kit v2 .DNA preparation was performed using the FastDNATo obtain Q-values by sequence, sequences that could be misidentified as ‘new species’ by Q-value were removed. Illumina next-generation sequencing (NGS) data were analyzed using Trimmomatic software, and other NGS data were analyzed using self-developed code. The Pandaseq software was used for bioinformatics, but this was not performed for single-end reads or PacBio’s CCS data . CJ BiosAll the sequences in the samples were grouped into multiple OTUs using various algorithms and programs. 1) Except for chimeras, sequences with ≥97% similarity were classified as species and each was assigned an OTU. 2) For sequences with <97% similarity, CD-hit and UCLUST programs were used to perform cluster analysis, and each cluster was assigned an OTU. 3) The OTUs from steps 1 and 2 were combined to obtain the final OTU information. 4) The OTU formed in step 2 as a single sequence was removed from the diversity analysis because it caused the diversity index to increase more than the actual value. It calculates various alpha diversity indices within a single sample using OTU information (the number of OTUs and the number of sequences in an OTU). Shannon, Simpson, and other species abundance and evenness statistics were included in alpha diversity.S. maritima were disinfected by soaking in 5 percent sodium hypochlorite for 5 minutes and wiping with 80 percent aqueous ethanol. The sterile leaves, stems, and roots of S. maritima were sliced into flakes and added to a chitin medium with sea salt , Czapek-Dox medium with sea salt , and A1 medium with sea salt  for 14 days to isolate endophytes from S. maritima. Each isolated endophyte was cultured in a modified K medium with sea salt .To remove exterior contaminants, whole parts of For identification of the isolated bacteria, DNA was extracted using the MagAttract HMW DNA Kit (Qiagen) according to the manufacturer’s instructions. The DNA extract was amplified with the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C, and extension at 68°C for 1.5 min, and a final extension at 68°C for 10 min. The purified PCR products were sequenced in the forward and reverse directions in separate reactions and duplicates. The PCR amplification primer sequences were as follows: 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) for the 16S rRNA. The purified PCR products were sequenced using two primer pairs: 785F (5′-GGA TTA GAT ACC CTG GTA-3′) and 907R (5′-CCG TCA ATT CMT TTR AGT TT-3′) for 16S rRNA and NS1 (5′-GTA GTC ATA TGC TTG TCT C-3′). Sequencing was performed using a Big Dye Terminator 3.1 cycle Sequencing Kit .S. costicola YSL5, were cultivated in a modified K liquid medium with sea salt for 3 days . Then, 10 ml of the bacterial culture was inoculated into diverse bacterial culture media, including K medium with salt , potato dextrose broth (PDB) medium with salt , potato dextrose yeast (PDY) media with salt , yeast extract-malt extract (YEME) medium with salt , and yeast peptone mannitol (YPM) medium with salt  in 250 ml Erlenmeyer flask . Each culture was extracted with ethyl acetate/water layer separation using a separatory funnel, and the ethyl acetate layer was concentrated in vacuo. The dried crude extract was dissolved in methanol at a concentration of 250 μg/ml and analyzed using liquid chromatography-mass spectrometry (LC-MS). High-performance liquid chromatography (HPLC) measurements were performed with an Agilent 1,290 ultra-high-performance liquid-chromatography (UHPLC) system  equipped with a 1,290 Infinity binary pump and a YMC-triart C18 column . The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with the following gradients: 10–100% B (0–20 min), 100% B (20.1–25 min), 100–10% B (25.1–30 min), and 10% B (30.1–35 min) with an injection in six replicates at a volume of 10 μL. Mass spectrometry was performed using an Agilent 6,530 quadrupole time-of-flight mass spectrometer  equipped with an electrospray ionization (ESI) source. The MS experiment was performed under the following conditions: source temperature, 100 °C; desolvation temperature, 300°C; desolvation gas flow rate, 500 L/h; cone gas flow rate, 30 L/h; capillary voltage +3.0 kV; positive and negative modes; cone voltage, 60 V; extractor voltage, 3 V.After isolating the single bacterial strains from a chitin medium with sea salt, the isolated Gram-negative bacteria see , S. costS. costicola YSL5 and the methanol extract of S. maritima. Based on the tandem mass spectrometry (MS/MS) data from an Agilent 6530 Q-TOF-MS, molecular networks of the secondary metabolites were created using the GNPS platform (http://gnps.ucsd.edu) -based Global Natural Product Social Molecular Networking (GNPS) analysis of the crude ethyl acetate extracts obtained from 3 days cultures of csd.edu) . The datsoftware .mzML files of six replicates of each medium, mass feature detection was performed using MZmine 2.53   .After the matrix data were normalized, the multivariate analysis comprised principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and the building of a heatmap showing the correlation between variables and samples. Finally, a hierarchical clustering dendrogram was obtained using a Euclidian distance measure and Ward clustering algorithm.S. costicola YSL5 preculture was transferred to 100 ml of modified K medium in a 250 ml Erlenmeyer flask. With continuous shaking at 28°C and 180 rpm for 3 days, 10 ml of the liquid culture was inoculated in a 2 L Erlenmeyer flask containing 1.2 L of the medium . The culture was extracted using ethyl acetate/water layer separation, and the ethyl acetate layer was subsequently concentrated in vacuo. The dried plant extracts were purified using a semi-preparative HPLC equipped with a 1,260 Infinity binary pump and YMC-triart C18 column  with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The conditions were 10% B (0–10 min), 10–50% B (10–30 min), 50–100% B (30–35 min), and UV detection at 210 nm. Nuclear magnetic resonance (NMR) data of the separated single compounds were recorded on a JEOL 600 MHz NMR spectrometer equipped with a 600 MHz ECZR narrow bore spectrometer. 1H NMR chemical shifts at δH 2.50 (DMSO-d6) and 13C NMR chemical shifts at δC 39.51 (DMSO-d6) were set as reference peaks, and the chemical structures were assigned by interpreting combinational data from two-dimensional (2D) NMR such as correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) experiments.A 10 ml liquid culture of S. maritima were dried at 45°C for 1 day with a dehydrator . Ground dried samples were extracted with methanol at 35°C for 120 min using an ultrasound-assisted extraction method. Methanol was used as the extraction solvent to obtain a diverse variety of compounds from plants. The extract was concentrated in vacuo and dissolved in methanol to a final concentration of 250 μg/ml. Metabolite analysis was conducted with MS/Q-TOF-MS with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The conditions were 10–100% B (0–20 min), 100% B (20.1–25 min), 100–10% B (25.1–30 min), and 10% B (30.1–35 min), with an injection volume of 10 μL and a flow rate of 0.3 ml/min. Mass spectrometry was performed using electrospray ionization (ESI) source in the positive mode. Extracted-ion chromatogram (m/z [M + H]+ and m/z [M + Na]+) and fragmentation patterns were analyzed to compare and detect compounds in both the methanol extract of S. maritima and liquid culture of S. costicola YSL5. The ethyl acetate extract was dissolved in methanol at a concentration of 1,000 μg/ml and the compound was isolated using a semi-preparative LC-MS system. HPLC measurements were performed using an Agilent 1,100 Series  equipped with a YMC-Triart C18 column . The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with the following gradients: 50–100% B (0–10 min), 100% B (10.1–15 min), 100–10% B (15.1–20 min), and 10% B (20.1–25 min). Mass spectrometry was performed using a single quadrupole mass spectrometer  equipped with an ESCi multimode ionization source. The MS experiment was performed under the conditions same as the analyses of S. costicola YSL5.Whole parts of S. maritima was 99.6%, indicating that the sample reached sufficient sampling depth . The values of alpha diversity, the species diversity existing in a particular location and a particular sample, for S. maritima were 522.6 ± 29.9 for ACE, 49.0 ± 27.0 for Chao1, and 552.0 ± 0 for Jackknife. The Shannon value, an index that reflects the number of species and the effect of evenness, for S. maritima was 2.544 ± 0.028. The phylogenetic diversity of S. maritima was calculated to be 879.0 by summing the shortest distance between the system diagram’s nodes. The detected OTUs were assigned to 15 phyla , 38 classes, 67 orders, 127 families, 248 genera, and 383 species. Of the identified 15 phyla, the most prominent phyla were Proteobacteria (79.4%), followed by Actinobacteria (15.6%), Bacteroidetes (2.0%), and Firmicutes (1.7%). All ten major phyla have been described as the dominant taxa in soil studies. In the Proteobacteria phylum, the Gammaproteobacteria group (68.2%) was the dominant bacterial group, followed by Alphaproteobacteria and Betaproteobacteria with a total percentage of reads of 6.4 and 4.6% at the class level  of V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using the genomic DNA extracted from the plant material. A total of 64772 amplicons were sequenced using the Illumina MiSeq platform, and 24004 quality-filtered reads were obtained . The number of reads that were identified at the species level based on 97% similarity was 59942 (92.5%), and 364 bacterial species were identified. Good’s coverage, calculated as the ratio of OTUs excluding singleton in the entire read, reflects the database coverage rate of the sample, and its value for ss level .S. maritima on the Chitin and Czapek-Dox media, four endophytes were isolated including Streptomyces xinghaiensis , Nocardiopsis aegyptia , and two Gram-negative bacteria, Salinivibrio costicola , and Alcaligenes aquatilis .From the fleshy leaves and roots of S. costicola YSL5, an endophyte of S. maritima, was cultivated for 3 days in modified K, PDB, PDY, YEME, and YPM liquid media with sea salt . HRMS2-based analysis of the YSL5 extract clustered structurally related molecules with GNPS, and the results were visualized using Cytoscape 3.9.0. (m/z 543.2 [M + Na]+ (1), m/z 629.2 [M + Na]+ (2), m/z 715.3 [M + Na]+ (3), m/z 801.3 [M + Na]+ (4), m/z 887.3 [M + Na]+ (5), m/z 973.4 [M + Na]+ (6), m/z 1,059.4 [M + Na]+ (7), m/z 1,145.5 [M + Na]+ (8), m/z 1,231.5 [M + Na]+ (10), m/z 1,317.5 [M + Na]+ (11), m/z 1,403.6 [M + Na]+ (12) made a large cluster.sea salt , and thesea salt . The bacse of 86 , displaye 3.9.0. . The clue 3.9.0. . Eleven m/z increasing by 86 among the media. A heatmap was generated based on the top 25 differentially expressed metabolites with the highest statistical difference and also exhibited distinct patterns of metabolites produced by S. costicola YSL5 in the five bacterial culture media  was 23.7% (PC1) and 18.9% (PC2), with a total variance of 42.6% . The disre media   was obtained at tR 33 min using semi-preparative HPLC. Compound 1 was isolated as a yellow oil; UV (MeOH) λmax (log ε) 218 and 290 nm. The molecular formula of compound 1 was determined to have a molecular formula of C24H40O12Na, based on high-resolution electrospray ionization (HR-ESI) mass spectrometry, which indicated five degrees of unsaturation with a molecular ion at m/z 543.2481 [M + Na]+. The structure was confirmed by 1H, 13C NMR, and 2D data : 1H NMR  δH 5.17-5.05 , 4.75 , 4.67 , 3.95 , 3.60 , 2.64-2.51 , 2.32 , 2.26 , 1.20 , 1.19 , 1.19-1.18 , 1.17 2-hydroxy-3.7,11,15-tetramethyl-5.9,13,17-tetraoxo-4.8,12,16-tetraoxaicosan-19-yl 3-hydroxybutanoate  five carbonyl carbons , four methoxy and two alcohol carbons , five methylene carbons , seven methyl carbons ; HR-ESI-MS m/z 543.2481 [M + Na]+ .1), is known to be an energy source for bacteria and plays important role in plant growth   were detected in both S. costicola YSL5 culture broth extract and S. maritima methanol extract using GNPS analysis (m/z 387.1 [M + H]+) demonstrated that a compound (12) was detected at the same retention time and mass pattern as YSL5 , with YSL5 being the only strain matching the metagenomic results of S. maritima at the family level. While moderate salinity of 15% is generally regarded to be a transitional salinity for marine microbes and moderate halophiles, the bacterial strain YSL5 grew in a non-saline bacterial culture medium. We cultivated YSL5 in diverse bacterial cultivation media to investigate secondary metabolites by upregulating the growth of the bacterium, and identified major secondary metabolites, PHB, using spectroscopic analysis. The crude extracts of YSL5 were compared with methanol extracts of S. maritima through GNPS analysis to detect accumulated secondary metabolites in the host produced by the endophytic bacterium YSL5. Using LC-MS and GNPS chemical profiling, two secondary metabolites were detected at identical retention times, showing the identical MS/MS patterns . After cultivating YSL5 on a large scale, the metabolite was identified as PHB with molecular weight (m/z 387.1787 [M + H]+). PHB, a high-molecular-weight polyester, is found in plants as a plant growth-promoting agent and accumulates as storage carbon in many bacterial species. In a hyperosmotic environment, PHB in the cytoplasm promotes the survival of bacterial cells exposed to osmotic stress. Although the mechanism of its protective action is unknown, the primary strategy of most bacteria under hyperosmotic stress is the accumulation of a diverse range of compatible solutes. The ability to adapt to changes in external osmolarity is critical for survival. Endophytic bacteria have developed various strategies to deal with osmotic pressure and changes in extracellular osmolarity in halophytes. Owing to the high osmotic potential of the soil solution and nutrient absorption limitations resulting from an oversupply of sodium and chloride ions, the high salinity of the water directly and effectively inhibits plant development (Azospirillum brasilense to osmotic stress and root colonization (S. costicola YSL5 produces PHB derivatives in the host halophyte S. maritima. These PHB derivatives potentially interact with one another to counter the osmotic stress, suggesting that PHB plays a role in cell protection under saline conditions.In this study, we used metagenomics analysis to track the bacterial endophytes of tilizers . Goel ettilizers . Similartilizers . Investitilizers . To achitilizers . Our invelopment . Nutrienelopment . Halophyelopment . In addielopment . Accordinization . Arora enization . Based oS. maritima using metagenomics and functionally associated traits of the endophytic bacterium YSL5 and its secondary metabolites. The results from this study may form the basis for the future development of new natural product discovery and their function using metagenomic techniques. Furthermore, this study demonstrates that halophytes in high-salinity environments, such as salterns, are an enormous and untapped resource of new microorganisms that could have practical benefits in environments with a high level of salinity.In conclusion, we explored the bacterial communities associated with the halophyte plant


The aim of this study was to investigate the effect of scanning origin location on the data accuracy of removable partial denture (RPD) abutment teeth region in digital impressions acquired by an intraoral scanner. A mandibular partially edentulous model including the following target abutment teeth was used: the left second molar (#37); left first premolar (#34); and right second premolar (#45). The following scanning strategies were tested: the strategy starting from #37 to mesial direction (37M); strategies starting from #34 to mesial (34M) and distal directions (34D), and strategies starting from #45 to mesial (45M) and distal directions (45D). The evaluated measures were trueness, precision, and linear accuracy. One-way and two-way ANOVA were performed for the comparison of trueness and linear accuracy, while Kruskal–Wallis test was performed for the precision comparison (α = 0.05). 45M and 45D showed significantly superior trueness of #34 to 37M and 34D. 45M also showed significantly superior trueness of #45 to 34. 45D showed significantly inferior linear accuracy of #34 and superior linear accuracy of #45 compared to other strategies. It was concluded that scanning origin location would have an impact on data accuracy of RPD abutment teeth region in digital impressions acquired by intraoral scanner. Recently, digital impression-taking by an intraoral scanner (IOS) has been clinically applied for the fabrication of removable partial dentures (RPDs) more widely than ever ,2,3. MeaThe data accuracy of remaining teeth regions in digital impressions can critically affect the fitting of RPD frameworks. The fitting of frameworks should be accurate to keep RPDs passive in the oral cavities to protect both the remaining teeth and denture-bearing mucosa . PreviouIn digital impression-taking by IOS, the scanning strategy specified by manufacturers is initiated at the most posterior region, followed by the scanning of occlusal surfaces. Subsequently, the lingual/palatal surfaces are scanned and finalized with the scanning of the buccal/labial surfaces . On the In this study, several digital impressions for a mandibular partially edentulous dental arch model were taken by an IOS with five different scanning strategies including several locations of the scanning origin. As measures of data accuracy, the trueness, precision, and linear accuracy were evaluated. This study aimed to investigate the effect of scanning origin location on the data accuracy of remaining teeth regions in digital impressions acquired by an IOS. The tested null hypothesis was “the scanning origin location would not affect the data accuracy of remaining teeth regions in digital impressions acquired by an IOS”.A mandibular Kennedy class II, modification 1 partially edentulous dental arch model  was used as the reference model in this study. In the reference model, the following four teeth were missing: the left second premolar (#35); left first molar (#36); right first molar (#46), and right second molar (#47). The target teeth to be evaluated were the following three teeth, supposing the RPD abutment teeth: the left first premolar (#34), left second molar (#37), and right second premolar (#45). The artificial teeth of reference model, including the target teeth, were made of melamine resin, while its residual ridge was made of epoxy resin. To calculate the linear accuracy, a stainless-steel spherule with a diameter of 3 mm was placed on the buccal cusp of each target tooth  and lingual frenum.n = 1). In accordance with the previous study, this scanner shows trueness of 3 µm for objects with a jaw size [n = 10). The trimmed STL files were used as the reference data.For evaluating the data accuracy of digital impression using an IOS, the reference data were acquired using a high-accuracy industrial scanner  (n = 10/scanning strategy). The sample size was determined according to the previous study [The digital impressions of the reference model were acquired by full-arch scanning using an IOS  to be evaluated. The five different scanning strategies performed in this study were defined based on the location of scanning origin and scanning direction as follows: scanning strategies (1) initiated at #37 and headed to the mesial direction (37M) (specified by the manufacturer ); (2) inus study .n is the total number of STL structural points of evaluated data; e,id is the value of measurement of ith structural point on evaluated data, and r,id is the value of measurement of ith structural point on reference data. The results of RMS calculations are shown as a color map image with the allocation of plus or minus signs based on the position of structural point on evaluated data relative to reference data, indicating that a positive RMS represents an externally positioned structural point on the evaluated data, while a negative RMS represents an internally positioned structural point on the evaluated data  value (µm) of deviation between two data was calculated using the following equation:ted data A.G), IOS data for full-arch model was superimposed on the reference data using best-fit algorithm of 3D analysis software  [G for each target abutment tooth (L), both the reference and IOS data were trimmed at each abutment tooth region beforehand, followed by data superimposition using best-fit algorithm. Then, the RMS value was calculated, representing TL for each target abutment tooth  . Then, tnt tooth B. Meanwhnt tooth C. In then = 10) by selecting all pairs from the 10 data was performed using best-fit algorithm. Then, the RMS value among each pair was calculated representing precision (n = 45/scanning strategy)  D. In theLA is the linear accuracy (%); ID is the distance from lingual frenum in IOS data (mm), and RD is the distance from lingual frenum in reference data (mm). A positive value of the linear accuracy indicated the distance between lingual frenum and each abutment tooth in IOS data was greater than that in reference data. Regarding the absolute value of linear accuracy, a higher value indicated inferior accuracy.To evaluate the linear accuracy, the distance between central coordinate of stainless-steel spherule placed on the lingual frenum and that on the buccal cusp of each target tooth was calculated both in reference and IOD data . The chaG and TL), precision, and linear accuracy were statistically compared among five scanning strategies. Shapiro–Wilk test indicated that all measures showed the normality of data, while Levene’s test indicated that all the measures except for precision showed the equality of variance of data. Therefore, a one-way ANOVA with a Tukey multiple comparison post hoc test was performed for the comparisons of trueness (TG and TL) and linear accuracy. Additionally, a two-way ANOVA based on the scanning strategy and target abutment tooth for their comparison. On the other hand, a Kruskal–Wallis test followed by pairwise comparisons with Bonferroni correction was performed for the comparison of precision. All statistical analyses were performed using statistical software  with a significance level set at 0.05.For each abutment tooth, the trueness  (µm) in #37 were 69.1 ± 5.7, 74.5 ± 13.5, 69.4 ± 6.1, 63.7 ± 9.4, and 64.7 ± 10.9 in IOS data acquired with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed no significant difference among the scanning strategies (p = 0.109). The mean values ± SD (µm) in #34 were 61.3 ± 5.8, 57.4 ± 5.7, 61.1 ± 5.1, 52.5 ± 4.9, and 52.4 ± 3.0 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed a significant difference among the scanning strategies (p < 0.001), and 37M and 34D revealed significantly higher TG than 45M  and 45D (p = 0.002 for both 37M and 34D). The mean values ± SD (µm) in #45 were 67.5 ± 4.1, 66.9 ± 2.8, 71.3 ± 4.2, 67.7 ± 2.8, and 68.9 ± 3.9 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed no significant difference among the scanning strategies (p = 0.070).L among five scanning strategies. The mean values ± SD (µm) in #37 were 40.6 ± 3.9, 45.1 ± 8.6, 42.9 ± 4.5, 40.6 ± 6.4, and 44.1 ± 8.6 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed no significant difference among the scanning strategies (p = 0.473). The mean values ± SD (µm) in #34 were 33.7 ± 3.6, 32.4 ± 1.6, 32.5 ± 1.1, 33.1 ± 1.3, and 30.6 ± 1.9 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed a significant difference among the scanning strategies (p = 0.024), and 37M revealed significantly higher TL than 45D (p = 0.015). The mean values ± SD (µm) in #45 were 74.3 ± 8.6, 70.6 ± 12.0, 81.0 ± 8.3, 66.5 ± 6.6, and 69.7 ± 8.7 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed a significant difference among the scanning strategies (p = 0.010), and 34D revealed significantly higher TL than 45M (p = 0.007).p = 0.431). The mean values ± SD (%) in #34 were −0.203 ± 0.035, −0.224 ± 0.056, −0.213 ± 0.029, −0.230 ± 0.035, and −0.305 ± 0.038 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed a significant difference among the scanning strategies (p < 0.001), and 45D revealed a significantly lower change ratio than 37M (p < 0.001), 34M (p < 0.001), 34D (p < 0.001), and 45M (p = 0.001). The mean values ± SD (%) in #45 were 0.475 ± 0.039, 0.493 ± 0.059, 0.482 ± 0.064, 0.490 ± 0.056, and 0.405 ± 0.041 with 37M, 34M, 34D, 45M, and 45D strategies, respectively. One-way ANOVA showed a significant difference among the scanning strategies (p = 0.003), and 45D revealed a significantly lower change ratio than 37M (p = 0.034), 34M (p = 0.005), 34D (p = 0.016), and 45M (p = 0.006).p = 0.542).Additionally, In this study, digital impressions of the mandibular partially edentulous dental arch model were taken by an IOS using five scanning strategies including three different scanning origins , followed by the comparisons of data accuracy  among scanning strategies. The overall results revealed that the location of scanning origin played a significant role in data accuracy, and scanning direction as well. Therefore, the null hypothesis was rejected.G) and local trueness based on individual abutment tooth superimposition (TL) were evaluated. TG was the RMS value calculated with data trimming at each target abutment tooth region after superimposition of the reference and IOS data, representing trueness mainly affected by locational condition of abutment tooth in whole dental arch. Previous studies indicated that tooth located at posterior region would show inferior data accuracy [G compared to #34 and #45 (L) was also evaluated, representing trueness mainly affected by local error in geometry. The results of this study revealed that scanning strategies initiated at #45 (45M and 45D) showed superior trueness both in TG and TL. This finding suggested that the scanning strategy specified by manufacturers would be not always recommended to achieve accurate digital impression data using an IOS. Nevertheless, it was also suggested that the scanning direction from scanning origin would not have an impact on the trueness of digital impression for partially edentulous dental arch. In this study, the highest RMS values in TG and TL were 74.5 µm  . AdditioIn the linear accuracy of #34, 45D showed most inferior accuracy . This fiPrevious study reported that the long distance from scanning origin would deteriorate data accuracy , suggestThis study had several limitations. First, a Kennedy class II, modification 1 mandibular model was the only model evaluated. Additionally, this study was conducted as an in vitro study using a resin model. Therefore, it is unclear whether our findings can be generalized for actual oral cavities with partially edentulous dental arches on the maxillary jaw and/or those with other Kennedy classifications. Second, we evaluated only RPD abutment teeth regions except for precision evaluation. Further studies are necessary to investigate how the scanning origin affects data accuracy of mucosal area and remaining teeth other than RPD abutment teeth. Third, there was the potential for a variation in number of images captured during scanning among evaluated five scanning strategies. A previous study indicated that number of images would affect data accuracy of digital impressions acquired using IOS . AlthougWithin the limitations of this study, it can be concluded that scanning origin has an impact on data accuracy of RPD abutment teeth region in digital impression acquired by an IOS, and scanning direction as well. To acquire superior data accuracy, it would be recommended to locate the scanning origin at end of remaining dental arch but not at remaining tooth isolated from other remaining teeth via mucosal area. Additionally, it was suggested that the scanning strategies directed from scanning origin directly to mucosal area would not be recommended.


S)-1,4-di(furan-2-yl)-2-hydroxybutane-1,4-dione (1) together with four known ones, rhizosolaniol (2), 5-hydroxymethylfurfural (3), 2-furoic acid (4) and (2-furyl) oxoacetamide (5), were isolated from the fungal strain Ceratobasidium sp. (GS2) inducing seed germination of the endangered medicinal plant Gymnadenia conopsea of Orchidaceae. The structure of new furanoid 1 was determined mainly based on HR-ESI-MS and NMR spectral data. Modified Mosher’s reactions were used to establish the stereochemistry of the hydroxyl group in 1, which was not stable in Mosher’s reagents and transformed into four analogs 6–9. These degraded products (6–9) were elucidated based on UPLC-Q-TOF-MS/MS analysis, and compound 8 was further isolated from the degraded mixture and its structure was characterized through NMR experiments. Therefore, the absolute configuration of compound 1 was determined by electronic circular dichroism combined with quantum-chemical calculations adopting time-dependent density functional theory. Compounds (1–5), and 8 showed weak antioxidant activities, and compounds (2–4) displayed phytotoxicity on punctured detached green foxtail leaves. In addition, compounds 3 and 4 strongly showed inhibition activities on the seed germination of G. conopsea. This was the first chemical investigation of the symbiotic fungus of G. conopsea.Five furanoids including a new analog ( R. Br. is a perennial herbaceous orchid plant that grows widely throughout Europe and in temperate and subtropical zones of Asia  were isolated from GS2 and further identified. Interestingly, when compound 1 was added into Mosher’s reagents, four other analogs (6–9) were formed, implying that the structure of 1 was not stable in the solvent of Mosher’s reagents. In this report, the isolation, structural elucidation, and biological evaluations of furanoids 1–5, and structural analysis of degraded products (6–9) were present  and 150 (13C) MHz. HR-ESI-MS were obtained using a UPLC-Q-TOF-MS/MS . Optical rotations were measured on a 241 polarimeter . CD spectra were recorded with a JASCO J-815 spectropolarimeter. UV-2102  was used to measure UV data. IR data were acquired using an FTIR-8400S spectrophotometer . Semi-preparative HPLC separation was performed on a Shimadzu LC-6 AD instrument packed with a YMC-Pack ODS-A column . Sephadex LH-20  and silica gel  were used for column chromatography.All G. conopsea and shown to promote seed germination and early seedling development under in vitro conditions  was previously isolated from the roots of nditions . The strnditions . Two 1 c3OH-H2O for 30 min, v/v, 2 ml/min) to afford 5 . Fr.5 (3.88 g) was separated by a silica gel CC eluted with petroleum ether-acetone  to get 6 subfractions (Fr5.1–Fr5.6). Fr5.6 (82 mg) was then purified by semi-preparative HPLC  to afford 2  and 1 . Fr.6 (1 g) was separated by a silica gel CC eluted with petroleum ether- acetone  to get 4 subfractions (Fr.6.1–Fr.6.4). Fr.6.2 (203 mg) was then purified by semi-preparative HPLC  to afford 3  and 4 .The fermented material was extracted with ethyl acetate for 3 times and soaked for 72 h each time, and the organic solvent was evaporated to dryness under vacuum to afford 100.0 g of extract. The crude extract (100.0 g) was subjected to a silica gel column chromatography (CC) eluting with petroleum ether-acetone  to obtain seven fractions (Fr.1-Fr.7). Fr.4 (647 mg) was separated by a silica gel CC eluted with petroleum ether- acetone  to get 5 subfractions (Fr.4.1–Fr.4.5). Fr.4.5 (49 mg) was then purified by semi-preparative HPLC -1,4-di(furan-2-yl)-2-hydroxybutane-1,4-dione (1): [α]D25 + 20.0 ; UV (MeOH) λmax (log ε) 224 (4.48) nm : λ(Δε) 250 (0.65), 290 (−0.40); IR (neat) νmax 3,444, 3,134, 1,668, 1,569, 1,466, 1,395, 1,288, 1,228, 1,162, 1,083, 1,033, 904, 883, 844, 769, 593 cm−1 (1H-NMR and 13C-NMR data see (4.48) nm ; CD : 1H-NMR ((CD3)2CO, 600 MHz) δH = 8.01 , 7.61 , 6.79 , 5.40 .Rhizosolaniol (3): 1H-NMR  δH = 9.61 , 7.22 , 6.53 , 4.73 .5-hydroxymethylfurfural (4): 1H-NMR  δH = 7.08 , 7.05 , 6.58 .2-furoic acid (5): 1H-NMR  δH = 8.15 , 7.78 , 6.64 , 0.98, 0.81.(2-furyl) oxoacetamide (8): 1H-NMR  δH = 7.79 , 7.73 , 7.45 , 6.65 .2,3-dihydroxy-1,4-furan-2-yl-1,4-butanedione (1 (2.0 mg) was transferred to two small clean bottles, respectively, and then pyridine-d5 (0.5 ml) and (R)-MTPACl/(S)-MTPACl (8.0 μl) were quickly added into the clean bottles. All contents were mixed thoroughly by shaking the clean bottles carefully. The reaction was performed at room temperature, and the solution was allowed to stand for 24 h. The modified Mosher’s reaction products (R and S) were analyzed through HPLC experiments , and four peaks (6–9) were observed, in which the main transformed product (8) was purified by semi-preparative HPLC .The powder of 1 and 6–9 were analyzed using a UPLC-Q-TOF-MS/MS system . Chromatographic analysis was carried out with a Waters Acquity UPLC-PDA system combined with an analytical reverse-phase C-18 column , and the detection wavelength of the PDA detector was 200 to 400 nm. The column temperature was set at 40°C. As the mobile phase, 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were used. The gradient conditions were as follows: 0–2 min, 10% B; 2–10 min, 10–80% B; 10–13 min, 80–100% B; 13–15 min, 100%; and 15–17 min, 10% B. The flow rate from the UPLC system into the UPLC-Q-TOF-MS/MS detector was 0.3 ml/min. The injection volume was 0.3 μl. Time-of-flight MS detection was performed with a Waters SYNAPT G2 HDMS  TOF mass spectrometer equipped with an ESI source in positive ion scan mode. The source temperature was 80°C, and the desolvation temperature was maintained at 450°C with desolvation gas flow at 900.0 l/h. The lock mass in all analyses was leucine-enkephalin ([M + H]+ = 556.2771), used at a concentration of 200 μg/ml and infused at a flow rate of 10 l/min. Raw data was acquired using the centroid mode and the mass range was set from m/z 100 to 1,000. The capillary voltage was set at 2.5 kV with 30 V of sample cone voltage. The collision energy was set as 6 eV for low-energy scan and a ramp from 30 to 50 eV for the high-energy scan. The instrument was controlled by MassLynx 4.1 software.Compounds The ability of scavenging free radicals of six compounds were assessed by reacting with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) . The comThe scavenging rate of DPPH according to the following formula:A0: the absorbance value of the control group; A1: the absorbance value of the experimental group.1–5 on green foxtail were tested by using leaf-puncture assay. The fresh leaves were cut into segments approximately 4 cm long to be placed on a glass slide in a sterile petri dish with one layer of absorbent paper wetted with sterile water. Filter paper disks of 5 mm diameter were prepared using a perforator and sterilized at 121°C for 30 min. A 1 mg amount of test compounds was dissolved in DMSO to produce the 1 mg/ml test solution. In the leaf-puncture assay, a small mild lesion was made from each other on each leaf segment with a nipper and covered with a filter paper disks. Then 20 μl of DMSO or 20 μl of test solution was added to each disk. All experiments were repeated at least three times. The effects were recorded after 3 days of observation  agar. Finally, the concentrations of the compounds in the medium were 0.01, 0.1, 1 mg/ml, respectively. To eliminate the effect of DMSO, plates with 0.5 ml DMSO were used as blank control. Seeds were distributed on each Petri dish as described above. A fresh mycelium (GS2) was also placed in each plate because the seeds of G. conopsea could not grow in the absence of the fungus. Each concentration was conducted in triplicate. The Petri dishes were placed in dark room at 25°C for 45 days. The growth of fungus and the number of seeds germination were recorded.1 was obtained as a pale yellowish oil, and its molecular formula was assigned as C12H10O5 based on HR-ESI-MS data (m/z 257.0426 [M + Na]+) with eight degrees of unsaturation. The IR spectrum showed absorption bands at 3444 and 1,668 cm−1, which implied the presence of hydroxyl and carbonyl groups. Analysis of the 1H-NMR, 13C-NMR . The planar structure of 1 was elucidated by extensive analysis of its 1H-1H COSY and HMBC spectral data  and C-2/2′ (152.0) indicated that there was an oxygen atom between C-5/5′ and C-2/2′ to form two furan rings -2-hydroxybutane-1,4-dione.Compound  13C-NMR  and HSQC 13C-NMR  for 1 reral data . The 1H-an rings ; the wea1 possessed a secondary hydroxyl at C-7, modified Mosher’s reaction was tried to determine its stereochemistry, whereas the reaction was not successful. After compound 1 was mixed with Mosher’s reagents, four main products (4 peaks were observed in the HPLC experiments) were produced, indicating that compound 1 was not stable in the solvent of Mosher’s reagents. To clarify the transformation process, the products were then analyzed based on UPLC-MS/MS experiments.Modified Mosher’s method was used to determine the absolute configuration of secondary hydroxyl group in different small molecules . As 1 po1 had transformed into four analogs 6–9 after mixed with Mosher’s reagents (deuterated pyridine as solvent). Based on high resolution mass and fragmentation mechanisms of the transformed products, the possible structures of 6–9 were tentatively elucidated. The high-resolution mass and fragment ions together with the elemental constituents were listed in 1 (tR = 4.68 min) according to UPLC-Q-TOF-MS/MS analysis was depicted in m/z 249) of 1 was not observed, whereas ions  were present in the UPLC-Q-TOF-MS/MS spectrum. This might be because the parent ion (m/z 249) of 1 was extremely unstable and easily lost H2O molecule to produce daughter ions at m/z 217. Then, this ion (m/z 217) could form the ion (m/z 189) by neutral loss of CO or produced ion m/z 149 by 1,3-rearrangement, which yielded other product ions at m/z 121 and 95 through neutral loss of CO and C2H2.UPLC-MS/MS analysis , 5 revea6  was the product of dehydration of compound 1 based on the UPLC-Q-TOF-MS/MS profile, which retention time was 5.51 min  as compound 1. The [M + H]+ parent ion at m/z 249 and the [M + Na]+ parent ion at m/z 257 of 7 together with high resolution mass suggested its molecular formula as C13H12O5 was 14 Daltons more than that of 1, implying that an additional methyl group was attached at the hydroxyl. The neutral loss of 32 Daltons to form the product ion m/z 217 confirmed this conclusion, and other daughter ions were same as those of 1. Compounds 6 (tR = 5.51 min) and 8 (tR = 6.35 min) was a pair of isomers which had the same molecular formula and similar MS/MS profiles. The cis-double bond in structure of 6 led to the steric hindrance between the two carbonyls, which would lead to blue shift compared with 8 in UV spectra  at m/z 433 ([M + H]+) was detected and its chemical formula was determined to be C24H16O8, indicating that compound 9 might be a dimer. The parent ion at m/z 433 easily lost furan rings and CO to produce daughter ion at m/z 269  combined with quantum-chemical calculations adopting time-dependent density functional theory  (3) (4) (5) (Four known furanoids were determined to be rhizosolaniol (2) , 5-hydro2) (3) , 2-furoi (3) (4)  and (2-f (4) (5)  based on1–5 and 8 were evaluated for the antioxidant activities by using DPPH scavenging assay. Compounds 1–5 and 8 showed scavenging efficiency with IC50 values of 145.5, 381.7, 417.5, 367.2, 480.8, 600.2 μg/ml, respectively, as standard acarbose having IC50 value of 13.07 μg/ml  were lower than the control  and the absolute configuration of 1 was determined by using quantum chemical electronic circular dichroism (ECD) calculations. Compound 1 was easy to transform into compounds 6–9 in Mosher’s reagents which were analyzed using the UPLC-Q-TOF-MS/MS technique in positive mode, from which their possible mass fragmentation patterns were suggested, besides neural loss and 1,3-rearrangement were the main clearage patterns. The bioactive evaluation revealed that compounds 1–5 and 8 displayed weak antioxidant activities, and compounds 2–4 possessed phytotoxicity against green foxtail leaves. In addition, compounds 3 and 4 displayed remarkable activities of inhibiting seed germination of G. conopsea. This was the first chemical investigation of the symbiotic fungus of G. conopsea, which could provide potent chemical clue to the symbiotic germination mechanism studies, but more experiments are needed to target the secondary metabolites with the abilities to promote the seed germination of G. conopsea.In this work, a new compound The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.XX and GD conceived and designed the study. LS, ZZ, QL, and YW performed the experiments. LS, LH, and GD performed the data collection and analyses. LH performed document processing. LS, GD, and XX wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The work was supported by the National Natural Science Foundation of China (grant no. 32170013) and CAMS Initiative for Innovative Medicine (grant no. 2021-I2M-1-031).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed β-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔmT values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions , compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from  Most of the reported Pels belong to the PL1 family, and they generally require additional Ca2+ for efficient enzymatic activity [Pectin, a complex heteropolysaccharide consisting of α-1,4-linked galacturonate chains, is a naturally ubiquitous constituent of the middle lamella of the primary cell wall in plants . Pectateactivity .Pels have many potentially important industrial applications, such as in food production and textile processing . AlkalinIn addition to the search for novel enzymes from nature, enzyme engineering, including randomized or irrational  and rational (structure based) approaches, has been used to improve the activity and stability of enzymes, including Pels, with varying degrees of success ,6,18,19.Bacillus clausii S10 (BacPelA) [In a previous study, we cloned, overexpressed, and characterized a novel thermostable and alkaline Pel belonging to family PL1 from BacPelA) . This enmT prediction were used to select mutation sites for the stability improvement of BacPelA. The mutations that were made also improved the activity of the enzyme. We anticipate that our findings will provide a strong foundation for the rational design of alkaline pectate lyase functional characteristics for industrial applications.In this work, we determined a high-resolution 3D structure of BacPelA. On the basis of the structure, the B-Fit Method and ΔBacillus sp. N16-5 (PDB ID code 3VMV) as the template. Detailed data collection and refinement statistics are presented in The sequence analysis showed that BacPelA has an N-terminal signal peptide of 23 amino acids. Recombinant mature BacPelA with an N-terminal His-tag was expressed and crystallized in the orthorhombic space group P43212. The crystal structure was solved and refined to 1.78 Å by using the method of molecular replacement with pectate lyase Bsp165PelA from n toroid [2+ was constructed by superimposing the structure of BacPelA and two highly homologous Pels: Bsp165PelA (PDB: 3VMW) containing pectate trisaccharide substrate . They typically have one of two core structure folds, a right-handed β-helix, or an (α/α)n toroid . Althougn toroid ,29,30,31ubstrate A or EcPeubstrate B. From tubstrate A, and onubstrate B than in members , while t members ,32. In tAll Pels have been reported to share a similar enzymatic mechanism (anti β-elimination), and the essential catalytic base and two supporting basic residues (Lys-Arg-Arg) are conserved in PL 1 pectate lyase ,33,34. SIn addition to the catalytic residues, some other residues interact with the substrate. R234 and Y269 in Bsp165PelA, corresponding to R245 and Y268 in EcPelC, interact with the substrate by hydrogen bond and stacking interactions, respectively . R223 in2+ is believed to be required for the pectolytic activity of all Pels [2+ ions or oligogalacturonate in Pel structures [2+ in Pel structures: primary Ca2+ which binds to the enzyme in the absence of substrate, and additional Ca2+, consisting of two or three Ca2+ ions, which bridge the enzyme and oligogalacturonate in the complex [2+ binding in BacPelA, which was confirmed by the crystal structure (2+ ion (the primary Ca2+), similar to Bsp165PelA, but different from EcPelC, which binds three additional Ca2+ ions in the presence of substrate. Although the locations and coordination numbers of the primary Ca2+ in the reported structures are the same, there are small differences in the binding sites. In EcPelC, the primary Ca2+ coordinates to residues D129, D131, E166, and D170 [2+, but the Asp, which is highly conserved in the other Pels and coordinates primary Ca2+, is substituted by T121. T121 cannot form an electrostatic interaction with the Ca2+ [Caall Pels . Previouructures ,28,30,35 complex ,35. Fromtructure . However2+ ions, in EcPelC, D160, and D162 participate in a loop interaction with 2Ca2+ and 3Ca2+ bridge substrate to both carboxyl oxygens of E166 [2Ca2+ binding. Therefore, we suspect that BacPelA does not bind additional Ca2+ at these two positions. Although the mutant R218K of EcPelC displayed 1Ca2+ binding to K218, 1Ca2+ cannot coordinate with any amino acid in the wild type (it binds only to the substrate). Thus, we considered that 1Ca2+ might be unimportant for catalysis [2+ ion (the primary Ca2+), which might be the reason that a low concentration of Ca2+ (0.1 mM) could induce the maximal catalytic activity of BacPelA. This low Ca2+ concentration dependence makes BacPelA more attractive for industrial applications.Considering the additional Ca of E166 . E166 isatalysis . Taken tmT values are effective for rationally choosing hot-spot residues to mutate to increase enzyme thermostability. The B-factor can be used to identify the flexibility of atoms, side-chains, or even whole regions of proteins, and to rationally identify appropriate  residues to increase thermostability [mT) using the experimental or modeled protein structure as the sole input, was recently applied in thermal stability engineering projects aimed at designing new proteins that feature increased heat resistance or remain active and stable in nonphysiological conditions [In a previous study, BacPelA showed high potential application in ramie degumming under alkaline conditions . Howevertability . Successtability ,43,44,45nditions ,47. Thisnditions ,49.mT values per residue was predicted by the HoTMuSiC algorithm (http://dezyme.com/ accessed on 11 September 2019). Six residues displaying the highest average B-factors and four residues with the highest sum of positive ΔmT values, which were located in unstable regions (loops or turns), were selected for site-directed saturation mutagenesis (SSM) library construction  and purified for further stability confirmation. After incubation with 0.1 mg mL−1 purified enzyme at 65 °C and pH 10.0 for 15 min, the residual activity of these eight mutant enzymes was determined. R150G, R216H, and A238C showed 33.2%, 42.6%, and 20.8% residual activity, respectively, while the other five mutants and the wild-type enzyme were almost inactivated. Thus, these three mutations resulted in significant improvement in the thermostability of BacPelA in alkaline conditions.After preliminary screening, a total of 16 potential mutants showing significant (>10%) higher residual enzyme activities than the wild-type were obtained from the SSM libraries: K139 (two mutants), R150 (four mutants), R216 (three mutants), A238 (five mutants) and R259 (two mutants). By sequencing, eight mutants, K139, R150G, R150S, R216H, R216G, A238C, A238V, and R259L, were identified. These eight mutants were then heterologously expressed in 5015T value  was used to determine the thermo-alkaline stability of the identified mutants in different conditions. As shown in T5015 value of the purified mutants R150G, R216H, and A238C at pH 10.0 increased by 6.5 °C, 6.0 °C, and 3.0 °C, respectively, compared with the wild-type enzyme. Addition of Ca2+ improves the thermo-alkaline stability of wide-type BacPelA. However, similar T5015 value increases were observed for the three mutants in the presence of 0.1 mM Ca2+ compared with its absence. The T5015 values of mutants R150G, R216H, and A238C also increased by 2.0 °C, 3.0 °C, and 5.0 °C, respectively, in neutral conditions without the addition of Ca2+. The residual activity after treatment in the enzymatic ramie degumming conditions for 4 h  was also assayed . Note also that the optimal reaction temperature of mutant R216H was changed to 65 °C, 5 °C lower than that for wild-type BacPelA. At 65 °C, the R216H mutant had a specific activity of 888.3 U mg−1, increased by approximately 24.8% compared with wild-type BacPelA at its optimal temperature (70 °C). Therefore, all three mutations not only improved the thermostability of the enzyme but also increased the specific activity.To elucidate the effects of these three substitutions on enzyme activity, the specific activities and steady-state kinetic parameters of wild-type BacPelA and mutants R150G, R216H, and A238C were determined at 70 °C. As shown in mK values of the R216H and A238C mutants were 0.47 and 0.48 g L−1, which were not substantially different from each other or the wild-type enzyme (0.44 g L−1). The mK value of R150G (0.56 g L−1) was slightly higher than that of the wild-type enzyme  . The spee enzyme . These de enzyme . Collect5015T values in alkaline conditions than in neutral conditions, which indicated that these residues might also be responsible for the alkaline stability of Pels. R150 is located in the T1 region, and R216 and A238 are located in the T3 region, which are flexible regions in the structure. Therefore, these three substitutions might enhance the rigidity of the protein, thus improving its thermostability. However, decreased conformational flexibility in thermophilic enzymes is generally thought to result in low enzyme activity, while increased flexibility in less stable enzymes is associated with enhanced enzyme activity [Bacillus sp. N16-5 [B. pumilus [Xanthomonas campestris [Paenibacillus sp. [Dickeya dadantii [To our knowledge, this is the first time that substitutions R150G, R216H, and A238C have been identified as contributing to the thermostability and thermo-alkaline stability of Pels. R150 and A238 are conservatively substituted residues in Pels, indicating that these sites might be critical for the thermostability of these enzymes. In addition, the R150G and R216H substitutions resulted in higher Δmpestris , Paenibadadantii , which i5015T values of the purified combination mutants R150G/R216H, R150G/A238C, and R216H/A238C at pH 10.0 increased by 11.0 °C, 4.5 °C, 5.5 °C, respectively, without the addition of Ca2+, and by 8.5 °C, 6.0 °C, 6.5 °C, respectively, with 0.1 mM Ca2+ added, compared with wild-type BacPelA. Furthermore, the T5015 values of these combination mutants increased by 4.0 °C, 6.5 °C, and 6.0 °C, respectively, under neutral conditions. The residual activity of these three combination mutants after treatment in the degumming conditions for 4 h was 50.9%, 75.6%, and 63.1%, 15.0-, 22.2- and 18.6-fold that of the wild-type BacPelA, respectively  compared with that of the wild type. Moreover, at their optimum reaction temperature of 65 °C, the mutants R150G/R216H and R150G/R216H/A238C showed activities of 854.9 U mg−1 and 925.3 U mg−1, respectively, which were 20.1% and 30.0% higher than that of wild-type BacPelA at its optimal reaction temperature of 70 °C. At the degumming temperature (60 °C), the specific activity of R150G/R216H, R150G/A238C, R216H/A238C and R150G/R216H/A238C mutants increased by approximately 34.4%, 75.8%, 37.7%, and 86.1%, respectively, compared with that of wild-type BacPelA  NaOH solution. Morphologically, the ramie fibers degummed by FCM were whiter, softer, and more dispersed than the fibers degummed by the wild-type enzyme or NaOH only . Each enzyme was used to degum ramie fibers at 60 °C and pH 10.0 for 4 h using the modified method reported previously . The weiaOH only B. Note tBacillus sp. MG-cp-2 [Amycolata sp. [B. pumilus DKS1 [B. subtilis 7-3-3 [B. pumilus ATCC7061 [Bacillus sp. N16-5 [D. dadantii after 2 h of degumming [w/v) NaOH solution, indicating its potential application in the substitution of chemical regents. However, the total gum content of the ramie fibers used in this experiment was around 34%, and only fibers with less than 2.5% residual gum can be directly used in further textile processes, and thus a weight loss of more than 30% is required [The enzymatic degumming activity of some other alkaline and thermostable Pels was reported previously. The polygalacturonase from  MG-cp-2  and pectlata sp.  and B. plus DKS1  showed ais 7-3-3 , B. pumiegumming . Thereforequired . In prevrequired . Our finE. coli BL21 (DE3)  and pET28a  were used as the host strain and vector for gene expression, respectively. Polygalacturonic acid (PGA) was purchased from Sigma-Aldrich . All enzymes for DNA manipulation were purchased from New England Biolabs . Isopropyl-β-D-thiogalactopyranoside (IPTG), kanamycin, and imidazole were from Amresco Inc. . All other chemicals were of reagent grade.N-terminal His-tag were expressed and purified as described previously [−1) as standard. Recombinant BacPelA was concentrated to 20 mg mL−1 for crystallization.Recombinant BacPelA and mutants with eviously , except 235 method). The reaction mixture consisted of 190 μL of 50 mM glycine–NaOH buffer (pH 10.5) containing 0.2% PGA (w/v) and 0.1 mM CaCl2 and 10 μL of appropriately diluted enzyme. The reaction was performed at 70 °C for 10 min and stopped by adding 300 μL of 30.0 mM H3PO4. Absorbance at 235 nm was measured with a Spectra Max 190 Microplate Reader . One unit of Pel activity was defined as the amount of enzyme producing 1 μmol of unsaturated bonds in 1 min with a molar extinction coefficient of 4600.0 M−1·cm−1. All activity measurements were repeated three times.The enzyme activity of purified BacPelA and mutants was determined by measuring the increase in produced unsaturated bonds at 235 nm  at the Shanghai Synchrotron Radiation Facility (SSRF). The crystals were mounted in a cryoloop, soaked with cryoprotectant solution  prior to data collection at 100 K. The diffraction images were processed using HKL2000 [Bacillus sp. N16-5 as the search model (PDB code 3VMW) [Concentrated BacPelA was subjected to crystallization screening using the sitting-drop vapor-diffusion method from Hampton Research  at 25 °C. In general, 1 μL of 6.5 mg mL HKL2000 . All the HKL2000  from the HKL2000  using thde 3VMW)  and Cootde 3VMW) . Prior tde 3VMW) .pelA), 1 μmol primer pairs, 0.2 mM dNTPs, and 1 U Pyrobest DNA polymerase in a final volume of 50 μL. The following PCR profile was used: 1 cycle of 94 °C for 4 min; 20 cycles of 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 7 min; followed by a final extension at 72 °C for 20 min. The PCR products were then digested by DpnI overnight. The products were transformed into E. coli BL21 (DE3) competent cells by electroporation transformation and plated onto LB-kanamycin (60 μg mL−1) agar plates for further screening. Single colonies from the plates were picked with sterilized toothpicks, plated in 160 μL LB medium containing kanamycin (50 μg mL−1) in sterilized 96-well microplates, and incubated overnight at 37 °C with shaking at 900 rpm. Fifty microliters of sterilized 60% glycerol were then added to each well, and the plates were stored at −70 °C to create the SSM libraries.SSM was performed using a previously published protocol . Degener−1) and IPTG (0.1 mM) prepared in a microplate using a sterile 96-pin replicator. After cultivation for 15 h at 37 °C, 10 μL of RIPA lysis buffer was added to each well, and the mixture was incubated at 37 °C for 30 min. Then 10 μL of lysate was added to 90 μL of 50 mM glycine–NaOH buffer (pH 10.0) in 96-well PCR plates, and heat-treated at 80 °C for 15 min. After incubation, residual enzyme activities were assayed immediately using the 3,5-dinitrosalicylic acid (DNS) method in new 96-well PCR plates. The reaction mixture in each well contained 70 μL of 50 mM glycine–NaOH buffer (pH 10.5), 0.2% PGA (w/v), and 0.1 mM CaCl2, and 10 μL of heat-treated variant enzyme. The reaction was performed at 70 °C for 10 min and stopped by adding 80 μL of DNS solution, followed by incubation at 98 °C for 8 min. Variants showing a significant increase in residual enzyme activities compared with the control  were regarded as candidate mutants with improved thermo-alkaline stability.Each mutant in the microplate was replicated in 200 μL LB medium containing kanamycin  with or without Ca2+ at different temperatures for 15 min. The residual activity was assayed using the A235 method under standard conditions.The 235 method at the optimal reaction temperature and pH. The substrate concentrations of PGA were 0.1–3.0 mg mL−1 for both wild-type BacPelA and mutant enzymes. All data are the averages of triplicate measurements. Kinetic parameters were calculated using GraphPad Prism 5.0 software and non-linear regression. All data are expressed as the means of triplicate measurements.The kinetic parameters of wild-type and mutant BacPelA were determined as previously described  using th−1 of purified wild-type or mutant enzyme was used to degum 1.5 g of ramie at 60 °C for 4 h in 30 mL of 50 mM glycine–NaOH buffer (pH 10.0) containing 0.1 mM CaCl2. Ramie fibers treated only with alkaline buffer (pH 10.0) or 0.5% (w/v) NaOH solution were used as negative and positive control, respectively. After enzymatic treatment, the fibers were washed twice with water after beating to remove residual gum from the surface of the ramie fibers and then dried to a constant weight at 105 °C. All experiments were repeated three times.The degumming of ramie fibers by wild-type BacPelA and its mutants was evaluated by measuring the weight loss of ramie fibers. The experiments were performed using modified Zhou’s method . ApproxiB. clausii S10 was solved at a high resolution (1.78 Å). The overall structure was similar to that of other members of the PL1 family, but there were some differences in the details of the secondary structure and Ca2+ binding sites. The structure suggests that BacPelA binds only to the primary Ca2+, which may explain why this enzyme shows low dependence on the Ca2+ concentration. This characteristic is attractive for industrial applications. On the basis of the crystal structure, BacPelA was engineered by applying B-factor and positive ΔmT analysis and SSM, aiming to improve the thermo-alkaline stability. Three key single substitutions, R150G, R216H, and A238C, were identified as improving the thermostability and thermo-alkaline stability of BacPelA. The mutant in which these three point mutations were combined, named FCM, exhibited a ΔT5015 value of 8.5 °C compared with that of the wild-type enzyme in thermo-alkaline conditions, and a 25.1% increase in catalytic activity. FCM was also more efficient than wild-type BacPelA in ramie degumming. Thus, our research has produced a thermo-alkaline stable and highly active pectate lyase with great potential for application in the textile industry. It also illustrates a rational design strategy for improving the thermo-alkaline stability and activity of pectate lyases. However, the molecular mechanism for thermo-alkaline stability and activity improvement of these key single substitutions is not very clear yet. So the molecular dynamics (MD) simulation and structural analysis need to be done in the future to reveal the mechanism. Moreover, the FCM-chemical degumming needs to be further evaluated in the ramie fiber degumming process.In this study, the crystal structure of the alkaline pectate lyase BacPelA from alkaliphilic


Lupinus mutabilis) oil is rich in monounsaturated (54.2%) and polyunsaturated (28.5%) fatty acids but has a ω-3:ω-6 ratio (1:9.2) above the recommended values for human health. Sacha inchi (Plukenetia volubilis) oil presents a high polyunsaturated fatty acid content (linolenic 47.2% and linoleic 34.7%), along a ω-3:ω-6 ratio (1:0.74) good for human consumption. The objective of this research was to study the physico-chemical properties and oxidative stability of tarwi and sacha inchi oil blends  with suitable ω-3:ω-6 ratios. All blends showed ω-3:ω-6 ratios between 1:0.8 and 1:1.9, acceptable from a nutritional point of view, and high total tocopherols’ content (1834–688 mg/kg), thanks to sacha inchi. The oxidative stability index (OSI) of the mixtures by the Rancimat method at 120 °C ranged from 0.46 to 8.80 h. The shelf-life of 1:1 tarwi/sacha inchi oil blend was 1.26 years; its entropy (−17.43 J/mol), enthalpy (107.04 kJ/mol), activation energy (110.24 kJ/mol) and Gibbs energy (113.76 kJ/mol) suggest low oxidation reaction rates and good stability. Hence, balanced blends of tarwi/sacha inchi oils can achieve optimal nutritional properties and enhanced shelf-life.Andean lupin ( For example, hydrogenation improves oxidative stability and texture, albeit may convert some n health . Interesn health ,3. Fractn health . On the n health . For exan health . The 80:n health . Supplemn health . Camellin health .Plukenetia volubilis L.) seeds have a high lipid content (33.4–54.3%), rich in polyunsaturated fatty acids (PUFA), α-linolenic acid (46–54%) and linoleic acid (33–37%) [Lupinus mutabilis Sweet), seeds contain 14–20% lipids [Sacha inchi ((33–37%) ,12,13,14(33–37%) . Andean % lipids , which a% lipids ,18.A diet with a balanced ω-3:ω-6 ratio affords numerous health benefits, because these PUFAs are precursors of eicosanoids  that intervene in many physiological processes such as blood clotting or inflammatory and immunological responses ,20. In f++), entropy (ΔS++), Gibbs free energy (ΔG++), Q10 and shelf life of different tarwi/sacha inchi oil blends, comparing them to pure tarwi and sacha inchi oils.The objective of this research was to determine the physico-chemical characteristics, oxidative stability, activation energy (Ea), enthalpy  for expeller press extraction performed at 70 °C . The sacLupinus mutabilis varieties [Lupinus mutabilis [Lupinus mutabilis oils (p < 0.05). A ω-3:ω-6 ratio between 1:2 and 1:3 plays a fundamental role in the secondary prevention of chronic metabolic and immunomodulatory diseases such as colon cancer and rheumatoid arthritis [The fatty acid composition of the oils is reported in arieties ,27, whilarieties . On the arieties ,15. The utabilis  and for utabilis . The M1,rthritis .p-anisidine value, acidity and total oxidation value . A similar profile, plus β-tocopherol, was reported for an Andenes lupin commercial sample extracted at higher temperature (70 °C) [Tarwi oil had a much lower α-, γ- and δ-tocopherols content than sacha inchi oil ( (70 °C) . Overall (70 °C) , possibl (70 °C) ,32. Neve (70 °C) . Sacha i (70 °C) , as well (70 °C) . In both (70 °C) , was the (70 °C) ,16,33. T (70 °C) ) and fla (70 °C) ). PUFA a (70 °C) .The carotenoid lutein, another well-known antioxidant molecule with beneficial health effects , was notIn spite of the high tocols’ content, at 120 °C the OSI of the PUFA-rich sacha inchi oil was lower than that of the MUFA-rich tarwi oil. Hence, the growing addition of tarwi oil in the blends increased their OSI from 0.46 (M1) to 8.80 h (M5) . The OSIThe tarwi oil results were not far from those  reported by Salvatierra-Pajuelo et al. , but werThe rate of auto-oxidation more than doubled for each 10 °C temperature increase; thus, the OSI values augmented sharply as the temperature decreased from 140 to 80 °C. The relevant concentrations of unsaturated fatty acids in the oils played a fundamental role in their oxidative instability, because lower OSI are correlated to high degrees of unsaturation . However++ and ΔS++ values (p ≤ 0.05) among treatments. The positive activation enthalpies (ΔH++ > 0) and negative entropies (ΔS++ ≤ 0) suggest an endothermic nature of the activated complex formation [++ > 0), suggesting that at these temperatures the auto-oxidation process is not spontaneous; sacha inchi oil had a higher ΔG++ value than the other samples, thus showing lower oxidation reaction rates.The ΔH+ values  showed sormation . During The activation energy (Ea) in the autoxidation process of oils denotes a delay in the initial oxidation process due to the rupture of the fatty acid chains and is influenced by the unsaturation levels. Hence, a high content of linoleic and/or linolenic acids should decrease the Ea, while a high oleic acid and saturated fatty acids percentage should increase it . However2: 0.97–1.00) between temperature and Log(OSI) for the tarwi, sacha inchi and M3 blend oils, with α values that range between −0.036 and −0.051. The shelf life of tarwi, M3 and sacha inchi oils at 25 °C was 2.57, 1.26 and 1.11 years, respectively. The sacha inchi shelf life was lower than the 1.79 years related by Rodríguez et al. [10 values varied between 2.26 (tarwi) and 2.62 (sacha inchi), i.e., were slightly superior to those of other vegetable oils such as chia (2.03), sesame (2.12), soybean (1.99–2.09), corn (2.01) and canola (2.01) [z et al.  for sacha (2.01) . Hence, Lupinus mutabilis seeds of the variety Andenes from the Ancash region , Peru, were obtained from the Programa de Leguminosas of the Universidad Nacional Agraria La Molina . After removing the bitter and toxic quinolizidine alkaloids by repeated washing [ washing , they wew:w), M2 (1:3), M3 (1:1), M4 (3:1) and M5 (4:1) were prepared and thoroughly mixed for 30 s with a vortex .The tarwi and the sacha inchi oils were extracted from the seeds by cold pressing (max temperature 50 °C), using a single-screw CA 59 G Komet expeller  at a screw speed of 35–40 rpm, clarified by centrifugation and stored at 4.0 ± 0.5 °C in dark flasks under nitrogen atmosphere. Five tarwi/sacha inchi oil blends, M1 . The operative conditions were: carrier He at 261.5 kPa and at 30 mL/min; oven temperature 100 °C for 4 min, increased by 3 °C/min to 240 °C, kept at 240 °C for 10 min; injection temperature 225 °C; flame ionization detector temperature 250 °C. The injection volume was 1 µL. Monounsaturated fatty acids (MUFA)/polyunsaturated fatty acids (PUFA), PUFA/saturated fatty acid (SFA) and ω-3:ω-6 ratios were computed from the fatty acids’ data.The fatty acids’ (FA) composition was determined as fatty acid methyl esters by gas chromatography after transesterification of the oils with 2 N KOH in methanol, according to Standard Method 2.302 . The chrp-anisidine value (p-AV), index of secondary oxidation, was analyzed following the method Cd 18-90 [p-AV [Peroxide value (PV), index of primary oxidation, was determined according to the method Cd 8-53  and exprCd 18-90  and acidCd 18-90 , while t90 [p-AV .v/v) at concentrations of 10 mg/mL and 100 mg/mL were filtered through a 0.2 µm PTFE and immediately analyzed by NP-HPLC under the following system and operating conditions: Adamas® Silica column 250 × 4.6 mm, 5 μm, and guard cartridge 10 × 4.6 mm, 5 μm ; mobile phase, hexane: ethyl acetate: acetic acid ; flow rate, 1.6 mL/min; pump L-2130 Elite LaChrom ; fluorimetric detector Jasco 821 FP Intelligent Spectrofluorometer (Japan) at excitation–emission wavelengths of 290 nm and 330 nm, respectively; connected to a computer with the software Empower 2  through the Waters e-SAT/IN module. The tocols’ standard curves were built using eleven concentrations (between 0.40 and 109.73 mg/L) of α-tocopherol standard , sixteen concentrations (between 0.20 and 23.20 mg/L) of γ-tocopherol standard  and eleven concentrations (between 0.05 and 9.35 mg/L) of δ-tocopherol standard . The total tocopherols were computed as the sum of the different homologues.The tocols were determined according to Rodríguez et al. . Brieflyv/v) at a concentration of 100 mg/mL. The mixes were filtered through a 0.2 μm PTFE filter, and analyzed immediately by NP-HPLC. The following system and operating conditions were used: Adamas® Silica column, 250 × 4.6 mm, 5 μm and guard cartridge 10 × 4.6 mm, 5 μm ; column oven at 20 °C L-2300 Elite LaChrom ; mobile phase, hexane: isopropyl alcohol (5%); flow rate, 1.5 mL/min; pump L-2130 Elite LaChrom . The carotenoids were detected at 450 nm by Diode Array Detector L2450 Elite LaChrom  set in the range of 200–650 nm. The HPLC system was controlled by the software EZChrom Client/Server version 3.1.7. For peak quantification, a calibration curve (between 0.3 and 3.0 mg/L) of the lutein standard  stock solution was used. The tocols and the carotenoids are reported as mg/kg oil. The carotenoids were assessed as outlined by Varas Condori et al.  on the oThe OSI (in hours) of the two oils and of their blends was evaluated according to method Cd 12b-92  using a The activation energy (Ea) was determined from the slope of the line representing the natural logarithm of the OSI values vs. the inverse of the absolute temperature (1/T) :(1)LnOSI++) and entropy (ΔS++) were determined as regression of the K/T logarithm vs. 1/T [−24 J s); kB is Boltzmann’s constant (1.380658 × 10−23 J/K) and R is the universal gas constant (8.314 J/mol). The Gibbs free energy (ΔG++) was computed as ΔG++ = ΔH++ − TΔS++. The shelf life was determined by extrapolating the linear correlation of the OSI logarithm at T = 25 °C [10 temperature coefficient, which indicates the increase in reaction rate after a 10 °C rise in temperature, was obtained from the (OSI at T)/(OSI at T + 10 °C) [The activation enthalpy (ΔH vs. 1/T :(2)LogKT = 25 °C : Log (OS+ 10 °C) .p ≤ 0.05, Fisher’s LSD at 95% significance level was computed. All analyses were performed using Statgraphics® Centurion XVI . Average values and standard deviations (SD) were computed with the Excel® program .The analyses were carried out on three independent samples (tocols and carotenoids in double on each sample) and the data underwent one-way ANOVA; when differences were found at Tarwi and sacha inchi oils are rich in monounsaturated and polyunsaturated fatty acids, respectively, while their blends show a more balanced ω-3:ω-6 compositions, nutritionally preferable for human consumption and potentially preventative against diseases such as rheumatoid arthritis and colon cancer. All the blends present very high tocopherol concentrations, low PV and acidity indices, mainly due to the sacha inchi oil. On the other hand, the oxidative stability of the blends improved with increasing the tarwi oil content. In particular, the 1:1 blend studied reached a shelf life of 1.26 years, very appealing for commercial development. In conclusion, balanced blends of tarwi/sacha inchi oils can achieve optimal nutritional properties along with enhanced shelf-life.


Introduction: Systematic gene knockout studies may offer us novel insights on cell metabolism and physiology. Specifically, the lipid accumulation mechanism at the molecular or cellular level is yet to be determined in the oleaginous yeast Y. lipolytica.Methods: Herein, we established ten engineered strains with the knockout of important genes involving in central carbon metabolism, NADPH generation, and fatty acid biosynthetic pathways.Results: Our result showed that NADPH sources for lipogenesis include the OxPP pathway, POM cycle, and a trans-mitochondrial isocitrate-α-oxoglutarate NADPH shuttle in Y. lipolytica. Moreover, we found that knockout of mitochondrial NAD+ isocitrate dehydrogenase IDH2 and overexpression of cytosolic NADP+ isocitrate dehydrogenase IDP2 could facilitate lipid synthesis. Besides, we also demonstrated that acetate is a more favorable carbon source for lipid synthesis when glycolysis step is impaired, indicating the evolutionary robustness of Y. lipolytica.Discussion: This systematic investigation of gene deletions and overexpression across various lipogenic pathways would help us better understand lipogenesis and engineer yeast factories to upgrade the lipid biomanufacturing platform. Yarrowia lipolytica is an oleaginous yeast with GRAS  status and has several significant attributes, such as a broad substrate spectrum, relatively well-defined genome annotations, diversified genetic manipulation methods, and abundant pool size of metabolic precursors, including acetyl-CoA, malonyl-CoA, and tricarboxylic acid (TCA) cycle intermediates  to alkyl groups (-CH2-CH2-) for growing fatty acid carbon backbone  cycle, and isocitrate-2-oxoglutarate shuttle in The strain po1fk obtained in our previous work was chosen as the starting strain in this study . MoreoveloxP based gene knockout method was used as previously reported (ylPFK (encoding phosphofructose kinase), pHyloxP-ylPYK (encoding pyruvate kinase), pHyloxP-ylPYC1 (encoding pyruvate carboxylase), pHyloxP-ylMAE1 , pHyloxP-ylIDH2 , pHyloxP-ylIDP2 (encoding cytosolic isocitrate dehydrogenase), pHyloxP-ylFAA1 , pHyloxP-ylACO1 (encoding aconitate hydratase), and pHyloxP-ylSNF1 (encoding AMP-activated protein kinase).The marker-free Cre-reported . In thisylPFK as an example. Firstly, the upstream and downstream sequences (both 1000 bp) of flanking gene ylPFK were obtained by the PCR-amplified reactions using primers ylPFK_UpF/ylPFK_UpR and ylPFK_DwF/ylPFK_DwR. Then, pYLXP’-loxP-Hyr containing loxP-Hyr-loxP cassette was digested by endonucleases AvrII and SalI to obtain loxP-Hyr-loxP cassette and plasmid backbone of pYLXP’-loxP-Hyr. Next, the upstream fragment ylPFK_Up, downstream fragments ylPFK_Dw, loxP-Hyr-loxP cassette and pYLXP’-loxP-Hyr backbone were joined by Gibson Assembly to generate the plasmid pHyloxP-ylPFK. The constructed plasmids were sequenced by Sangon Biotech Co., Ltd. .Here, we took the process of constructing the plasmid pHyloxP-SnaBI and KpnI, obtaining the linearized pYLXP’. Then, the linearized pYLXP’ was ligase with the DNA fragment of gene PFK (that was obtained by the PCR-amplified reaction using primers PFK-F and PFK-R) by Gibson assembly method to generate plasmid pYLXP’-ylPFK, which was sequenced by Sangon Biotech Co., Ltd. .The pYLXP’, a YaliBrick plasmid, was used for gene expression . For exaY. lipolytica transformation have been reported  contains ammonium sulfate 1.1 g/L, YNB 1.7 g/L, acetate 41.0 g/L, CSM-Leu 0.74 g/L, and L-leucine 0.20 g/L.Seed culture medium of CSM (the yeast complete synthetic media) contains ammonium sulfate 5.0 g/L, YNB (yeast nitrogen base without ammonium sulfate) 1.7 g/L, glucose 20.0 g/L, CSM-Leu 0.74 g/L, and L-leucine 0.20 g/L. The CSM fermentation medium (C/N = 80) contains ammonium sulfate 1.1 g/L, YNB 1.7 g/L, glucose 40.0 g/L, CSM-Leu 0.74 g/L, and L-leucine 0.20 g/L. Specifically, phosphoric buffer solution (PBS), including 0.2 M Na600). The OD600 value could be converted to dry cell weight (DCW) according to Eq. 1 OD600 = 0.35 g/L. The concentrations of glucose and acetate were measured under a flow rate of 0.6 mL/min of the mobile phase (5 mM of H2SO4) at 40°C by high-performance liquid chromatography (HPLC) equipped with a HPX-87H column  and a refractive index detector through Shimadzu Prominence HPLC LC-20 A. To determine lipid composition, 4 OD units of cells were directly saponified with 0.5 M sodium methoxide with vortexing at 1,200 rpm for 2 h, and then neutralized with 40 μL 98% sulfuric acid to facilitate transesterification. Next, 400 μL hexane was added to extract fatty acid methyl ester for GC analysis. Moreover, to quantitatively determine lipid titer, 100 μL of 2 g/L of tridecanoate methyl ester and 2 g/L of glyceryl trihepta-decanoate were added as internal standard at the beginning, to benchmark the transesterification and saponification efficiency, respectively. Triplicate samples were taken and results were reported as average ± standard deviations. Lipid samples were taken at 120 h of shaking incubation. The method of GC analysis is consistent with previously reported method , ylPYK , and ylAC O 1 , and further constructed three strain po1fk_ylPFK, po1fk_ylPYK, and po1fk_ylACO1.Noticeably, cytosolic acetyl-CoA is a direct precursor for lipid synthesis , which i3-CO-) originating from acetyl-CoA is reduced to alkyl groups (-CH2-CH2-) when growing fatty acid carbon backbone, which needs to consume the reducing equivalent NADPH , mitochondrial isocitrate dehydrogenase , cytosolic isocitrate dehydrogenase , and glucose-6-phosphate dehydrogenase . Particularly, the POM cycle . Thus, these five genes in the NADPH metabolism were deleted to obtained strains po1fk_ylMAE1, po1fk_ylIDH2, po1fk_ylIDP2, po1fk_ylZWF1, and po1fk_ylPYC1, respectively. Moreover, we also investigated the influences of deleting lipogenesis gene ylFAA1  on the lipid synthesis  was also knocked out, which is a cellular carbon metabolism regulator in Y. lipolytica to control the transition from growth phase to oleaginous production phase  to measure the lipid titers and cell growth. As a result, the maximal biomasses of strains po1fk_ylPYC1, po1fk_ylACO1, and po1fk_ylMAE1 were slightly decreased compared with po1fk . Notably, gene ylPYK encodes pyruvate kinase, which converts the reaction of phosphoenolpyruvate to pyruvate in glycolysis. Therefore, deletion of gene ylPYK could block the metabolic flux from the glycolytic pathway to TCA cycles. On the other hand, glucose-6-phosphate dehydrogenase ZWF1 catalyzes glucose 6-phosphate to 6-phospho-glucono-1,5-lactone, providing the primary source of cytosolic NADPH in Y. lipolytica. It should be noted that NADPH functions as reducing equivalents to maintain cellular redox homeostasis and serves as important electron donors in the anabolic metabolism (ylZWF gave a 76.9% reduction (0.18 ± 0.05 g/L with the yield of 13.01 mg/g Glucose) in the lipid synthesis, indicating that the OxPP pathway is the primary NADPH source for lipogenesis. However, an interesting phenomenon to note is that the surface wrinkle of po1fk_ylZWF1 is disappeared. We speculated that the reduced surface area of po1fk_ylZWF1 colony may be attributed to the less oxygen demand for the ZWF1 deficient strain.To confirm whether cell growth and lipid levels of engineered th po1fk , and deltabolism . In thisylFAA1, po1fk_ylPYC1, po1fk_ylACO1, and po1fk_ylMAE1 are 0.49 ± 0.11, 0.59 ± 0.10, 0.62 ± 0.02, and 0.67 ± 0.02 g/L, decreased by 38.0%, 25.3%, 21.5%, and 15.2% relatively to po1fk (0.79 ± 0.10 g/L), respectively, suggesting that genes ylFAA1, ylPYC1, ylAC O 1, and ylMAE are necessary for the lipid synthesis in Y. lipolytica. However, the deletion of gene ylIDH2 increased the lipid titer, reaching .87 g/L with the yield of 44.8 mg/g Glucose, which were 1.10-fold and 1.19-fold of that in the strain po1fk, respectively. It is incomprehensible because the mitochondrial NAD+ isocitrate dehydrogenase IDH2 is important for TCA cycle, catalyzing isocitrate to 2-oxoglutarate. To explain this result, we performed a KEGG pathway analysis and found that the mitochondrial NAD+ isocitrate dehydrogenases have two isoenzymes in Y. lipolytica, encoded by genes YALI0E05137g and YALI0D06303g, respectively. Therefore, we speculate that deletion of IDH2 downregulated the activity of mitochondrial NAD+ isocitrate dehydrogenases, which strengthened the accumulation of citrate to promote the synthesis of lipids. Besides, the deletion of carbon catabolite repressor gene ylSNF1 significantly increased the lipid titer, reaching 2.45 ± 0.02 g/L with the yield of 100.1 mg/g Glucose, which was 3.10-fold and 2.65-fold of that of po1fk, respectively. This result is consistent with the previous research  was used to track the change of cultivation pH , respectively. These results indicate that the evolutionary robustness of cells that enable them adapt to alternate carbon sources when central metabolism is compromised, and most importantly, acetate could be used as a shortcut nutrient to support cell growth. However, the cell growth of po1fk_ylPYC1 was repressed in the CSM-acetate medium, and its maximum biomass (OD600) was 5.72 ± 0.50, which could be attributed to the fact that PYC is important for cell to replenish gluconeogenesis when the cells were grown on the acetate media. The maximal lipid titer was produced by po1fk_ylPFK, reaching 1.04 ± 0.23 g/L, which is 1.09-fold of that of po1fk (0.95 ± 0.04 g/L). Interestingly, the lipid titer of po1fk_ylSNF1 was close to that of po1fk using acetate as carbon source. Studies have reported that downregulation of Snf1 can activate the activity of acetyl-CoA carboxylase ACC1, which could facilitate the flux from acetyl-CoA to malonyl-CoA . As suggested by the stoichiometry, the yield of lipid synthesized from acetate (0.294 g/g Acetate) is higher than that from glucose (0.271 g/g Glucose), indicating that acetate is a dominant carbon source for lipid synthesis. As a result, strain po1fk_ylPYK produced 0.96 ± 0.06 g/L of lipid using acetate as the carbon source.In  pathway . TherefoY. lipolytica. However, in the CSM-acetate medium, the cell growth of po1fk_ylPYC1 was repressed, suggesting that the deficiency of pyruvate carboxylase PYC1 would lead to the disruption of gluconeogenesis. Moreover, it is reported that the distributions of the cellular reducing equivalent are highly compartmentalized in eukaryotes, which is attributed to the specific localization of metabolic pathways and impermeabilities of organelles membranes , which could help us to understand the influences of NADPH supplement on lipid synthsis.The glucose-6-phosphate dehydrogenase ZWF is a key enzyme that catalyzes glucose 6-phosphate to 6-phosphogluconolactone with generating NADPH. NADPH provides reducing power for synthesizing biological macromolecules  and directly affects cell growth, protein expression, and other secondary metabolites synthesis (polyketides or triterpenoids) . We founembranes . Specifiembranes . Our resY. lipolytica. Herein, deletion of genes ylSNF1 significantly increased the lipid titer in the CSM-leu medium, reaching 2.45 ± 0.02 g/L with the yield of 100.0 mg/g Glucose and the productivity of 0.289 g Lipid/L/g DCW (In addition, protein kinase ylSNF1 is a crucial regulator of glucose signal transduction, which is a negative regulator of the fatty acid biosynthesis pathway and acetyl-CoA carboxylase. It has been identified that SNF1 plays a vital role in the transition from growth to oil production in /L/g DCW , which i/L/g DCW .Y. lipolytica. Moreover, the knockout of mitochondrial isocitrate dehydrogenase IDH1 and the carbon catabolite repressor SNF1 could facilitate the lipid synthesis. Besides, acetate is a more favorable carbon source for the lipid synthesis when glycolysis step is impaired, indicating this could be alternate carbon source that reach metabolic optimality in lipid synthesis. This systematic investigation of gene deletions and overexpression across various lipogenic pathways would help us better understand lipogenesis and engineer yeast factories to upgrade the lipid biomanufacturing platform.In this work, we established ten engineered strains with deleting the important gene involved in the central carbon, NADPH, and lipid metabolism, and systematically investigated the influence of such gene deletions on the lipid synthesis and cell growth. As a result, our study demonstrated that NADPH sources for lipogenesis include the OxPP pathway, POM cycle, and isocitrate-2-oxoglutarate shuttle in