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.

Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function.

BACKGROUND: Cardiac metabolic dysfunction is a hallmark of heart failure (HF). Estrogen-related receptors ERRα and ERRγ are essential regulators of cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for HF. However, in vivo studies demonstrating the potential usefulness of ERR agonist for HF treatment are lacking, because compounds with pharmacokinetics appropriate for in vivo use have not been available. METHODS: Using a structure-based design approach, we designed and synthesized 2 structurally distinct pan-ERR agonists, SLU-PP-332 and SLU-PP-915. We investigated the effect of ERR agonist on cardiac function in a pressure overload-induced HF model in vivo. We conducted comprehensive functional, multi-omics (RNA sequencing and metabolomics studies), and genetic dependency studies both in vivo and in vitro to dissect the molecular mechanism, ERR isoform dependency, and target specificity. RESULTS: Both SLU-PP-332 and SLU-PP-915 significantly improved ejection fraction, ameliorated fibrosis, and increased survival associated with pressure overload-induced HF without affecting cardiac hypertrophy. A broad spectrum of metabolic genes was transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function. Metabolomics analysis showed substantial normalization of metabolic profiles in fatty acid/lipid and tricarboxylic acid/oxidative phosphorylation metabolites in the mouse heart with 6-week pressure overload. ERR agonists increase mitochondria oxidative capacity and fatty acid use in vitro and in vivo. Using both in vitro and in vivo genetic dependency experiments, we show that ERRγ is the main mediator of ERR agonism-induced transcriptional regulation and cardioprotection and definitively demonstrated target specificity. ERR agonism also led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocytes. CONCLUSIONS: ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload-induced HF in vivo. Our results provide direct pharmacologic evidence supporting the further development of ERR agonists as novel HF therapeutics.

Rh(I)-Catalyzed [5 + 2]/[2 + 2] Cycloaddition Cascade to Access a Cyclobutane-Fused [4-5-6-7] Tetracyclic Framework.

A Rh(I)-catalyzed [5 + 2]/[2 + 2] cycloaddition cascade has been developed to afford a complex and highly strained [4-5-6-7] tetracyclic framework in good yields and excellent diastereoselectivities. During this transformation, three rings, three C-C bonds, and four contiguous stereocenters were formed efficiently. Mechanistically, the rare sterically congested multisubstituted cyclobutanes are constructed readily through Michael addition and a Mannich reaction cascade.

The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

Comparative lipidomics of Pichia pastoris using constitutive promoter reveals lipid diversity and variability at different growth phases.

Pichia pastoris is an expression platform widely used for foreign protein expression, while it is unknown how the global lipid profiles changed during the cultivation process, which is crucial for fermentation optimization and chassis design. Therefore, this study aimed to reveal the diverse lipid profiles of P. pastoris controlled by constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase gene and to unravel their change in the lag, logarithmic, stationary, and death phases, using ultra-performance liquid chromatography/nano-electrospray ionization-tandem mass spectrometry. Two hundred forty lipid species across 11 lipid classes were detected, including various glycerolipids, glycerophospholipids, and sphingolipids. Pichia cells displayed high diversity and variability of lipids in lipid profile, relative intensity, phosphatidylinositol/phosphatidylserine ratio, fatty acid chain length, and unsaturation degree. Notably, increase of unsaturated triacylglycerol level was accompanied by rise of malondialdehyde level under oxidative stress. The increased ceramide with long fatty acid chain could be a key feature at death phase. This work deepened our understanding of the physiology of P. pastoris during cultivation and provided valuable information for further improvement of the P. pastoris expression system.

Comparative lipidomics of methanol induced Pichia pastoris cells at different culture phases uncovers the diversity and variability of lipids.

Pichia pastoris is an attractive eukaryotic host widely employed in industrial biotechnology for protein production and biocatalysis, and oxidative stress and other harsh conditions were frequently encountered during the cultivation cycle, however, the global lipidomic profile change needed to be revealed. The present study aimed to discover the variation in P. pastoris lipids at different stages (lag, logarithmic, stationary, induction and decline phases) by a sensitive ultra-performance liquid chromatography/nanoelectrospray-mass/mass spectrometry. We identified 253 lipid species across 11 lipids classes, including glycerophospholipids, glycerolipids and sphingolipids. High diversity and flexibility of lipids (including the composition and relative intensity) were observed during different phases, especially when glycerol was shifted to methanol. Especially, the unsaturated-double-bonds containing lipids showed a close relationship with the change of carbon source, which also led to increase of oxidative stress. Additionally, the relative intensity of sphingolipids was increased obviously in decline phase, likely associated with cell apoptosis. The current study expanded our understanding of the cell physiology of P. pastoris through the lipid profile change and lay the foundation for chassis design strains in the future by engineering microbial membrane.

Understanding a defensive response of methicillin-resistant Staphylococcus aureus after exposure to multiple cycles of sub-lethal blue light.

Blue light (BL) has shown bactericidal effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), one of the major clinical pathogens with antibiotic resistance. Bacteria likely respond to the oxidative stress induced by BL; however, the defensive response is still unclear. This study aimed to reveal the phenotypic change in MRSA after being exposed to 15 cycles of sub-lethal BL illumination. The comparative transcriptomic results showed that the expression of peptidoglycan (PG) synthesis gene glmS was significantly upregulated in the cells after the multiple cycle light treatment, and the biochemical analysis determined that the content of PG synthesized was increased by 25.86% when compared with that in control cells. Furthermore, significant thickening of the cell wall was observed under a transmission electron microscope (P < .05). The light sensitivity of the tested MRSA strain was reduced after the multiple cycle light treatment, indicating the possibility of MRSA being more adaptive to the BL stress. The present study suggested that multiple cycles of sub-lethal BL could change the light susceptibility of MRSA through thickening the cell wall.

Light-driven selective aerobic oxidation of (iso)quinoliniums and related heterocycles.

Selective C1-H/C4-H carbonylation of N-methylene iminium salts, catalyzed by visible-light photoredox and oxygen in the air, has been reported. A ruthenium complex acts as a chemical switch to conduct two different reaction pathways and to afford two different kinds of products. In the absence of the ruthenium complex, the Csp2-H bonds adjacent to the nitrogen atoms are oxidized to α-lactams by the N-methyleneiminium substrates themselves as photosensitizers. In the presence of the ruthenium complex, the oxidation reaction site of quinoliniums is switched to the C4 region, resulting in the formation of 4-quinolones. The use of two transformations directly introduces oxygen into the nitrogen heterocyclic skeletons under an air atmosphere.