Response of Root-Associated Bacterial Communities to Different Degrees of Soft Rot Damage in Amorphophallus konjac Under a Robinia pseudoacacia Plantation

Bacterial soft rot is a destructive disease that restricts the development of the konjac (Amorphophallus konjac K. Koch ex N.E.Br) industry. The objective of this study was to investigate how soft rot disease affects bacterial communities associated with the roots of konjac plants growing under a pure Robinia pseudoacacia plantation. Three sampling sites affected by different degrees of soft rot damage were selected based on the disease incidence [0%, non-diseased (ND); 4.2%, moderately diseased (MD); and 18.6%, highly diseased (HD)]. The variation in soil and root bacterial diversity and community composition among the sampling sites was determined by Illumina HiSeq sequencing of the V3–V4 hypervariable regions of the bacterial 16S rRNA gene. The results showed that the contents of soil organic matter and available nutrients (N, P, and K) increased with increasing damage degree, whereas higher damage degree resulted in lower soil pH and enzymatic activity (sucrase, urease, catalase, and polyphenol oxidase). The composition of root-associated bacterial communities differed among the three sampling sites. Proteobacteria was the most dominant bacterial phylum in all soil and root samples. Pseudomonas, Bacillus, Rhizobium, and Streptomyces were the most abundant in all samples from the ND sites, whereas Pectobacterium carotovorum and Serratia were predominant in the samples from the MD and HD sites. The abundance and alpha diversity of root-associated bacteria were significantly higher (p < 0.05) in the ND sites than in the diseased sites. The results suggested pronounced differences in the abundance, alpha diversity, and community composition of bacteria associated with the roots of konjac plants affected by different degrees of soft rot damage. Such differences in bacterial community structure were related to dynamic changes in soil variables, especially soil available potassium content, sucrase activity, and urease activity. Analysis of the dominant root-associated bacterial taxa offers an approach to predict the damage degree due to soft rot in konjac and provides evidence for the prevention of this soil-borne disease via microecological regulation.

Modular biosynthesis of plant hemicellulose and its impact on yeast cells

Background The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past 5 years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes. Results Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana. Conclusion The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins. Graphic abstract

Amorphophallus konjac: A Novel Alternative Flour on Gluten-Free Bread

The demand for gluten-free products is rising, but their production with similar quality as their gluten counterparts is challenging. This study aimed to develop gluten-free bread samples using different concentrations of Amorphophallus konjac flour (0%, 12.5%, 25%, 37.5%, and 50% of the total flour content) and to evaluate their nutritional and physicochemical properties. Proteins, lipids, carbohydrates, moisture, ash content, fibers, resistant starch, firmness, specific volume, and color were evaluated using official methods. Protein varied from 2.95% to 4.94%, the energy value from 347.93 to 133.55 kcal/100 g, dietary fiber from 8.19 to 17.90%, and resistant starch from 0.67% to 0.75% on wet basis. The addition of konjac flour positively influenced the specific volume. Higher concentrations of konjac flour in the formulations led to lower calories of the bread due to the significant addition of water to the dough. The bread samples with konjac showed high fiber content due to the composition of the flour. They had lower levels of carbohydrates, which can positively influence the glycemic index. Konjac flour provided dough mold, growth, and better texture for gluten-free bread. The best formulations were prepared in concentrations up to 37.5% konjac. The 50% konjac bread showed slightly reduced specific volume and pale color.

Rapid, modular biosynthesis of plant hemicellulose and its impact on yeast cells

Interest in the engineering of polysaccharide-based biomaterials has emerged in recent years. Despite impressive advances on bacterial cellulose, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes. Here, we optimized the cultivation of the yeast Pichia pastoris for the orthogonal production of plant polysaccharides, and enhanced heteromannan (HM) production by assembling modular cellulose synthase-like A (CSLA) enzymes. Chimeric proteins swapping the domains of a plant mannan synthase and a glucomannan synthase led, in three cases, to higher yields or improved growth compared to the parental CSLA enzymes. Prolonged expression of a glucomannan synthase from Amorphophallus konjac (AkCSLA3) was toxic to yeast cells, as demonstrated by reduced biomass accumulation and elevated uptake of dyes that are normally restricted to the extracellular matrix. However, no growth inhibition was observed for CSLA variants producing relatively pure mannan or a CSLC glucan synthase. The toxicity of AkCSLA3 was reduced by swapping its C-terminal region with that of a mannan synthase. HM production was further boosted by co-expressing chimeric CSLA proteins with the MANNAN-SYNTHESIS-RELATED1 (MSR1) putative glycosyltransferase. Interestingly, Pichia cells either increased or decreased in size depending on the CSLA variant expressed, and most of them remained viable even producing copious amounts of hemicellulose. Therefore, yeast modified with non-toxic plant polysaccharides could represent a modular chassis to produce and protect sensitive cargo such as therapeutic proteins.

Structure-dependent absorption of atypical sphingoid long-chain bases from digestive tract into lymph

Background Dietary sphingolipids have various biofunctions, including skin barrier improvement and anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are expected to be important for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear. Methods In this study, five structurally different LCBs were prepared from glucosylceramides (GlcCers) with LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH moieties derived from konjac tuber (Amorphophallus konjac), from GlcCers with an LCB 18(9Me):2(4E,8E);2OH moiety derived from Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and from ceramide 2-aminoethyphosphonate with LCB 18:3(4E,8E,10E);2OH moiety and LCB 18(9Me):3(4E,8E,10E);2OH moiety derived from giant scallop (Mizuhopecten yessoensis), and their absorption percentages and metabolite levels were analyzed using a lymph-duct-cannulated rat model via liquid chromatography tandem mass spectrometry (LC/MS/MS) with a multistage fragmentation method. Results The five orally administered LCBs were absorbed and detected in chyle (lipid-containing lymph) as LCBs and several metabolites including ceramides, hexosylceramides, and sphingomyelins. The absorption percentages of LCBs were 0.10–1.17%, depending on their structure. The absorption percentage of LCB 18:2(4E,8Z);2OH was the highest (1.17%), whereas that of LCB 18:3(4E,8E,10E);2OH was the lowest (0.10%). The amount of sphingomyelin with an LCB 18:2(4E,8Z);2OH moiety in chyle was particularly higher than sphingomyelins with other LCB moieties. Conclusions Structural differences among LCBs, particularly geometric isomerism at the C8–C9 position, significantly affected the absorption percentages and ratio of metabolites. This is the first report to elucidate that the absorption and metabolism of sphingolipids are dependent on their LCB structure. These results could be used to develop functional foods that are more readily absorbed.

Effect of Konjac Glucomannan (KGM) on the Reconstitution of the Dermal Environment against UVB-Induced Condition

Skin layers serve as a barrier against unexpected critical changes in the body due to environmental factors. Excessive ultraviolet (UV) B exposure increases the levels of age-related factors, leading to senescent cells and damaged skin tissues. Widely used as a dietary supplement, konjac (Amorphophallus konjac) glucomannan (KGM) has shown skin regeneration potential in patch or sheet form with anti-inflammatory or immunosuppressive effects. However, the ability of KGM to reconstitute senescent/damaged skin following UV radiation has not been explored. Here, we demonstrate that KGM alleviates skin damage by increasing the proportion of young cell populations in UVB-exposed senescent human epidermal primary melanocytes. Young cell numbers increased depending on KGM dosage, but the senescent cells were not removed. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis showed that mRNA and protein levels of age- and pigmentation-related factors decreased in a manner dependent on the rate at which new cells were generated. Moreover, an analysis of mRNA and protein levels indicated that KGM facilitated youth by increasing cell proliferation in UVB-damaged human fibroblasts. Thus, KGM is a highly effective natural agent for maintaining skin homeostasis by promoting the reconstitution of the dermal environment against UVB-induced acute senescence or skin damage.