Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications.

BACKGROUND: Pichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities. RESULTS: Two novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in ß-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage. CONCLUSIONS: Two novel P. pastoris chassis hosts with impaired ß-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Development of a novel ß-1,6-glucan-specific detection system using functionally-modified recombinant endo-ß-1,6-glucanase.

ß-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring ß-1,6-glucan, another primary ß-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for ß-1,6-glucan using a modified recombinant endo-ß-1,6-glucanase having diminished glucan hydrolase activity. The purified ß-1,6-glucanase derivative bound to the ß-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this ß-1,6-glucan probe by demonstrating its ability to detect cell wall ß-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long ß-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular ß-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro We also used this assay to measure ß-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for ß-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

Conformation and cooperative order-disorder transition in aqueous solutions of ß-1,3-d-glucan with different degree of branching varied by the Smith degradation.

ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.

Differentiation of the seed coat and composition of the mucilage of Lepidium perfoliatum L.: a desert annual with typical myxospermy.

Myxospermy is an important feature in seeds of many plant species grown in desert region. Fertilization can initiate differentiation of the seed coat epidermis into a specialized cell type with mucilage production. In the present study, comprehensive analyses were performed on the seed coat differentiation, mucilage production and composition, and seed germination in Lepidium perfoliatum (Brassicaceae), a desert annual with typical myxospermy in China. First, results indicated that mucilage was secreted uniformly at the outer tangential wall, resulting in compression of the cytoplasm to the bottom of the epidermal cells. Secondly, the inner tangential wall and two radial walls of the subepidermal cells were apparently thickened by production of a secondary cell wall material, which resulted in a 'typical' palisade appearance. Thirdly, immunohistochemical staining combined with the enzymatic digestion and infrared spectrum analysis of the mucilage indicated that, while one important component of the seed coat mucilage in L. perfoliatum was pectin, it also contained ß-1,3-d-glucan and xyloglucan. Finally, seed germination showed that seeds with mucilage displayed significantly higher germination percentage than that of demucilaged seeds in abundant or excess water conditions. These results suggest that the possible ecological role of mucilage in L. perfoliatum is in the adaptation to habitats with well-watered and water-logged conditions, rather than water stress.

Performance of Multiplex PCR and ß-1,3-D-Glucan Testing for the Diagnosis of Candidemia.

Bloodstream infections caused by Candida yeasts (candidemia) are associated with high morbidity and mortality. Diagnosis remains challenging, with the current gold standard­isolation from blood culture (BC)­being limited by low sensitivity and long turnaround time. This study evaluated the performance of two nonculture methods: PCR and ß-1,3-D-glucan (BDG) testing. The sera of 103 patients with BC-proven candidemia and of 46 controls were analyzed with the Fungiplex Candida Real-Time PCR and the Wako ß-Glucan Test. The BDG assay demonstrated higher sensitivity than the multiplex PCR (58% vs. 33%). This was particularly evident in ICU patients (60% vs. 28%) and in C. albicans candidemia (57% vs. 37%). The earlier prior to BC sampling the sera were obtained, the more the PCR sensitivity decreased (46% to 18% in the periods of 0−2 and 3−5 days before BC, respectively), while BDG testing was independent of the sampling date. No positive PCR results were obtained in sera sampled more than five days before BC. Specificities were 89% for BDG and 93% for PCR testing. In conclusion, BDG testing demonstrated several advantages over PCR testing for the diagnosis of candidemia, including higher sensitivity and earlier diagnosis. However, BC remains essential, as BDG does not allow for species differentiation.

Metabolic flux and transcriptome analyses provide insights into the mechanism underlying zinc sulfate improved ß-1,3-D-glucan production by Aureobasidium pullulans.

The effects of zinc sulfate at various concentrations on ß-1,3-D-glucan (ß-glucan) and pullulan production were investigated in flasks, and 0.1 g/L zinc sulfate was found to be the optimum concentration favoring increased ß-glucan production. When batch culture of Aureobasidium pullulans CCTCC M 2012259 with 0.1 g/L zinc sulfate was carried out, the maximum dry biomass decreased by 16.9% while ß-glucan production significantly increased by 120.5%, compared to results obtained from the control without zinc sulfate addition. To reveal the mechanism underlying zinc sulfate improved ß-glucan production, both metabolic flux analysis and RNA-seq analysis were performed. The results indicated that zinc sulfate decreased carbon flux towards biomass formation and ATP supply, down-regulated genes associated with membrane part and cellular components organization, leading to a decrease in dry cell weight. However, zinc sulfate increased metabolic flux towards ß-glucan biosynthesis, up-regulated genes related to glycan biosynthesis and nucleotide metabolism, resulting in improved ß-glucan production. This study provides insights into the changes in the metabolism of A. pullulans in response to zinc sulfate, and can serve as a valuable reference of genetic information for improving the production of polysaccharides through metabolic engineering.

[House Dust and Its Adverse Health Effects].

In this review, we examine house dust and its effect on inhabitants' health. Residential house dust includes components from plants, pollens, microorganisms, insects, skin flakes, hairs and fibers. It also includes materials contaminated with chemicals from combustion, furniture, interior materials, electronics, cleaning agents, personal care products. Nowadays, most people spend their time indoors. Thus, dust is an important medium of exposure to pollutions. According to United States Environmental Protection Agency Exposure Factors Handbook, the estimated amount of dust ingestion is 30 mg/day for adults, and 60 mg/day for children over 1 year of age. Since 2003, we have been conducting epidemiological studies to find the association between the indoor environment and the inhabitants' health. The levels of mite allergens, endotoxins, and ß-1,3-d-glucan in house dust were measured as biological factors. Semi volatile organic compounds (SVOC) such as phthalates and phosphate flame retardants (PFRs) in dust were also analyzed. As a result, we found that the ORs (95%CI) of nasal and optical symptoms of sick building syndrome (SBS) were 1.45 (1.01-2.10) and 1.47 (1.14-1.88), respectively, when there was a 10-fold increase in the levels of mite allergens. There was no association of mite allergens with allergies. Endotoxins and ß-1,3-d-glucan did not show any association with SBS. Regarding SVOC, increased levels of phthalates and PFR increased the risk of allergies. The association between phthalates and increased risk of allergies was clearer among children than adults. There were no gold standards of dust sampling and pretreatment methods. Thus, caution is needed when comparing findings of various studies. Methods should accurately reflect exposure levels.

Influence of exposure to coarse, fine and ultrafine urban particulate matter and their biological constituents on neural biomarkers in a randomized controlled crossover study.

BACKGROUND: Epidemiological studies have reported associations between air pollution and neuro-psychological conditions. Biological mechanisms behind these findings are still not clear. OBJECTIVES: We examined changes in blood and urinary neural biomarkers following exposure to concentrated ambient coarse, fine and ultrafine particles. METHODS: Fifty healthy non-smoking volunteers, mean age 28years, were exposed to coarse (2.5-10µm, mean 213µg/m3) and fine (0.15-2.5µm, mean 238µg/m3) concentrated ambient particles (CAPs), and filtered ambient and/or medical air. Twenty-five participants were exposed to ultrafine CAP (mean size 59.6nm, range 47.0-69.8nm), mean (136µg/m3) and filtered medical air. Exposures lasted 130min, separated by ≥2weeks, and the biological constituents endotoxin and ß-1,3-d-glucan of each particle size fraction were measured. Blood and urine samples were collected pre-exposure, and 1-hour and 21-hour post-exposure to determine neural biomarker levels. Mixed-model regressions assessed associations between exposures and changes in biomarker levels. RESULTS: Results were expressed as percent change from daily pre-exposure biomarker levels. Exposure to coarse CAP was significantly associated with increased urinary levels of the stress-related biomarkers vanillylmandelic acid (VMA) and cortisol when compared with exposure to filtered medical air [20% (95% confidence interval: 1.0%, 38%) and 64% (0.2%, 127%), respectively] 21hours post-exposure. However exposure to coarse CAP was significantly associated with decreases in blood cortisol [-26.0% (-42.4%, -9.6%) and -22.4% (-43.7%, -1.1%) at 1h and 21h post-exposure, respectively]. Biological molecules present in coarse CAP were significantly associated with blood biomarkers indicative of blood brain barrier integrity. Endotoxin content was significantly associated with increased blood ubiquitin C-terminal hydrolase L1 [UCHL1, 11% (5.3%, 16%) per ln(ng/m3+1)] 1-hour post-exposure, while ß-1,3-d-glucan was significantly associated with increased blood S100B [6.3% (3.2%, 9.4%) per ln(ng/m3+1)], as well as UCHL1 [3.1% (0.4%, 5.9%) per ln(ng/m3+1)], one-hour post-exposure. Fine CAP was marginally associated with increased blood UCHL1 when compared with exposure to filtered medical air [17.7% (-1.7%, 37.2%), p=0.07] 21hours post-exposure. Ultrafine CAP was not significantly associated with changes in any blood and urinary neural biomarkers examined. CONCLUSION: Ambient coarse particulate matter and its biological constituents may influence neural biomarker levels that reflect perturbations of blood-brain barrier integrity and systemic stress response.

Endotoxin and ß-1,3-d-Glucan in Concentrated Ambient Particles Induce Rapid Increase in Blood Pressure in Controlled Human Exposures.

Short-term exposure to particulate matter (PM) is associated with increased blood pressure (BP) in epidemiological studies. Understanding the impact of specific PM components on BP is essential in developing effective risk-reduction strategies. We investigated the association between endotoxin and ß-1,3-d-Glucan-two major biological PM components-and BP. We also examined whether vascular endothelial growth factor, a vasodilatory inflammatory marker, modified these associations. We conducted a single-blind, randomized, crossover trial of controlled human exposure to concentrated ambient particles with 50 healthy adults. Particle-associated-endotoxin and ß-1,3-d-Glucan were sampled using polycarbonate-membrane-filters. Supine resting systolic BP and diastolic BP were measured pre-, 0.5-hour post-, and 20-hour postexposure. Urine vascular endothelial growth factor concentration was determined using enzyme-linked immunosorbant assay and creatinine-corrected. Exposures to endotoxin and ß-1,3-d-Glucan for 130 minutes were associated with increases in BPs: at 0.5-hour postexposure, every doubling in endotoxin concentration was associated with 1.73 mm Hg higher systolic BP (95% confidence interval, 0.28, 3.18; P=0.02) and 2.07 mm Hg higher diastolic BP (95% confidence interval, 0.74, 3.39; P=0.003); every doubling in ß-1,3-d-Glucan concentration was associated with 0.80 mm Hg higher systolic BP (95% confidence interval, -0.07, 1.67; P=0.07) and 0.88 mm Hg higher diastolic BP (95% confidence interval, 0.09, 1.66; P=0.03). Vascular endothelial growth factor rose after concentrated ambient particle endotoxin exposure and attenuated the association between endotoxin and 0.5-hour postexposure diastolic BP (Pinteraction=0.02). In healthy adults, short-term endotoxin and ß-1,3-d-Glucan exposures were associated with increased BP. Our findings suggest that the biological PM components contribute to PM-related cardiovascular outcomes, and postexposure vascular endothelial growth factor elevation might be an adaptive response that attenuates these effects.

A high throughput colorimetric assay of ß-1,3-D-glucans by Congo red dye.

Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, ß-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for ß-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and ß-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (>20 nm) in UV-Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 µg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. ß-D-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of ß-1,3-D-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of ß-1,3-D-glucans was investigated in several mushroom species.